C180-E059C

C180-E059C
Shimadzu Analysis Guidebook

Food Product Analyses
CONTENTS
H O 1. Food Product Components 2. Food Additives

1. 1 Analysis of Fatty Acids in Fish (1) - GC/MS/MS .............................................1 2. 1 Analysis of Food Preservatives (1) - GC/MS ................................................58
Analysis of Fatty Acids in Fish (2) - GC/MS/MS .............................................2 Analysis of Food Preservatives (2) - GC/MS ................................................59
1. 2 Analysis of Fatty Acids in Butter Using GC × GC/MS - GC/MS......................3 2. 2 High Speed Analysis of Benzoic Acid and Sorbic Acid - LC ........................60
1. 3 Quantitative Analysis of Trans Fatty Acid by FTIR (1) - FTIR .........................4 2. 3 Analysis of Natamycin in Cheese - LC ........................................................61
Quantitative Analysis of Trans Fatty Acid by FTIR (2) - FTIR .........................5 2. 4 Analysis of Food Antioxidants (1) - GC/MS ..................................................62
1. 4 Analysis of 3-MCPD Fatty Acid Diesters in Palm Oil (1) - LC/MS/MS ............6 Analysis of Food Antioxidants (2) - GC/MS .................................................63
Analysis of 3-MCPD Fatty Acid Diesters in Palm Oil (2) - LC/MS/MS ............7 2. 5 High Speed Analysis of Ascorbic Acid and Erythorbic Acid - LC .................64
1. 5 Quantitative Analysis of Fat in Milk by UV-Vis-NIR Reflectance 2. 6 High Speed Analysis of Tocopherols - LC ...................................................65
Spectroscopy and Multivariate Analysis (1) - UV ...........................................8 2. 7 Simultaneous Determination of Sweeteners in Food (1) - LC .....................66
Quantitative Analysis of Fat in Milk by UV-Vis-NIR Reflectance Simultaneous Determination of Sweeteners in Food (2) - LC .....................67
Spectroscopy and Multivariate Analysis (2) - UV ...........................................9 2. 8 Analysis of Cyclodextrins in Food (1) - LC ..................................................68
1. 6 Analysis of Orotic Acid in Yogurt - LC ...........................................................10 Analysis of Cyclodextrins in Food (2) - LC ..................................................69
1. 7 High Speed Analysis of Organic Acids - LC .................................................11 2. 9 Analysis of Sugar Alcohols in Food (1) - LC ................................................70
1. 8 Analysis of Amino Acids Contained in Vegetable Juice (1) - GC/MS............12 Analysis of Sugar Alcohols in Food (2) - LC ................................................71
Analysis of Amino Acids Contained in Vegetable Juice (2) - GC/MS............13 2.10 High Speed Analysis of Artificial Colorings (1) - LC .....................................72
1. 9 High Speed Analysis of Pre-Column Derivatized Amino Acids by High Speed Analysis of Artificial Colorings (2) - LC .....................................73
SIL-30AC Auto-sampler (1) - LC ..................................................................14
2.11 Analysis of Curcumin in Turmeric - LC .........................................................74
High Speed Analysis of Pre-Column Derivatized Amino Acids by
SIL-30AC Auto-sampler (2) - LC ..................................................................15 2.12 Analysis of Impurities in Curcumin (1) - LC/MS ............................................75
1.10 High Speed Analysis of Pre-Column Derivatized Amino Acids in Alcoholic Analysis of Impurities in Curcumin (2) - LC/MS ............................................76
Beverage (1) - LC .........................................................................................16 2.13 Analysis of Sudan Dyes in Foods - LC .........................................................77
High Speed Analysis of Pre-Column Derivatized Amino Acids in Alcoholic 2.14 Analysis of Sudan Dyes and Para Red - LC/MS ..........................................78
Beverage (2) - LC .........................................................................................17 2.15 Analysis of Essential Oil - GC ......................................................................79
1.11 Ultra High-Sensitivity Analysis of Water-Soluble Vitamins - LC ....................18 2.16 Flavor Analysis by Fast - GC/MS (1) - GC/MS .............................................80
1.12 High-Sensitivity Analysis of Retinol Acetate and Retinol Palmitate - LC ......19 Flavor Analysis by Fast - GC/MS (2) - GC/MS .............................................81
1.13 Analysis of Water-Soluble Vitamins with Multi-Mode ODS Column (1) - LC/MS ......20 2.17 Analysis of Benzoyl Peroxide in Foods - LC ................................................82
Analysis of Water-Soluble Vitamins with Multi-Mode ODS Column (2) - LC/MS ..................21
1.14 High Speed Analysis of Nucleobases, Nucleosides, and Nucleotides (1) - LC.....22
High Speed Analysis of Nucleobases, Nucleosides, and Nucleotides (2) - LC.....23
1.15 Analysis of Oligosaccharides in Beer - LC ...................................................24 3. Aromas and Odors
1.16 Determination of Fructo-oligosaccharides in Food - LC ...............................25 3. 1 Analysis of Aroma Compounds in Peach Juice (1) - GC/MS........................83
1.17 Analysis of Saccharides Using Post-Column Derivatization System (1) - LC ....26 Analysis of Aroma Compounds in Peach Juice (2) - GC/MS........................84
Analysis of Saccharides Using Post-Column Derivatization System (2) - LC ....27 3. 2 Analysis of Aroma Compounds in Cheese (1) - GC/MS ...............................85
1.18 Analysis of Ethyl Į -D-Glucoside in Japanese Sake - LC..............................28 Analysis of Aroma Compounds in Cheese (2) - GC/MS ...............................86
1.19 Analysis of Alliin in Garlic - LC......................................................................29 3. 3 Higher Sensitivity Analysis of 2-Methoxy-3-Isobutylpyrazine (MIBP) in
1.20 High Speed Analysis of Catechins in Green Tea (1) - LC .............................30 Wine (1) - GC/MS/MS ..................................................................................87
High Speed Analysis of Catechins in Green Tea (2) - LC .............................31 Higher Sensitivity Analysis of 2-Methoxy-3-Isobutylpyrazine (MIBP) in
1.21 High Speed Analysis of Resveratrol in Wine - LC ........................................32 Wine (2) - GC/MS/MS ..................................................................................88
1.22 High Speed Analysis of Phenolic Acids (1) - LC ...........................................33
High Speed Analysis of Phenolic Acids (2) - LC ...........................................34
1.23 High Speed Analysis of Į -Acids and ȕ -Acids in Hops (1) - LC ...................35
High Speed Analysis of Į -Acids and ȕ -Acids in Hops (2) - LC ...................36
4. Residual Pesticides
1.24 High Speed Analysis of Iso-Į -Acids and Į -Acids in Beer (1) - LC ................37 4. 1 Analysis of Organophosphorus Pesticides in Baby Foods (1) - GC/MS/MS....89
High Speed Analysis of Iso-Į -Acids and Į -Acids in Beer (2) - LC ................38 Analysis of Organophosphorus Pesticides in Baby Foods (2) - GC/MS/MS....90
1.25 Analysis of Lycopene and ȕ -Carotene in Tomato - LC .................................39 4. 2 Analysis of Pesticides in Baby Foods (1) - GC/MS/MS ................................91
1.26 Determination of Cyanidin-3-Glucoside in Black Soybeans - LC .................40 Analysis of Pesticides in Baby Foods (2) - GC/MS/MS ................................92
1.27 Analysis of Capsaicinoids in Spices - LC .....................................................41 4. 3 Analysis of Pesticides in Citrus Oil (1) - GC/MS/MS ....................................93
1.28 High Speed Analysis of Isothiocyanates and Sinigrin - LC ...........................42 Analysis of Pesticides in Citrus Oil (2) - GC/MS/MS ....................................94
1.29 High Speed Analysis of Isoflavones in Soy - LC ..........................................43 4. 4 Simultaneous Analysis of Residual Pesticides in Foods via the
1.30 Analysis of Flavonoids in Ginkgo Biloba Extract (1) - LC .............................44 QuEChERS Method (1) - GC/MS/MS...........................................................95
Analysis of Flavonoids in Ginkgo Biloba Extract (2) - LC .............................45 Simultaneous Analysis of Residual Pesticides in Foods via the
QuEChERS Method (2) - GC/MS/MS...........................................................96
1.31 Analysis of Terpenoids in Ginkgo Biloba - LC...............................................46
4. 5 Scan/MRM Analysis of Residual Pesticides in Foods (1) - GC/MS/MS........97
1.32 Analysis of Ginkgolic Acids in Ginkgo Biloba Extract (1) - LC ......................47
Scan/MRM Analysis of Residual Pesticides in Foods (2) - GC/MS/MS........98
Analysis of Ginkgolic Acids in Ginkgo Biloba Extract (2) - LC ......................48
4. 6 Screening of Residual Pesticides in Food with Two Different Columns (1)
1.33 High Speed Analysis of Gingerol and Shogaol in Ginger (1) - LC ................49 - GC/MS .......................................................................................................99
High Speed Analysis of Gingerol and Shogaol in Ginger (2) - LC ................50 Screening of Residual Pesticides in Food with Two Different Columns (2)
1.34 High Speed Analysis of Flavanones in Citrus Juice (1) - LC ........................51 - GC/MS .....................................................................................................100
High Speed Analysis of Flavanones in Citrus Juice (2) - LC ........................52 4. 7 Easy Screening for Residual Pesticides in Processed Foods (1)
1.35 High Speed Analysis of Ginsenosides in Ginseng (1) - LC ..........................53 - GC/MS/MS ...............................................................................................101
High Speed Analysis of Ginsenosides in Ginseng (2) - LC ..........................54 4. 7 Easy Screening for Residual Pesticides in Processed Foods (2)
1.36 High Speed Analysis of Lutein and Zeaxanthin (1) - LC ...............................55 - GC/MS/MS ...............................................................................................102
High Speed Analysis of Lutein and Zeaxanthin (2) - LC ...............................56 4. 8 High Throughput LC-MS/MS Analysis of Carbendazim in Orange Juice (1)
- LC/MS/MS ................................................................................................103
1.37 Determination of Coenzyme Q10 in Food - LC ............................................57
High Throughput LC-MS/MS Analysis of Carbendazim in Orange Juice (2)
- LC/MS/MS ................................................................................................104
CONTENTS
Cd
Pb 9DULHW\,GHQWLILFDWLRQ*HQHWLF
Ca 5. Inorganic Metals $QDO\VLV,UUDGLDWHG)RRG
5. 1 Measurement of Lead in Sugar (1) - AA .....................................................105
,GHQWLILFDWLRQRI5LFH9DULHWLHVí0&( ...................................................152
Measurement of Lead in Sugar (2) - AA .....................................................106
,GHQWLILFDWLRQRI5LFH9DULHWLHVí0&( ...................................................153
5. 2 Measurement of Minerals in Dietary Supplements (1) - AA........................107
,GHQWLILFDWLRQRI7KXQQXVí0&( ...........................................................154
Measurement of Minerals in Dietary Supplements (2) - AA........................108
,GHQWLILFDWLRQRI7KXQQXVí0&( ...........................................................155
5. 3 Measurement of Cadmium and Lead in Food Additives (1) - AA................109
9. 3 Spectroscopic Measurement and Multivariate Analysis for Classification of
Measurement of Cadmium and Lead in Food Additives (2) - AA................110 %HHU7\SHVí89 ...................................................................................156
5. 4 Measurement of Heavy Metals (Cd, Pb) in Pet Food (1) - AA .................... 111 Spectroscopic Measurement and Multivariate Analysis for Classification of
Measurement of Heavy Metals (Cd, Pb) in Pet Food (2) - AA ....................112 %HHU7\SHVí89 ...................................................................................157
5. 5 Measurement of Cadmium in Brown Rice (1) - AA .....................................113 9. 3 Spectroscopic Measurement and Multivariate Analysis for Classification of
Measurement of Cadmium in Brown Rice (2) - AA .....................................114 %HHU7\SHVí89 ...................................................................................158
'HWHFWLRQRI$OOHUJHQLF6XEVWDQFHVí0&( ..........................................159
'HWHFWLRQRI$OOHUJHQLF6XEVWDQFHVí0&( ..........................................160
'HWHFWLRQRI)RRG3RLVRQLQJ5HODWHG*HQHVí0&(............................161
7R[LQ,QRUJDQLF3RLVRQ 'HWHFWLRQRI)RRG3RLVRQLQJ5HODWHG*HQHVí0&(............................162
4XDOLWDWLYH$QDO\VLVRI*HQHWLFDOO\0RGLILHG&RUQí0&(.....................163
6. 1 Analysis of Diarrhetic Shellfish Poison (DSP) (1) - LC/MS .........................115
4XDOLWDWLYH$QDO\VLVRI*HQHWLFDOO\0RGLILHG&RUQí0&(.....................164
Analysis of Diarrhetic Shellfish Poison (DSP) (2) - LC/MS .........................116
'HWHFWLRQRI0ROGDQG<HDVW*HQHVí0&(..........................................165
6. 2 High Speed Analysis of Aflatoxins in Food (1) - LC ....................................117
'HWHFWLRQRI0ROGDQG<HDVW*HQHVí0&(..........................................166
High Speed Analysis of Aflatoxins in Food (2) - LC ....................................118
'HWHFWLRQRI,UUDGLDWHG)RRGVí*&06 ...............................................167
6. 3 Analysis of Aflatoxin B1, B2, G1 and G2 at High Sensitivity (1) - LC .............119
'HWHFWLRQRI,UUDGLDWHG)RRGVí*&06 ...............................................168
Analysis of Aflatoxin B1, B2, G1 and G2 at High Sensitivity (2) - LC .............120
Analysis of Aflatoxin B1, B2, G1 and G2 at High Sensitivity (3) - LC .............121
6. 4 High Speed Analysis of Nivalenol and Deoxynivalenol (1) - LC .................122
High Speed Analysis of Nivalenol and Deoxynivalenol (2) - LC .................123
6. 5 Analysis of Arsenic in Foods (1) - EDX ......................................................124
10. )RRG&RQWDLQHUV3DFNLQJ0DWHULDOV
Analysis of Arsenic in Foods (2) - EDX ......................................................125 /HDFK7HVWIRU3KHQROLQ5XEEHU1LSSOHVí89.....................................169
/HDFK7HVWIRU3KHQROLQ5XEEHU1LSSOHVí89.....................................170
$QDO\VLVRI(SLFKORURK\GULQ'LVVROYHGIURP0HWDO)RRG&DQVí*&..........171
10.3 $QDO\VLVRI7ULHWK\ODPLQHDQG7ULEXW\ODPLQHLQ3RO\FDUERQDWH3ODVWLFVí*& ...172
7. )RUHLJQ0DWWHUV2IIHQVLYH2GRUV $QDO\VLVRI7ULHWK\ODPLQHDQG7ULEXW\ODPLQHLQ3RO\FDUERQDWH3ODVWLFVí*& ...173
7. 1 High Sensitivity Analysis of 2,4,6-Trichloroanisole in Wine (1) - GC/MS/MS ...126 10.4 $QDO\VLVRI9LQ\OLGHQH&KORULGHLQ3RO\YLQ\OLGHQH&KORULGH3ODVWLFVí*& ...174
High Sensitivity Analysis of 2,4,6-Trichloroanisole in Wine (2) - GC/MS/MS ...127 $QDO\VLVRI9LQ\O&KORULGHLQ3RO\YLQ\O&KORULGH3ODVWLFVí*&...................175
7. 2 Rapid Determination of Melamine in Food (1) - LC ....................................128 10.6 $QDO\VLVRI0HWK\O0HWKDFU\ODWHLQ3RO\PHWK\O0HWKDFU\ODWH3ODVWLFVí*& ...176
Rapid Determination of Melamine in Food (2) - LC ....................................129 $QDO\VLVRI+HDY\(OHPHQWVLQ7DEOHZDUHí,&3$(6 ..........................177
7. 3 Analysis of Contaminant Adhering to Frozen Pizza (1) $QDO\VLVRI+HDY\(OHPHQWVLQ7DEOHZDUHí,&3$(6 ..........................178
- FTIR/EDX/EPMA......................................................................................130 $QDO\VLVRI+HDY\(OHPHQWVLQD&XSí('; .............................................179
Analysis of Contaminant Adhering to Frozen Pizza (2) ;5D\&72EVHUYDWLRQRI6HDOVRI)RRG3URGXFW3DFNDJLQJí1', .....180
- FTIR/EDX/EPMA......................................................................................131 ;5D\&72EVHUYDWLRQRI6HDOVRI)RRG3URGXFW3DFNDJLQJí1', .....181
7. 4 Introduction of a Search System for Contaminants in Tap Water (1) ;5D\&72EVHUYDWLRQRI6HDOVRI)RRG3URGXFW3DFNDJLQJí1', .....182
- FTIR/EDX .................................................................................................132
Introduction of a Search System for Contaminants in Tap Water (2)
- FTIR/EDX .................................................................................................133
7. 5 Identification of Contaminants (Animal Hair) (1) - MCE..............................134
Identification of Contaminants (Animal Hair) (2) - MCE..............................135
7. 6 Analysis of Odor in Frozen Shrimp (1) - GC/MS ........................................136
Analysis of Odor in Frozen Shrimp (2) - GC/MS ........................................137
8. Food Properties
8. 1 Melting of Chocolate - TA ...........................................................................138
8. 2 Gelatinization of Starch - TA .......................................................................139
8. 3 DSC Measurement of Liquor - TA...............................................................140
8. 4 DSC Measurement of Fish Meat - TA.........................................................141
8. 5 Protein Denaturation and Texture Analysis for Chicken (1) - TA.................142
Protein Denaturation and Texture Analysis for Chicken (2) - TM................143
8. 6 Texture Analysis of "Soumen" Japanese Vermicelli (1) - TM ......................144
Texture Analysis of "Soumen" Japanese Vermicelli (2) - TM ......................145
8. 7 Measurement of Texture Characteristics of Rice - TM ...............................146
8. 8 Measurement of Texture of Pork - TM ........................................................147
8. 9 Texture Evaluation of Care Food (1) - TM ..................................................148
Texture Evaluation of Care Food (2) - TM ..................................................149
8.10 Particle Size Distribution Measurement of Chocolate (1) - PT ...................150
Particle Size Distribution Measurement of Chocolate (2) - PT ...................151
C
H O
1. Food Product Components
1.1 Analysis of Fatty Acids in Fish (1) - GC/MS/MS
Q Explanation
While some fatty acids, such as the n-3 fatty acids EPA 200 mg of Pulverized Edible Flesh
and DHA, are beneficial to human health because they
2 mL of extraction liquid added
lower the amount of blood-borne neutral fat, too much
Agitated and then centrifuged
intake of saturated fatty acids raises the risk of some
diseases. For this reason, there is a need for the batch 500 μL of Extracted Liquid
analysis of these fatty acids in the life sciences and food
Dried with nitrogen gas
engineering sectors. Despite requiring methylation, GC-
MS has gained attention because of its suitability for Dried Residue
multicomponent batch analyses. In fatty acid analyses
500 μL each of reagents A and B added
utilizing GC-MS, the EI (electron ionization) method
Left standing 1 hour at 37 $C
is used for ionization. With the EI method, there are
500 μL of reagent C added
many types of fragment ions, making it easy to select
Left standing 20 minutes at 37 $C
an m/z to enable separation by mass from impurities.
2 mL of extraction liquid added
However, because of the large number of fragment ions,
Centrifuged
the sensitivity of the individual ions is reduced, making
it difficult to detect trace quantities of fatty acids. In Organic Phase
contrast, with the PCI (positive chemical ionization)
1 mL deionized water
method, protonated molecular ions can be detected, from
Agitated and then centrifuged
which molecular weight data can be obtained. Since
there is only a small number of fragment ion types, the Organic Phase
sensitivity is increased. This means, however, that the
ion types that can be selected for monitoring are limited
and there may not be any ions that can be separated by GC/MS and GC/MS/MS Measurements
mass from impurities. Here we introduce the results of an
investigation of separation from impurities based on the
EISIM, PCI-SIM, EI-MRM, and PCI-MRM methods. Fig. 1.1.1 Pretreatment of Saury
Q Pretreatment Method QAnalysis Results

Saury (fish) was used to investigate the separation The sample extracted from the saury was measured in
from impurities in each analysis mode. The fatty acid each analysis mode, and the separation from impurities
methylation kit (P/N: 06482) sold by Nacalai Tesque was investigated. Most of the fatty acid methyl esters could be
utilized for the pretreatment. The pretreatment method is separated from the impurities regardless of the analysis
shown in Fig. . . . The edible esh from the saury was mode. However, a portion of the fatty acids was hard
collected and pulverized with a mill, after which 200 mg to completely separate from the impurities, both with
was measured out. After adding 2 mL of the extraction EI-SIM and EI-MRM. Fig. 1.1.2 shows examples of
liquid and agitating, the mixture was centrifuged, and measuring fatty acid methyl esters for which the degree of
500 μL of extracted liquid was obtained. The extracted separation from impurities varied signi cantly depending
liquid was dried under a nitrogen ow, and 500 μL each of on the analysis mode. Methyl linolenate;(Z)18:3n-3 and
reagents A and B were added. After leaving the mixture to methyl cis-11,14,17-Icosatrienoate;(Z)20:3n-3 were hard
stand for 1 hour at 37 °C, 500 μL of reagent C was added, to separate from the impurities, both with EI-SIM and
and it was left to stand at 37 °C for a further 20 minutes. EI-MRM. Some degree of separation was possible with
Afterward, 2 mL of the extraction liquid was added, PCI-SIM, but there was only one kind of monitoring m/z,
and after centrifuging, the organic phase was collected. so problems with peak identi cation could be expected.
Deionized water was used to clean 1 mL of the organic In contrast, with PCI-MRM, mass separation excluded
phase, resulting in the test solution. Refer to the next page impurities eluted nearby, making peak identi cation easy.
for the analytical conditions for the EI-SIM, PCI-SIM,
EI-MRM, and PCI-MRM methods. Analysis methods
included in the GC/MS Metabolite Database Ver. 2 were
used for the analysis conditions and monitoring m/z.
1
1.1 Analysis of Fatty Acids in Fish (2) - GC/MS/MS
QAnalytical Conditions
Instrument : GCMS-TQ8030
Column : SP-2560
/HQJWKPPP,'GI ƫP [MS]
Glass Insert : 6SOLWOHVVLQVHUWZLWKZRRO31 Interface Temp. : 250 °C
[GC] Ion Source Temp. : 200 °C
Injection Temp. : 250 °C Measurement Mode :
Column Temp. : &PLQ-&PLQ-&PLQ GC/MS : SIM
Injection Mode : Split GC/MS/MS : MRM
Split Ratio : 1:10 Ionization Method : EI and PCI methods
Carrier Gas Control : /LQHDUYHORFLW\FPVHF PCI Reagent Gas : Isobutane
Injection Volume : ƫ/ PCI Reagent Gas Pressure: 70 kPa
Methyl linolenate;(Z)18:3n-3
EI-SIM EI-MRM PCI-SIM PCI-MRM
(×1,000,000) (×10,000) (×1,000,000) (×100,000)
7.0 1.25
2.0 292.20 292.30>135.10 293.20
236.00 293.30>261.30
6.0 236.20>175.10 1.00 293.30>243.20
1.00
1.5 5.0
0.75
4.0 0.75
1.0 3.0 0.50
0.50
2.0
0.5 0.25 0.25
1.0
51.75 52.00 52.25 52.50 51.75 52.00 52.25 52.50 52.75 53.00 53.25 53.50 53.00 53.25 53.50
Methyl cis -11,14,17- Icosatrienoate;(Z)20:3n-3

EI-SIM EI-MRM PCI-SIM PCI-MRM
(×1,000,000) (×100,000) (×100,000) (×10,000)
320.30 320.30>121.10 321.30 321.30>289.30
7.5 264.00 6.0 1.50 321.30>271.30
264.30>136.10
4.0
5.0 1.25
5.0 3.0 4.0 1.00
2.0 3.0 0.75

2.5 2.0 0.50
1.0
1.0 0.25
54.50 54.75 55.00 55.25 54.50 54.75 55.00 55.25 55.75 56.00 56.25 56.50 55.75 56.00 56.25 56.50
Fig. 1.1.2 Mass Chromatograms for Methyl linolenate;(Z)18:3n-3 and Methyl cis-11,14,17-Icosatrienoate;(Z)20:3n-3
Contained in an Extract of Saury Measured in Individual Analysis Modes
We analyzed fatty acids in sh to investigate mass separation from impurities and sensitivity for the EI-SIM, EI-MRM,
PCI-SIM, and PCI-MRM analysis modes. The results revealed that the PCI method is the most sensitive, and for unsaturated
fatty acids in particular, provides more sensitive detection than the EI method. Also, PCI-MRM was found to be the most
ideal for mass separation from impurities, making peak identi cation easy. It is thus evident that the PCI-MRM method is
effective for multicomponent batch analyses of fatty acids.
2
C Food Product Components
H O
1.2 Analysis of Fatty Acids in Butter Using GC × GC/MS - GC/MS
Q Explanation
Fig. 1.2.1 shows the results from GC GC-MS analysis of methyl esteri ed lipids extracted from commercial butter
by the Folch method. It con rms the main components, palmitic acid (C16) and oleic acid (C18 1ω6), as big blobs (see
Fig 1.2.1). C18 fatty acids include a variety of components and isomers, but these can be separated into their respective
components for highly accurate qualitative and quantitative results by using a second column with high polarity.
GC × GC Modulator : ZX1-GC × GC Modulator
GC-MS : GCMS-QP2010 Ultra
[GC × GC] [MS]
Column : VW'%P/ðPP,'ƫP Interface Temp. : 240 °C
QG5W[:$;P/ðPP,'ƫP Ion Source Temp. : 200 °C
Injection Volume : ƫ/ Solvent Elution Time : 15.5 min
Injection Mode : 6SOLWVSOLWUDWLR Data Sampling Time : 16 min to 80 min
Injection Temp. : 250 °C Measurement Mode : Scan
Column Temp. : &PLQ&PLQ&&min Mass Range : m/z 45-330
&PLQ Event Time : 0.02 sec
Control Mode : &RQVWDQWSUHVVXUHN3D
Modulation Time : 8 sec
Hot Pulse Time : VHF&
Gamma linolenic
acid
Linoleic acid
Elaidic acid
Palmitic acid
Oleic acid
Stearic acid
Fig. 1.2.1 2-Dimensional Image of GC × GC-MS Analysis Results for Butter
3
1.3 Quantitative Analysis of Trans Fatty Acid by FTIR (1) - FTIR
Q Explanation
Trans fatty acids are a kind of fat that is found in foods.
Excessive ingestion of this type of fat is associated
with elevated LDL cholesterol (low density lipoprotein
cholesterol) levels, which increases the risk of heart
disease1). In Japan, there is currently, no obligation to
display trans fatty acid content on food labels, nor is
there a specified content limit in Japan. However, food
entrepreneurs have been taking measures for some time
by independently developing low trans fatty acid foods
and selling them on the market. On the other hand, nations
Fig. 1.3.1 PIKE MIRacle A ATR Accessory with Optional
such as the United States and Denmark are obligated to Heated Crystal Plate
display the trans fatty acid content on food labels and to
adhere to content restrictions on trans fatty acids. Here we
introduce a quantitative analysis study we conducted for Cis Unsaturated Fatty Acid Trans Unsaturated Fatty Acid
trans fatty acids by the ATR and transmission methods. H2
H
C
QMeasurement of Trans Fatty Acid by Single C C
H2 H H2
5HÀHFWLRQ$756SHFWURVFRS\ H2
C C C C
C H
According to the AOCS method, analysis of trans C C C
fatty acid content by FTIR is to be conducted by the C
transmission method 2) and the ATR 3) method. Here we H2 C H2 H H2
used the latter method, which features easier operation. H2

Fig. 1.3.1 shows the MIRacle A (ZnSe prism) single
re ectance ATR (Attenuated Total Re ectance) accessory
Fig. 1.3.2 Structures of Cis and Trans Fatty Acids
that was used to conduct measurement of the trans fatty
acid content. A temperature controller was used during
Table 1.3.1 Analytical Conditions
measurement to maintain the prism temperature at 65 °C
(±2 °C). Unsaturated fatty acids are characterized as Analytical Instrument : IRPrestige-21, MIRacle A
being either a cis type or trans type of isomer, as shown KHDWLQJSODWH
in Fig. 1.3.2. The samples used for analysis consisted of Resolution : 4 cm-1
triolein (Glyceryl Trioleate, Sigma Corp.), with a single Accumulation : 45
cis double bond, and trielaidin (Tokyo Chemical Industry, Detector : DLATGS
Co., Ltd.), having a trans type double bond. Table 1.3.1
Abs
shows the analytical conditions used, and Fig. 1.3.3
0.14 Triolein Ɏ
shows the infrared spectra of triolein and trielaidin. In Trielaidin Ɏ
the infrared spectrum of trielaidin, the characteristic 0.12
trans-vinylene peak of the trans-type unsaturated fatty 0.1
acid is seen at 966 cm-1. Next, using triolein as a base,
0.08
we prepared sample solutions spiked with trielaidin at
concentrations of 0.1, 0.2, 0.5, 1.0, 2.0, 5.0, and 10.0 %, 0.06
respectively, to generate a trans fatty acid calibration 0.04
curve. The infrared spectra were measured similarly as
0.02
described above. Fig. 1.3.4 shows the trans fatty acid peak
in the vicinity of 966 cm-1 which was used to generate the 0
calibration curve. An excellent correlation coef cient of 4000 3000 2000 1500 1000 750
0.9999 was obtained with respect to the calibration curve 1/cm
generated from this, as shown in Fig. 1.3.5.
Fig. 1.3.3 Infrared Spectra of Triolein and Trielaidin
4
H O
1.3 Quantitative Analysis of Trans Fatty Acid by FTIR (2) - FTIR
Abs Abs
Trielaidin 10.0 % content 2 Trielaidin (non-spiked)
Trielaidin (10.0% spiked)
1.5
0.5
Trielaidin 0.1 % content 0
1000 990 980 970 960 950 940 930 1/cm 4000 3000 2000 1500 1000 750 500 1/cm
Fig. 1.3.4 Trans Fatty Acid Peak Region Selected for Calibration Curve Fig. 1.3.6 Infrared Spectra of Unspiked Olive Oil and Olive Oil
Spiked with Trielaidin
Abs Trielaidin (10.0 % spiked)
Trielaidin (non-spiked)
1000 990 980 970 960 950 940 930 1/cm

Fig. 1.3.5 Calibration Curve of Trans Fatty Acid by Single
Re ection ATR Spectroscopy Fig. 1.3.7 Trans Fatty Acid Peak Region Selected for Calibration Curve
Q Measurement of Trans Fatty Acid by

Transmission Spectroscopy
We also conducted measurement by the transmission method.
Detection with good S/N ratio is possible by selecting a fixed
thickness cell with an appropriate wavelength. Here we used a
0.1 mm- xed thickness cell (window plate material: KBr). This
time, using as a base a commercially available olive oil in which
the principle substance is a cis isomer, trielaidin-spiked samples
were prepared at concentrations of 0.1, 0.5, 1.0, 2.0, 5.0, and 10.0 %
respectively. Measurement was conducted by the ATR method
using the same conditions as shown in Table 1.3.1.
The infrared spectra obtained from measurement of unspiked olive
oil and 10.0 % trielaidin-spiked olive oil are shown in Fig. 1.3.6,
Fig. 1.3.7 shows the trans fatty acid peak in the vicinity of 966 cm-1
which was used to generate the calibration curve. An excellent
correlation coef cient of 0.9999 was obtained with respect to the Fig. 1.3.8 Calibration Curve of Trans Fatty Acid by Transmission
calibration curve generated from this, as shown in Fig. 1.3.8. Spectroscopy
[Reference]
1) Information on Trans Fatty Acids, Ministry of Agriculture, Forestry QConclusion
and Fisheries Web Page A calibration curve was generated using ATR method
http://www.maff.go.jp/j/syouan/seisaku/trans_fat/
2) AOCS Of cial Method Cd 14-95
and transmission method for trielaidin-spiked samples
Isolated trans Isomers infrared Spectrometric Method prepared at concentrations from 0.1 % to 10.0 %. Using
3) AOCS Of cial Method Cd 14d-99 either method, an excellent correlation coefficient of
Rapid Determination of Isolated trans Geometric Isomers in Fats 0.9999 was obtained, con rming that these methods are
And oils by Attenuated Total Re ection Infrared Spectroscopy effective for quantitative analysis of trans fatty acids.
5
1.4 Analysis of 3-MCPD Fatty Acid Diesters in Palm Oil (1) - LC/MS/MS
Q Explanation GC/MS following derivatization with phenylboronic acid (DGF

3-MCPD (3-monochloropropane-1,2-diol) is a byproduct that Standard methods 2009, Section C-Fats) has traditionally been
is formed in the production of condiments such as soy sauce used for analysis of 3-MCPD fatty acid esters, yet direct analysis
when hydrochloric acid is used to accelerate the hydrolysis of by LC/MS/MS without derivatization is gaining attention as an
vegetable proteins such as defatted soybean and wheat gluten. attractive alternative method. Significant amounts of 3-MCPD
According to the risk assessment of 3-MCPD by the Joint FAO/ fatty acid esters are present in numerous natural vegetable oils,
WHO Expert Committee on Food Additives (JECFA), 3-MCPD and their concentration is particularly high in palm oil. Here, we
is not considered to be genotoxic or carcinogenic. However, introduce the quantitative analysis of 3-MCPD fatty acid esters in
animal tests have indicated that it adversely affects the kidneys if palm oil using LC/MS/MS.
ingested in large quantities over a long period of time. In Japan, QAnalysis of Standard Samples
it has been con rmed that there is no 3-MCPD present in honjozo Synthetic 3-MCPD-dipalmitoyl ester and 3-MCPD-dioleoyl ester
(authentically-brewed) soy sauce produced by a traditional were used as standard samples. Electrospray ionization (ESI)
method, which accounts for 85 % of the soy sauce produced in was used as the ionization method and the 3-MCPD-di-fatty acid
Japan. The general dietary intake of 3-MCPD that can be ingested esters were detected as NH4+ adduct ions due to the addition of
without causing problems is not regulated in Japan. However, ammonium acetate in the mobile phase. The MS/MS spectra
measures have been implemented to improve upon production obtained using the adduct ions as the precursor are shown in
methods and limit the inclusion of 3-MCPD. Recently, the Fig. 1.4.1. Varying the collision energies (CE) produced the MS/
presence of 3-MCPD fatty acid esters have been reported in many MS spectra in Fig. 1.4.1 with the top, middle, and bottom spectra
foods containing refined edible oils. The toxicity of 3-MCPD generated by 10, 30, and 40 V, respectively. As each one of the fatty
fatty acid esters has not yet been clari ed, therefore the analysis acids is desorbed, it is detected as a product ion. Fig. 1.4.2 shows
of 3-MCPD fatty acid ester is very important. The application of the MRM chromatograms of the standard samples (1 μg/L).
O O
C35H67ClO4 C39H71ClO4
A O
O
O
Exact Mass: 586.47 B O
O
O
Exact Mass: 638.50
Mol. Wt.: 587.36 Mol. Wt.: 639.43
Cl Cl
Inten.(×100,000) Inten.(×100,000)
10 V [M+H-C16H32O2]+ 1.0 10 V [M+H-C18H34O2]+

1.0 604 656
331 [M+NH4]+ [M+NH4]+
0.5 357
0.0 0.0
100 200 300 400 500 600 m/z 100 200 300 400 500 600 m/z
Inten.(×100,000) Inten.(×100,000)
1.5 1.5
30 V 331 30 V 357
1.0 1.0
0.5 0.5
0.0 0.0
100 200 300 400 500 600 m/z 100 200 300 400 500 600 m/z
Inten.(×10,000) Inten.(×10,000)
40 V 331 40 V 357
5.0 5.0
57
85 6995
0.0 0.0
100 200 300 400 500 600 m/z 100 200 300 400 500 600 m/z
Fig. 1.4.1 MS/MS Spectra of the Synthetic Samples (A: 3-MCPD-Dipalmitoyl Ester, B: 3-MCPD-Dioleoyl Ester)
604.40>331.10(+) 30 656.50>357.20(+)
40
5.985
6.083
35 A 25 B
30
20
25
15
20
15 10
10
5
5
0 0
4.0 5.0 6.0 7.0 min 5.0 6.0 7.0 8.0 min
Fig. 1.4.2 MRM Chromatograms of the Synthetic Samples (1 μg/L, A: 3-MCPD-Dipalmitoyl Ester, B: 3-MCPD-Dioleoyl Ester)
6
H O
1.4 Analysis of 3-MCPD Fatty Acid Diesters in Palm Oil (2) - LC/MS/MS
QCalibration Curve QAnalytical Conditions

The calibration curves for 3-MCPD-dipalmitoyl ester Column : Shim-pack XR-ODS 1Ⅱ
and 3-MCPD-dioleoyl ester are shown in Fig. 1.4.3A and PP/ðPP,'ƫP
Fig. 1.4.3B, respectively. Excellent linearity was obtained Mobile Phase A : Methanol with 3 mmol/L Ammonium
over a wide range from 1–1000 μg/L, with correlation
Acetate / Acetonitrile = 9/1
coef cient (R 2) values greater than 0.999.
The repeatability using 6 repeat measurements of Mobile Phase B : Acetone / Methanol with 3 mmol/L
3-MCPD-dipalmitoyl ester and 3-MCPD-dioleoyl ester Ammonium Acetate / Acetonitrile = 8/1/1
was 15.47 and 19.64 area %RSD, respectively, at 1 μg/L, Gradient Elution Method
and 6.54 and 9.32, respectively, at 10μg/L. Time Program : %PLQ ń PLQ ń
PLQńPLQ
QAnalysis of Palm Oil
Fig. 1.4.4 shows an example of analysis of palm oil. 169.9 mg Flowrate : 0.4 mL/min
of palm oil was weighed out, dissolved in 1 mL of hexane and Column Temp. : 40 °C
diluted 100 to 1 with acetone (588.6 times dilution), and then Injection Volume : ƫ/
analyzed. 3-MCPD dipalmitoyl ester and 3-MCPD-dioleoyl ester Probe Voltage : N9(6,3RVLWLYH0RGH
were detected in this diluted solution at approximately 10 μg/L Nenulizing Gas Flow : 1.5 L/min
(Fig. 1.4.4A and Fig. 1.4.4B). This corresponds to a concentration
in palm oil of about 6 mg/L of 3-MCPD-dipalmitoyl ester and Drying Gas Flow : 20 L/min
3-MCPD-dioleoyl ester, respectively. Thus, it is possible to use DL Temp. : 300 °C
a triple quadrupole mass spectrometer for detection of 3-MCPD Block Heater Temp. : 400°C
fatty acid esters using a simple pretreatment procedure that is DL / Q-array Voltage : Using default values
limited to sample dilution.
Area Area
125000
175000 A B
150000 100000
125000
75000
100000
75000 50000
50000
25000
25000
R2=0.9992873 R2=0.9998887
0 0
0 250 500 750 0 250 500 750
Concentration Concentration
Fig. 1.4.3 Calibration Curves (1–1000 μg/L, n = 6)
1:604.40>331.10(+) 200 2:656.50>357.20(+)

350 A B
6.108
6.020
175
300
150
250
125
200
100
150 75
100 50
50 25
0 0
4.0 5.0 6.0 7.0 min 5.0 6.0 7.0 8.0 min
Fig. 1.4.4 MRM Chromatograms of 3-MCPD Fatty Acid Diesters in Palm Oil (A:3-MCPD-Dipalmitoyl Ester, B:3-MCPD-Dioleoyl Ester)
7
1.5 4XDQWLWDWLYH$QDO\VLVRI)DWLQ0LONE\899LV1,55HÀHFWDQFH
Spectroscopy and Multivariate Analysis (1) - UV
Q Explanation Integrating
sphere
Milk is one of the most popular drinks among humans.
In recent years, however, there has been a great increase
in the sales of milk products with adjusted fat content.
Milk fat content is typically measured using the Roese-
Gottlieb or the Gerber method, but measurement by
these methods is ext remely time-consuming. We Securing jig
therefore investigated the use of the spectral re ectance
method as a simpler quantitative method. By applying a
combination of re ectance measurement using a screw-
top glass tube in conjunction with multivariate analysis, Fig. 1.5.1 Sample Set in Integrating Sphere
we found that the fat content could be determined quite
easily. As multivariate analysis methods, the multiple
R%
linear regression method, PLS method, and support 80.0
vector regression method (SVR) were used to conduct

a comparative analysis of the quantitative accuracy of 60.0
these quantitative methods. The results indicated that
the support vector regression method provided the best 40.0
quantitative accuracy. The results are introduced in this
paper. 20.0
Q7RWDO/XPLQRXV5HÀHFWDQFH0HDVXUHPHQWRI0LON 0.0
900.0 1500.0 2000.0 2300.0
Nine types of measurement samples with differing nm
levels of fat content (3 types of high-fat milk, 3 types of Fig. 1.5.2 Total Luminous Re ection Spectra of Samples
medium-fat milk, 3 types of low-fat milk) were used. The Red: High-Fat Milk, Black: Medium-Fat Milk, Blue:
fat content values displayed on the various milk cartons Low-Fat Milk
are shown in Table 1.5.1. To ensure that all of the samples
were positioned identically, measurement was conducted Table 1.5.1 Measurements of 9 Types of Milk Samples
with the integrating sphere mounted in a securing jig.
Sample Fat Content as Listed on Milk Carton (g/200 mL)
Each sample was transferred to a screw-top glass tube High-fat 1 9.3
which was then set in the integrating sphere as shown in High-fat 2 9.4
Fig. 1.5.1, and the total light reflectance was measured High-fat 3 9.5
twice for each sample by transferring the same sample to Medium-fat 1 7.6
Medium-fat 2 7.8
a different screw-top glass tube. Thus, a total of 18 data Medium-fat 3 7.6
points were obtained (9 × 2 = 18). The disposable screw- Low-fat 1 0.2
top glass tubes were discarded after each use. In addition, Low-fat 2 1.0
a Spectralon® re ectance standard (U. S. Labsphere, Inc.) Low-fat 3 2.0
was used for the re ectance measurements.
The measurement results are shown in Fig. 1.5.2. In QAnalytical Conditions
Fig. 1.5.2, the redcolored trace lines correspond to high- Instrument : UV-3600 UV-visible-near-infrared
fat milk, the black trace lines to medium-fat milk, and the spectrophotometer
blue trace lines to low-fat milk. The data clearly indicate MPC-3100 Large sample compartment
that the lower the fat content, the lower the reflectance ZLWKEXLOWLQLQWHJUDWLQJVSKHUH
becomes overall. An enlarged view of a portion of Measurement : 900 nm – 2300 nm
Fig. 1.5.2 is shown in Fig. 1.5.3. Looking at the blue-trace Wavelength Range
spectra of the low-fat milk, each of the two respective Scan Speed : Medium
repeat measurement results overlap nearly perfectly, Sampling Pitch : 1.0 nm
suggesting that exchanging the screw-top glass tube has Photometric Value : 5HÁHFWDQFH
little effect. The blue line spectra are clearly divided into Slit Width : QP
3 groups, corresponding to the descending order of fat
content from 2.0, 1.0 and 0.2 (g/200 mL).
8
H O
1.5 4XDQWLWDWLYH$QDO\VLVRI)DWLQ0LONE\899LV1,55HÀHFWDQFH
Spectroscopy and Multivariate Analysis (2) - UV
QResults of Quantitative Analysis R%
70.0
The multiple linear regression method of multivariate analysis,
the PLS method, and support vector regression method (SVR) Fat content 2.0 (g/200 mL)
60.0
were applied to the acquired data to quantify the fat content.
The rst and second samples of the high-fat, medium-fat, and Fat content 1.0 (g/200 mL)
low-fat samples of Table 1.5.1 were used as standard samples to

generate a calibration model. The fat content values displayed on
the milk cartons were taken as the true fat content values of the 40.0
standard samples. As for the support vector regression method,

this can be considered as a type of quantitative method of kernel
multivariate analysis which can also be applied to non-linear
data. In addition, the number 3 samples of the respective fat 20.0
content samples of Table 1.5.1 were used as veri cation samples

to confirm the prediction accuracy of the calibration models. Fat content 0.2 (g/200 mL)
The fat content prediction results for the verification samples
using each of the calibration models are shown in Table 1.5.2. 0.0
1000.0 1200.0 1400.0 1600.0 1800.0 1900.0
nm
The values in parentheses represent the error of the predicted
value with respect to the fat content displayed on the milk Fig. 1.5.3 Expanded Spectra of Fig. 1.5.2
carton. By comparing the amount of error among the various Red: High-Fat Milk, Black: Medium-Fat Milk,
techniques, it was determined that the best results were obtained Blue: Low-Fat Milk
using the support vector regression method. It is thought that the Note:
relationship between the re ectance spectrum and concentration Calculations using the multiple linear regression method were conducted with
is not a linear relationship (proportional relationship). Compared respect to four wavelengths, 1100 nm, 1200 nm, 1500 nm and 1800 nm. As
to the multiple linear regression method and PLS method, which for the PLS method, all the data from 1100 to 1500 nm were used and mean
demonstrate their effectiveness with linear data, the support centering was conducted to conduct the calculations. As for the support vector
regression method, all the data from 1100 nm to 1500 nm were used, and the
vector regression method may provide better results because parameters C = 1 and Ķ = 0.08 were calculated using a linear kernel function.
it can also handle non-linear data effectively. Calculations 1) The Unscrambler is a trademark or registered trademark of CAMO
associated with the PLS method and the support vector Software.
regression method were conducted using The Unscramber® 1) 2) Excel is a t rademark or registered t rademark of Microsof t
Corporation.
multivariate analysis software of CAMO Software company.
In addition, regression analysis calculations for the multiple
linear regression method were conducted using the "Regression
Analysis" feature the Microsoft Excel® 2) spreadsheet software.
Table 1.5.2 Concentration of Fat for Samples and Results Calculated by Multiple Linear Regression, PLS Regression and Support
Vector Regression
Fat Content Listed on Milk Predicted Results by Multiple Predicted Results by PLS Predicted Results by Support
Sample
Carton (g/200 mL) Linear Regression Method Method Vector Regression Method
High fat 3 (1st) 9.5 8.87（6.6 %） 9.57（0.7%） 9.72（2.3 %）

High fat 3 (2nd) 9.5 8.89（6.4 %） 9.67（1.6 %） 9.47（0.3 %）
Medium fat 3 (1st) 7.6 8.04（5.8 %） 8.40（10.5 %） 7.88（3.7 %）
Medium fat 3 (2nd) 7.6 7.59（0.1 %） 7.71（1.4 %） 7.31（3.8 %）
Low fat 3 (1st) 2.0 2.24（12.0 %） 1.78（11.0 %） 2.01（0.5 %）
Low fat 3 (2nd) 2.0 2.36（18.0 %） 1.72（14.0 %） 1.86（7.0 %）
QConclusion spectral transmission measurement of liquid samples

We conducted quantitation of fat content in various milk typically involves time-consuming washing of the cell after
products by applying multivariate analysis to reflectance each measurement, the current method which combines the
data. The result of a compar ison of th ree t y pes of use of the spectral re ectance method with disposable screw-
multivariate analysis including the multiple linear regression top glass tubes eliminates the need for troublesome cleaning,
method, the PLS method, and the support vector regression while also saving time. The results obtained here suggest that
method indicated that the support vector regression method this method is effective for the determination of fat content in
offered the best quantitative accuracy. In addition, while highly turbid samples such as milk.
9
1.6 Analysis of Orotic Acid in Yogurt - LC
Q Explanation QAnalytical Conditions

Orotic acid, a heteroaromatic compound discovered in Column : Hydrosphere C18 (150 mm L. × 4.6 mm I.D.,
whey, is also referred to as orotate, and uracil-6-carboxylic ƫP<0&&R/WG
acid. In the past, it was also called vitamin B13 , but
Mobile Phase : PPRO/6RGLXP Phosphate Buffer
since it is synthesized in vivo, it is no longer considered
to be a vitamin. Orotic acid is a major precursor of the S+
nucleic acid pyrimidine, and has been attracting attention Flowrate : 1.0 mL/min
due to its diverse physiological effects. In Japan, it has Column Temp. : 40 °C
been formulated into cosmetics and pharmaceuticals Injection Vol. : ƫ/
sold as "class 3 OTC drugs", namely OTC drugs with Detection : SPD-20A at 280 nm
minimum risk of side effects. On January 23, 2012, the
Pharmaceutical and Food Safety Bureau of the Japanese QAnalysis of Yogurt
Ministry of Health, Labour and Welfare published a Fig. 1.6.3 shows a chromatogram of yogurt, and Fig. 1.6.4
noti cation (No. 0123-3) regarding "a partial revision of shows that of a yogurt drink. After ltering the sample
the standards for a range of pharmaceutical products", through an ultra ltration membrane and then diluting it
stating that orotic acid, orotic acid potassium salt, and ten times with water, a 10 μL injection was made. The
magnesium salt were added to the list of items speci ed orotic acid content in the yogurt was found to be about
as "ingredients that are not considered as drugs unless 75 mg/L, and in the yogurt drink, about 35 mg/L.
drug efficacy is advocated". As a result, its increased
use in health foods, nutritional supplements, etc. can be mAU
25
expected. Here, we introduce an example of analysis of Peak 1
orotic acid in yogurt by reversed-phase chromatography 1. Orotic Acid
using a Shimadzu Prominence HPLC system. 20
15
10
Fig. 1.6.1 Structure of Orotic Acid 0 1 2 3 4 5 6 7 8 9 min
Fig. 1.6.3 Chromatogram of Yogurt (10 μL injected)

mAU
35.0
Peak 1 mAU
30.0 1. Orotic Acid 25
Peak
1. Orotic Acid
25.0 20
20.0
15
15.0 1
10
10.0
5.0 5
0.0 0
0 1 2 3 4 5 6 7 8 9 min 0 1 2 3 4 5 6 7 8 9 min
Fig. 1.6.2 Chromatogram of Standard Solution of Orotic Acid Fig. 1.6.4 Chromatogram of Yogurt Drink (10 μL injected)
(10 mg/L, 10 μL injected)
10
H O
1.7 High Speed Analysis of Organic Acids - LC
Q Explanation QAnalysis of Soft Drinks

HPLC analysis of organic acids (short-chain fatty acids) in food Figs. 1.7.2 and 1.7.3 show chromatograms of commercially
products is generally conducted using ion exclusion chromatography available soft drinks. After soft drink A and soft drink B
with post-column derivatization detection. However, this approach were diluted fifty-fold and twenty-fold, respectively, with
is limited from the standpoint of high-speed analysis. Here we mobile phase, and then filtered through a membrane filter
show an example of high-speed, high-resolution analysis of organic (pore diameter 0.2 μm), 4 μL of ltrate was injected for each
acids with the “Prominence UFLC” ultra-fast HPLC system using
reversed phase HPLC and an absorbance detector. analysis.
mAU
QAnalysis of a Standard Solution Q Peak 1
3.0
Retention of organic acids like acetic acid and citric acid on a 1. Citric acid
high-polarity, reversed phase column requires the use of 100 % 2.5
aqueous mobile phases. However, such a mobile phase tends
to adversely affect the longevity of silica type reversed phase 2.0
columns. With that in mind, we conducted an investigation using
the “Phenomenex Synergi Hydro- RP” ODS column (particle 1.5
diameter 2.5 μm), in which the polar group is endcapped to
1.0
not only strengthen the retention of polar compounds, but to
better withstand 100 % aqueous mobile phases. Fig. 1.7.1 shows 0.5
the chromatogram obtained from analysis of an organic acid
standard mixture (formic acid, malonic acid, acetic acid, and 0.0
citric acid, each 100 mg/L adjusted with mobile phase), using a 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 min
4 μL injection. Acidic phosphate buffer was used as the mobile
phase, and ultraviolet detection was carried out at 210 nm. With Fig. 1.7.2 Soft Drink A (50 × dilution, 4 μL injected)
these analytical conditions, the organic acids were separated in
under 2 minutes. mAU
1 ■ Peaks
QAnalytical Conditions 4.5 1. Citric acid
Column : 3KHQRPHQH[6\QHUJLƫP+\GUR53c 4.0
(100 mm L. × 3.0 mm I.D., ƫP 3.5
Mobile Phase : PPRO/6RGLXP3KRVSKDWH%XIIHUS+ 3.0
Flowrate : 0.8 mL/min 2.5
Column Temp. : 30 °C 2.0
Injection Volume : ƫ/ 1.5
Detection : SPD-20A at 210 nm
1.0
Flow Cell : Semi-micro cell
0.5
mAU 0.0
1
2 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 min
8
7 4
Fig. 1.7.3 Soft Drink B (20 × dilution, 4 μL injected)
6
*Cautions Regarding This Analysis
5 Sample injection
3 Since a buffered solution is used as the mobile phase, the sample
4
solvent and injection volume may affect the results. For example, if
3 a large volume of an alcoholic drink is injected, peak distortion may
occur.
2
Detection selectivity
1 Since detection is conducted at UV 210 nm, the results are easily
affected by impurities. Analysis may be dif cult if large quantities of
0
impurities are present in the sample.
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 min Column washing
If 100 % buffer solution is used as the mobile phase, elution of the
■ Peaks organic acids may gradually occur earlier. In this situation, conduct
1. Formic acid, 2. Malonic acid
3. Acetic acid, 4. Citric acid
column washing with mobile phase containing some amount of an
organic solvent (example: water/acetonitrile = 1/1). This washing
is also effective for removing hydrophobic constituents from the
Fig. 1.7.1 Chromatogram of a Standard Mixture of 4 Organic
sample.
Acids (100 mg/L each, 4 μL injected)
11
1.8 Analysis of Amino Acids Contained in Vegetable Juice (1) - GC/MS
Q Explanation
Amino acids contained in vegetable juice were treated with EZ: faast TM (Phenomenex, Inc.), which enables easy
pretreatment, and then analyzed with a GC-MS system. Two kinds of vegetable juice were treated with EZ: faast.
Norvaline was added as an internal standard. A GCMS-QP2010 Ultra (with high-power oven) was used for the
measurements. The analysis conditions were in conformity with the “Amino Acid Analysis Methods” in the “GC/MS
Metabolic Components Database.”
Instrument : *&06438OWUDZLWKKLJKSRZHURYHQ
Column : =%$$$OHQJWKPPP,'3KHQRPHQH[,QF
[GC] [MS]
Injection Volume : ƫ/ Interface Temp. : 280 °C
Injection Temp. : 280 °C Ion Source Temp. : 200 °C
Column Temp. : &&PLQ& Solvent Elution Time : 0.4 min
Control Mode : &RQVWDQWSUHVVXUHN3D Data Sampling Time : 0.5 min to 7 min
Injection Mode : Split Measurement Mode : Scan
Split Ratio : 1:15 Mass Range : m/z XVHF
Carrier Gas : He Event Time : 0.15 sec
Fig. 1.8.1 Total Ion Current Chromatogram (TIC) for Amino Acid Derivatives in Vegetable Juice A
The numbers for each component follow the serial numbers in the “GC/MS Metabolic Components Database.”
1 Alanine 8 Leucine 14 As paragine 23 Glutam ine

3 Glycine 10 Is oleucine 16 As partic acid 26 Lys ine
4 alpha-aminobutyric acid 11 Threonine 17 Methionine 27 Histidine
5 Valine 12 Serine 19 Glutam ic acid 29 Tyros ine
7 Norvaline(I.S.) 13 Proline 20 Phenylalanine 31 Tryptophan
12
H O
1.8 Analysis of Amino Acids Contained in Vegetable Juice (2) - GC/MS
Fig. 1.8.2 Total Ion Current Chromatogram (TIC) for Amino Acid Derivatives in Vegetable Juice B
The numbers for each component follow the serial numbers in the “GC/MS Metabolic Components Database.”
1 Alani ne 8 Leucine 14 As paragi ne 23 Glutam i ne

3 Glycine 10 Is oleucine 16 As partic acid 26 Lys ine
4 alpha-aminobutyric acid 11 Threonine 17 Methionine 27 Histidine
5 Valine 12 Serine 19 Glutam ic acid 29 Tyros ine
7 Norvaline (I.S.) 13 Proline 20 Phenylalanine 31 Tryptophan
QSummary
Pretreatment using the EZ: faast kit, following by analysis using the GCMS-QP2010 Ultra, which is equipped with a
high-speed scanning function, enabled rapid analysis of amino acids. With this combination, it took only 15 minutes per
sample from pretreatment to analysis.
13
1.9 High Speed Analysis of Pre-Column Derivatized Amino
Acids by SIL-30AC Auto-sampler (1) - LC
Q Explanation
Amino acid analysis is required in a wide range of Vial
elds, including foods and pharmaceuticals, and various
methods of derivatization have been devised to improve 0HUFDSWR3URSLRQLF$FLGѥ/
sensitivity and selectivity when conducting amino 23$ѥ/
acid analysis by HPLC. Here, using the RF-20Axs
6DPSOHѥ/
uorescence detector and the SIL-30AC autosampler with
its automated pretreatment functions, we introduce the Mix
analysis of amino acids using pre-column derivatization
with OPA and FMOC.
Wait 1.0 min
QSimultaneous Determination of 22 Amino Acids )02&ѥ/

Using the automated pretreatment functions of the
Nexera SIL-30AC autosampler, primary and secondary Mix
amino acids were automatically der ivatized into
f luorescent substances within the autosampler using Wait 2.0 min
o-phthalaldehyde (hereafter, OPA) and 9-f luorenyl
methyl chloro formate (hereafter, FMOC), respectively.
,QMHFWLRQWR+3/&ѥ/
After separation of the derivatized amino acids using
the ultra-high speed YMC-Triart C18 column (1.9 μm,
YMC Co., Ltd.), high-sensitivity detection was conducted Fig. 1.9.1 Flowchart of Automated Pre-Column Derivatization
using the RF-20Axs f luorescence detector. Since the with SIL-30AC
OPA-derivatized amino acids and FMOC-derivatized
amino acids are detected at different wavelengths, Table 1.9.1 Derivatization Reagents
simultaneous analysis was conducted utilizing the
ŏ Mercaptopropionic Acid
automatic wavelength switching feature. Table 1.9.1
3-Mercaptopropionic Acid 10 μL in 0.1 mol/L Borate Buffer (pH 9.2) 10 mL
shows the derivatization reagents used in this method,
ŏ o -Phthalaldehyde Solution
and Fig. 1.9.1 shows the reagent addition and mixing steps
o-Phthalaldehyde 10 mg in 0.1 mol/L Borate Buffer (pH 9.2) 5 mL
used to conduct the automated derivatization using the
ŏ Fluorenyl Methyl Chloro Formate - Acetonitrile Solution
SIL-30AC autosampler. Since a constant reaction time can
9-Fluorenyl Methyl Chloro Formate 4 mg in Acetonitrile 20 mL
be maintained with the automated derivatization using
the autosampler, excellent repeatability can be obtained
compared with pre-column derivatization by manual QAnalytical Conditions
operation. Fig. 1.9.2 shows the chromatogram. Column : <0&7ULDUW&ƫP
PP/ðPP,'ƫP<0&&R/WG
Mobile Phase: $ PPRO/ 3RWXVVLXP 3KRVSKDWH
%XIIHUS+
B : 45/40/15 Acetonitrile / Methanol / Water
Gradient Elution Method
Time Program: B 11 % APLQ
APLQAPLQ
A PLQA PLQ
APLQ
Flowrate : 0.8 mL/min
Column Temp.: 35 °C
Injection Volume : ƫ/
Detection : RF-20Axs Ex. at 350 nm, Em. at 450 nm
A([DWQP(PDWQPPLQ
Cell Temp. : 20 °C
Flow Cell : Conventional cell
14
H O

Acids by SIL-30AC Auto-sampler (2) - LC
mV
22
200
150
100
12 13 15
1 10 11
18 20
4 7 9 17 19
50 3 5
2 8
6 14 16 21
0.0 2.5 5.0 7.5 min
Peaks
1. Aspartic Acid 2. Glutamic Acid 3. Asparagine 4. Serine 5. Glutamine 6. Histidine 7. Glycine 8. Threonine 9. Citruline
10. Arginine 11. Alanine 12. GABA 13. Tyrosine 14. Cys-Cys 15. Valine 16. Methionine 17. Tryptophan 18. Phenylalanine
19. Isoleucine 20. Leucine 21. Lysine 22. Proline
Fig. 1.9.2 Chromatogram of 22 Amino Acids (10 μmol/L Each, 1 μL Injection)
QLinearity and Repeatability QAnalysis of Actual Samples

Using calibration curves generated with a concentration Fig. 1.9.3 shows a chromatogram of analysis of a
range from 1-100 μmol/L for each amino acid, excellent commercially available soft drink using this method.
linearity was obtained, with a ratio of contribution (R 2 The sample was analyzed after filtering it through a
value) greater than 0.999 in all cases. Table 1.9.2 shows the 0.2 μm membrane lter.
peak area repeatability for all 22 amino acids obtained in
repeat analysis (n = 6).
mV
450 Peaks
1. Aspartic Acid 2. Glutamic Acid 3. Serine
400 4. Histidine 5. Glycine 6. Threonine 7. Arginine 8. Alanine
Table 1.9.2 Repeatability 9. Tyrosine 10. Valine 11. Methionine 12. Tryptophan
350 13. Phenylalanine 14. Isoleucine 15. Leucine 16. Lysine
Area%RSD Area%RSD 17. Proline
300
Asp 0.50 GABA 0.41
Glu 0.48 Tyr 0.55 250
Asn 0.51 Cys-Cys 0.46 5 17
200
Ser 0.41 Val 0.71
150 14
Gln 0.56 Met 0.71 11
8 9 10
His 0.57 Trp 0.70 100 15
Gly 0.29 Phe 0.73 6 7 13
50 3 16
2 4 12
Thr 0.55 Ile 0.63 1
Cltruline 0.46 Leu 0.55 0
Arg 0.45 Lys 0.56 0.0 2.5 5.0 7.5 min

Ala 0.46 Pro 2.35
Fig. 1.9.3 Chromatogram of Soft Drink
15
Acids in Alcoholic Beverage (1) - LC
Q Explanation reagents used in this method and the pretreatment
In the previous section we introduced the analysis of program for the SIL-30AC, refer to the previous section.
amino acids that are obtained by hydrolysis primarily Fig. 1.10.1 shows the chromatogram obtained from
of proteins. Amino acids were prepared by automated analysis of a standard mixture of 26 amino acids in
precolumn derivatization using the SIL-30AC. solution.
However, for amino acid analysis applications which
require the search for functional constituents, etc. in QAnalytical Conditions
foods, monitoring of even more types of amino acids is Column : <0&7ULDUW&ƫP
becoming necessary. Here, we introduce an example of the (100 mm /ðPP,'ƫPPDQXIDFWXUHG
determination of 26 amino acids using a different column E\<0&&R/WG
size and different mobile phase conditions than were Mobile Phase : $PPRO/3RWXVVLXP3KRVSKDWH
used in the previous section. The automatic pretreatment %XIIHUS+
feature of the SIL-30AC was utilized for derivatization B : 45/40/15 Acetonitrile/Methanol/Water
of the amino acids during analysis, thereby enabling the Gradient Elution Method
overall analysis time to be substantially shortened. Time Program : %PLQAPLQ
APLQAPLQ
QSimultaneous Determination of 26 Amino Acids APLQAPLQ
The automatic pretreatment features of the Nexera APLQAPLQ
SI L -30AC autosampler were ut ilized to conduct
Column Temp. : 35 °C
automated derivatization of primary amino acids using Injection Volume: ƫ/
o-phthalaldehyde (hereafter, OPA) and secondary amino Detection : RF-20Axs, Ex. at 350 nm, Em. at 450 nm
acids, such as proline, etc., using 9-f luorenyl methyl A([DWQP(PDWQPPLQ
chloro formate (hereaf ter, FMOC), to produce uorescent Cell Temp. : 25 °C
substances within the autosampler. For the derivatization Flow Cell : Semi-micro cell
mv
13
14
300
15
250
12
11
200 1 9
26
150 16
5
3 10
100 2 18 20
4 19 23
78
21 22
50 6 24 25
17
0
0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

■ Peaks
1. Aspartic Acid 2. Glutamic Acid 3. Asparagine 4. Serine 5. Glutamine 6. Histidine 7. Glycine 8. Threonine 9. Citlulline
10. Arginine 11. ћ -Alanine 12. Alanine 13. Taurine 14. Theanine 15. GABA 16. Tyrosine 17. Cystine 18. Valine
19. Methionine 20. Tryptophan 21. Phenylalanine 22. Isoleucine 23. Leucine 24. Ornithine 25. Lysine 26. Proline
Fig. 1.10.1 Chromatogram of Standard Mixture Solution of 26 Amino Acids (10 μmol/L Each)
16
H O

Acids in Alcoholic Beverage (2) - LC
QApplication of Overlapping Injection
Pretreatment Feature Sample
試料注入
injection
Sample
試料注入
injection
Sample
試料注入
injection
The overlapping injection function of the SIL-30AC allows
preparation of the next sample to occur during the current Analysis
分析
洗浄
･ Derivatization
誘導体化 Analysis
分析
洗浄
･ Derivatization
誘導体化 Analysis
分析
analysis operation, thereby shortening the time necessary for 平衡化
Rinse
平衡化
Rinse
Equilibration
a series of analyses. In the amino acid analysis presented here,
Equilibration
Typical Analysis Cycle

the time required to complete an analysis cycle is shortened by
conducting sample derivatization (reagent addition and mixing) of Sample Sample Sample Sample
試料注入試料注入試料注入試料注入
the subsequent sample during the current analysis. A derivatized injection injection injection injection
sample is injected into the column, and when the analysis starts, 洗浄洗浄洗浄洗浄
the autosampler begins to prepare the next sample by adding Analysis

分析
平衡化
･ Analysis
分析
平衡化
･
Rinse
Analysis
分析
平衡化
Rinse
･ Analysis
分析
平衡化
Rinse
･
reagent and mixing the contents. Fig. 1.10.2 shows a ow diagram

Rinse
Equilibration Equilibration Equilibration Equilibration
description of the overlapping injection analysis cycle. Derivatization Derivatization Derivatization Derivatization
誘導体化誘導体化誘導体化誘導体化
QAnalysis of Alcoholic Beverages

Two commercially available alcoholic beverages were Analysis Cycle with Overlapping Injection
analyzed using the automated pre-column derivatization. Fig. 1.10.2 Automated Derivatization Using Overlapping Injection
Fig. 1.10.3 shows each of the chromatograms. After diluting
each of the samples with 0.1 mol/L HCl, they were filtered
through a 0.2 μm membrane lter, and then derivatized by the
SIL-30AC pretreatment procedure. 1 μL of each was injected.
mV
100 13 23
1
12
14 (A)
15
75
10 11
50 7
2
18 20
25 3 8 16
4 5 6 17 19 22
9
0
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 min
mV
90
13
23
80 10 (B)
70 12 15
60
2
50 1
11
40
30 3 7
22
20 8 20
4 6 14 16
21
10 5 9 18 19
17
0
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 min
■ Peaks
1. Aspartic Acid 2. Glutamic Acid 3. Asparagine 4. Serine 5. Glutamine 6. Histidine 7. Glycine 8. Threonine 9. Citlulline 10. Arginine
11. ћ -Alanine 12. Alanine 13. GABA 14. Tyrosine 15. Valine 16. Methionine 17. Tryptophan 18. Phenylalanine 19. Isoleucine
20. Leucine 21. Ornithine 22. Lysine 23. Proline
Fig. 1.10.3 Chromatograms of Alcoholic Beverages: (A) Beer (B) White Wine
17
1.11 Ultra High-Sensitivity Analysis of Water-Soluble Vitamins - LC

The Nexera SR is a high-end model within the Nexera X2 series Column : .LQHWH[ƫP&c
of ultra high performance liquid chromatographs. It features the (100 mm L. ×PP,'ƫP
SPD-M30A high-sensitivity photodiode array detector which Mobile Phase : $PPRO/6RGLXP3KRVSKDWH
incorporates the newly designed capillary SR-Cell (Sensitivity %XIIHUS+PPRO/6RGLXP
and Resolution Cell). Optimization of the optical path length 1-Hexanesulfonate
and diameter results in both high sensitivity and low noise.
B: Mobile PhaseA/Acetonirtile = 2/3
Introduced here is an example of high-speed, high-sensitivity Gradient Elution Method
simultaneous analysis of water-soluble vitamins using the
Time Program : %PLQAPLQ
Nexera SR ultra high performance liquid chromatograph with
high-sensitivity cell (option). APLQ
QSimultaneous Analysis of 6 Water-Soluble Injection Volume : ƫ/
Vitamins
High-sensitivity cell (option) of the Nexera SR ultra high
performance liquid chromatograph incorporates 85 mm optical
path length. Low noise levels and long optical path length
have achieved excellent S/N, not only high signal response. In
this simultaneous analysis of water-soluble vitamins, S/N has
increased by 7.0 times compared to the previous instrument.
High sensitivity detection is achieved even for compounds with
low molar absorptivity.
mV
275 nm Current model
4500 SPD-M30A (High-sensitivity cell)
4000
S/N 7.0 times greater !
3500
6
3000
2500
4
2000 3
5
1500
1
1000 2
500
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 min
Peaks: :
1. Niacin
2. Nicotinamide
3. Pyridoxine
4. Riboflavinphosphate
phosphate
5. Thiamine
6. Riboflavin
Fig. 1.11.1 Chromatogram of a Standard Mixture Solution of 6 Water-Soluble Vitamins
18
H O
1.12 High-Sensitivity Analysis of Retinol Acetate and

Retinol Palmitate - LC
In everyday use, the term "Vitamin A" is synonymous with Column : Shim-pack FC-ODS
retinol. Because retinol is a substance that is easily oxidized, (75 mm L. × 4.6 mm ,'ƫP
highly stable derivatives of retinol are used in such products Mobile Phase : Methanol
as foods, medicines and cosmetics. In HPLC analysis of stable Flowrate : 1.2 mL/min
retinol derivatives such as retinol acetate and retinol palmitate, Column Temp. : 35 °C
a f luorescence detector offers both selectivity as well as Injection Volume : ƫ/
highsensitivity analysis. Here we introduce examples of high- Detection : RF-20AXS Ex. at 350 nm, Em. at 480 nm
sensitivity analysis of retinol acetate and retinol palmitate, in Cell Temp. : 25 °C
addition to ultra-high speed analysis of retinol palmitate using Flow Cell : Conventional cell
the Prominence RF-20A XS uorescence detector.
QAnalysis of Standard Solution QAnalysis of Multivitamin Tablets

Fig. 1.12.1 shows the structures of retinol, retinol acetate, and Fig. 1.12.3 shows an example of analysis of the multivitamin
retinol palmitate. Fig. 1.12.2 shows an example of analysis tablets. The multivitamin tablets were crushed, extracted
of a retinol acetate and retinol palmitate standard solution in methanol (ultrasonic extraction), centrifuged, and then
(1.2 μg/L and 1.1 μg/L in methanol solvent). This con rmed diluted in methanol. After filtering through a membrane
that the Prominence RF-20A XS is capable of detecting these lter, 10 μL was injected.
retinol derivatives at concentrations as low as 10 pg.
H H H H mV
OH Retinol 3.50
■ Peak
H H
1. Retinol palmitate
3.00
H H H H 2.50
O Retinol acetate
2.00
H H O 1
1.50
H H H H
Retinol palmitate
O 1.00
7
H H O
0.50
Fig. 1.12.1 Structures of Retinol, Retinol Acetate and Retinol Palmitate
0.00
mV 0.0 2.0 4.0 6.0 8.0 10.0 min
1.40 1 ■ Peaks
1. Retinol acetate (12 pg) Fig. 1.12.3 Chromatogram of Multivitamin Tablets (10 μL injected)
1.20 2. Retinol palmitate (11 pg)
1.00
0.80
0.60
0.40
2
0.20
0.00
0.0 2.0 4.0 6.0 8.0 10.0 min
Fig. 1.12.2 Chromatogram of a Standard Mixture of Retinol Acetate

(1.2 μg/L) and Retinol Palmitate (1.1 μg/L) (10 μL injected)
19
1.13 Analysis of Water-Soluble Vitamins with Multi-Mode
ODS Column (1) - LC/MS
Q Explanation
Dietary guidelines and nutritional supplements are a significant Here we present an example of analysis of 8 water-soluble vitamins
concern to health conscious consumers. Upper and lower limits using a cation exchange-anion exchange multi-mode ODS column,
of daily intake of such functional foods have been speci ed for 12 (Scherzo SM-C18, Imtakt Corporation) in conjunction with the
vitamins and 2 minerals. Water-soluble vitamins are one class of LCMS2020 mass spectrometric detector. The gradient consisted
nutrients whose measurement is important to the food and nutritional of formic acid / ammonium formate buffer and acetonitrile mobile
supplement industries. Due to their high polarity, their retention phases, components typical for high sensitivity reverse-phase LC/MS
is extremely weak when using reversed-phase chromatography. analysis. Fig. 1.13.1 shows the chromatograms of the 8 water-soluble
Historic use of ion-pair reagents when conducting LC/MS analysis vitamins, Fig. 1.13.2 shows the mass spectra, and Fig. 1.13.3 shows the
of such weakly retained analytes has resulted in reduced sensitivity. respective calibration curves. The linearity in all cases was excellent,
with coef cient of repeatability values all greater than R 2 = 0.99.
Column : Imtakt Scherzo SM-C18
(150 mm /ðPP,'ƫP
Mobile Phase : A: 5 mmol/L Ammonium Formate +
0.1 % Formic Acid-Water
B: Acetonitrile
Time Program : %PLQAPLQA
PLQ
Injection Volume : 2ƫ/
Probe Voltage : N9(6,3RVLWLYH0RGH
N9(6,1HJDWLYH0RGH
Nebulizing Gas Flow: 1.5 L/min
Drying Gas Flow : 10 L/min
DL Temp. : 250 °C
Block Heater Temp. : 450 °C
Fig. 1.13.1 Chromatograms of 8 Water-Soluble Vitamins DL/Q-array Voltage : Using default values
Fig. 1.13.2 Mass Spectra of Water-Soluble Vitamins
20
H O
1.13 Analysis of Water-Soluble Vitamins with Multi-Mode

ODS Column (2) - LC/MS
Fig. 1.13.3 Calibration Curves of Water-Soluble Vitamins
QQuantitative Analysis of Water-Soluble

Vitamins from Cereal
One gram of a commercial functional food product was
prepared according the procedure shown in Fig. 1.13.4,
and quantitation of the water-soluble vitamins was
conducted. Fig. 1.13.5 shows the SIM chromatograms
obtained from analysis of the cereal extract solutions. The
content of ⑥ cyanocobalamin (vitamin B12) in the sample
was below the method detection limit. Actual sample
analysis requires confirmation of extraction efficiency,
daily quality control etc., but this analysis con rmed that
quantitation was possible without almost any interference Fig. 1.13.4 Sample Preparation
from impurities and demonstrated the LCMS-2020 to be a
suitably selective and sensitive detector.
Fig. 1.13.5 SIM Chromatograms of Extract from Cereal
21
1.14 High Speed Analysis of Nucleobases, Nucleosides,
and Nucleotides (1) - LC
Nucleic acids are biological macromolecules consisting of Column : .LQHWH[ƫP&c
linear chains of nucleotides, each of which is made up of PP/ðPP,'ƫP
a base, a sugar, and a phosphate group, and are important Mobile Phase : 200 mmol/L Sodium Perchlorate,
components that bear an organism’s genetic code. In PPRO/6RGLXP3KRVSKDWH%XIIHU
addition, nucleic acid-related compounds, including S+ DT
nucleobases, nucleosides, and nucleotides have a variety Flowrate : 0.7 mL/min
of functions. Here, using the Nexera UHPLC (Ultra Column Temp. : 40 °C
High Performance Liquid Chromatography) System, and Injection Volume : ƫ/
the Shim-pack XR-ODS Ⅲ and Phenomenex Kinetex Detection : SPD-20AV at 260 nm
C18 high-speed, high-resolution columns, we introduce Flow Cell : Semi-micro cell
examples of ultra-high-speed analysis and ultra-high-
resolution analysis of nucleic acid-related compounds.
mAU
QAnalysis of Nucleobases and Nucleosides 40
4
We prepared a sample solution consisting of a standard
35
mixture of 10 nucleic acid-related substances, including 2
5 nucleobases (adenine, guanine, uracil, thymine, 30
cytosine) and 5 nucleosides (adenosine, guanosine, 1
uridine, thymidine, cytidine), each at a concentration of 25 3
10 mg/L, and conducted analysis using the Phenomenex 5
Kinetex C18 column (particle size 1.7 μm, 100 mm L. 20
7
× 2.1 mm I.D.). The Phenomenex Kinetex C18 is a 6
15 8
Core-shell column consisting of a 1.25-μm solid core
coated with a bonded 0.23 μm multilayer of porous lm. 10 9
Fig. 1.14.1 shows the chromatogram obtained using a 1 μL 10
injection of the prepared standard mixture. This analysis, 5
which took 30 minutes to complete using conventional
conditions, took about 1/10 as long (3 minutes) using these 0
analytical conditions. The system back pressure during

this analysis was about 75 MPa. 0.0 1.0 2.0 min
■ Peaks
1. Cytosine, 2. Uracil, 3. Guanine, 4. Adenine, 5. Cytidine,
6. Uridine, 7. Thymine, 8. Adenosine, 9. Guanosine, 10. Thymidine
Fig. 1.14.1 Chromatogram of a Standard Mixture of Nucleobases

and Nucleosides (10 mg/L each)
Note:
When using a 100 % aqueous mobile phase or a composition close to that, as indicated in the analytical conditions in this document the retention
times may become smaller by temporarily stopping solvent delivery, and then restarting. To prevent the occurrence of this phenomenon, after
completion of the analysis, it is recommended to replace the mobile phase with one containing an organic solvent (example: water/acetonitrile = 1/1)
before stopping solvent delivery. In addition, if the retention times gradually become faster, perform a rinse using the same mobile phase.
22
H O
1.14 High Speed Analysis of Nucleobases, Nucleosides,

and Nucleotides (2) - LC
QAnalysis of ATP-related Compounds mAU
30
We prepared a sample solution consisting of a standard mixture
of 6 ATP-related substances (hypoxanthine, inosine, IMP, AMP,
1
ADP, ATP)*, each at a concentration of about 10 mg/L, and 25
conducted analysis using the Shim-pack XR-ODS Ⅲ column
(1.6 μm particle size, 50 mm L. × 2.0 mm I.D.). Fig. 1.14.2 20
shows the chromatogram obtained using a 1 μL injection of the
prepared standard mixture. 15
This analysis, which took 25 minutes to complete using
conventional conditions, took about 1/10 as long (2.5 minutes)
10
using these analytical conditions. The system back pressure 4
3
during this analysis was about 83 MPa.
5 2
QAnalytical Conditions 5
6
Column :Shim-pack XR-ODSⅢ 0
PP/ðPP,'ƫP
Mobile Phase :100 mmol/L Phosphoric Acid, 150 mmol/L
0.0 0.5 1.0 1.5 2.0 min
7ULHWK\ODPLQHDT$FHWRQLWULOH YY
Flowrate : 0.9 mL/min ■ Peaks
1. Hypoxanthine, 2. IMP, 3. Inosine, 4. AMP, 5. ADP, 6. ATP
Injection Volume: ƫ/ Fig. 1.14.2 Chromatogram of a Standard Mixture of ATP-Related
Detection : SPD-20AV at 260 nm Compounds
Flow Cell :Semi-micro cell
mAU
QAnalysis of Nucleotides 35
We prepared a sample solution consisting of a standard 30 1

mixture of 18 nucleotides (AMP, ADP, ATP, GMP, GDP, GTP,
UMP, UDP, UTP, TMP, TDP, TTP, CMP, CDP, CTP, IMP, 25
IDP, ITP)*, each at a concentration of about 50 mg/L, and 2
4
conducted analysis using the high-resolution Shim-pack XR- 20 10
ODS Ⅲ column (2.2 μm particle size, 200 mm L. × 2.0 mm I.D.).
Fig. 1.14.3 shows the chromatogram obtained using a 1 μL 15 3
injection of the prepared standard mixture. Analysis of these 18 56 8
substances was achieved at high speed and with high resolution 10 11
using these conditions, and the system back pressure during the 16
7 9 12 13
analysis was about 78 MPa. 5 14
15
18
17
Column : Shim-pack XR-ODS Ⅲ
PP/ðPP,'ƫP 0.0 5.0 10.0 15.0 min
Mobile Phase : A: 100 mmol/L Phosphoric Acid, 150 mmol/L ■ Peaks
7ULHWK\ODPLQHDT 1. CMP, 2. UMP, 3. CDP, 4. GMP, 5. IMP, 6. UDP, 7. CTP, 8. GDP,
%0RELOH3KDVH$$FHWRQLWULOH YY 9. IDP, 10. AMP, 11.TMP, 12. UTP, 13. GTP, 14. ITP, 15. TDP,
$% YY 16. ADP, 17. TTP, 18. ATP
Column Temp. : 50 °C Fig. 1.14.3 Chromatogram of a Standard Mixture of Nucleotides
Detection : SPD-20AV at 260 nm
* AMP: Adenosine 5'-monophosphate, ADP: Adenosine 5'-diphosphate, ATP: Adenosine 5'-triphosphate, GMP: Guanosine 5'-monophosphate,
GDP: Guanosine 5'-diphosphate, GTP: Guanosine 5'-triphosphate, UMP: Uridine 5'-monophosphate, UDP: Uridine 5'-diphosphate, UTP:
Uridine 5'-triphosphate, TMP: Thymidine 5'-monophosphate, TDP: Thymidine 5'-diphosphate, TTP: Thymidine 5'-triphosphate, CMP: Cytidine
5'-monophosphate, CDP: Cytidine 5’-diphosphate, CTP: Cytidine 5’-triphosphate, IMP: Inosine 5’-monophosphate, IDP: Inosine 5’-diphosphate,
ITP: Inosine 5’-triphosphate
23
1.15 Analysis of Oligosaccharides in Beer - LC

Combining the g radient elution method with the Column : Asahipak NH2P - 50 4E
ELSD-LT enables efficient separation when analyzing PP/ðPP,'
oligosaccharides. Fig. 1.15.1 shows the result of analyzing Mobile Phase : $FHWRQLWULOH:DWHU YY
oligosaccharides in beer using the isocratic elution )LJ
method and the gradient elution method. 10 μL of beer $$FHWRQLWULOH
was injected after ltering with a membrane lter. There B: Water
are branched oligosaccharides (1¤6 glycosidic linkage), Linear Gradient B 30 % ¤ 60 %
linear oligosaccharides (1¤ 4 glycosidic linkage), and )LJDQG)LJ
other types oligosaccharides. In general, the different Flowrate : 1.0 mL/min
types of oligosaccharide are mixed together when eluted. Column Temp. : 40 °C
The elution times for monosaccharides as well as linear Detection : ELSD-LT
disaccharides, trisaccharides, and heptasaccharides are Temperature : 35 °C
indicated in the chromatogram. It can be seen that the GAIN :7
gradient elution method enables the ef cient separation Nebulizer Gas : N2
and detection of oligosaccharides up to 20-mer. The Gas Pressure : 350 kPa
results of analyzing commercial beers under the same
gradient elution conditions are shown in Fig. 1.15.2 and
Fig. 1.15.3.
2500
■ Elution times
1. Glucose
2000
2. Maltose
3. Maltotriose
4. Maltoheptaose
1500
Gradient Elution
1000 1 2 3 4
500
Isocratic Elution
-500
12 3 4
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
min
Fig. 1.15.1 Analysis of Oligosaccharides in Beer
mV mV
1200 1200
1000 1000
800 800
600 600
400 400
200 200
0 0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
min min
Fig. 1.15.2 Chromatogram of beer A Fig. 1.15.3 Chromatogram of beer B
24
H O
1.16 Determination of Fructo-oligosaccharides in Food - LC

Fructo-oligosaccharides are types of oligosaccharides Column : Asahipak NH2P-50 4D
consisting of fructose and glucose. They have lower calories PP/ðPP,'
than sucrose, and recently, have received attention related Guard Column : Asahipak NH2P-50G
to their contribution to the proliferation of bi dobacteria, PP/ðPP,'
possibly because they reach the large intestine without Mobile Phase : :DWHU$FHWRQLWULOH YY
decomposition by enzymes. Recently, many food products Flowrate : 1.0 mL/min
with fructo-oligosaccharides have reached the market. Column Temp. : 30 °C
Here we introduce the simultaneous analysis of sugars and Detection : Refractive Index Detector
fructo-oligosaccharides in food using HPLC. 3RODULW\&HOOWHPSÝ&
Glucose Sucrose HOH2C

H O H
HOH2C HOH2C
H
O H O H OH H
H H
H H HO
OH H OH H n =1 1-Kestose
HO OH HO O
H HO n =2 Nystose
H HO H HO O HOH2C O n =3 Fructofuranosylnystose
HOH2C O
H HO
H HO H CH2
H CH2OH
OH H O
Fructose HO H
n
HOH2C O
HOH2C O HO
H HO
H HO H CH2OH
H CH2OH
OH H
HO H
Fig. 1.16.1 Structural Formulas
QAnalysis of Standard Solution QAnalysis of Syrup

Fig. 1.16.1 shows the structural formulas of sucrose, Fig. 1.16.3 shows the chromatogram of a commercially
f r ucto - oligosacchar ides kestose, nystose and available syrup containing fructo-oligosaccharides. 1.0 g
fructofuranosylnystose, as well as glucose and fructose, of the syrup was diluted and made 200 mL with distilled
w h ic h a r e t h e c o m p o n e n t s of t h e s e s u g a r s . T h e water. This solution was then ltered using a 0.45 μm pore
chromatogram obtained from analysis of a standard mixture membrane lter.
of these six compounds (each 5.0 g/L) is shown in Fig. 1.16.2.
ѥRIU ѥRIU
7.5
1 ■ Peaks ■ Peaks
30 1. Fructose 1. Fructose
2. Glucose 2 2. Glucose
2
25 3 3. Sucrose 3. Sucrose
4. Kestose 5.0 4. Kestose
5. Nystose 5. Nystose
20 6. Fructofuranosylnystose 6. Fructofuranosylnystose
4
4
15 5
5 2.5 3
10
6 1
5 6
0.0
0
0.0 5.0 10.0 15.0 min 0.0 5.0 10.0 15.0 min
Fig. 1.16.2 Chromatogram of a Standard Mixture of Fructo- Fig. 1.16.3 Chromatogram of Oligosaccharide Syrup (10 μL injected)
oligosaccharides (5.0 g/L each, 10 μL injected)
25
1.17 Analysis of Saccharides Using Post-Column
Derivatization System (1) - LC
The Shimadzu Prominence reducing sugar analysis <Separation>
system is a post-column derivatization system; after Column : Shim-pack ISA-07/S2504
separation of saccharides by the column, an arginine/ PP/ðPP,'
boric acid reagent solution is continuously added to the Guard Column : Shim-pack Guard Column ISA
column eluent to convert the saccharides to fluorescent PP/ðPP,'
derivatives for detection. This system allows detection Mobile Phase : A: 0.1 mol/L Potassium Borate Buffer (pH
of saccharides at high sensitivity and with excellent %PRO/3RWDVVLXP%RUDWH%XIIHUS+
selectivity, furthermore with the new Prominence RF- A ¤ B Linear Gradient Elution
20A XS , even higher sensitivity is achieved. Additionally, Flowrate : 0.6 mL/min
the temperature controlled f lowcell in the RF-20A XS Column Temp. : 65 °C
allows highly reliable analysis that is unaffected by Injection Volume : ƫ/
ambient temperature uctuations. Here we present some <Detection>
examples of analyzing saccharides using the reducing Reaction Reagent : 10 g/L Arginin, 30 g/L Boric Acid
sugar analysis system (anion exchange mode) with the Flowrate : 0.5 mL/min
Prominence RF-20A XS . Reaction Coil : SUS, 10 m L. × 0.8 mm I.D.
Reaction Temp.: Ý&
QAnalysis of Standard Solution Cooling Coil : SUS, 6 m L. × 0.3 mm I.D.
Detection : RF-20AXS Ex. at 320 nm, Em. at 430 nm
Fig. 1.17.1 shows a f low diagram of the Prominence
Cell Temp. : Ý&
reducing sugar analysis system, and Fig. 1.17.2 shows the
chromatogram of a standard solution of 11 saccharides.
mV
6
6
5
1 5 10
2
4
4
3 8
3 9
7
2 11
0.0 25.0 50.0 75.0 min

Q Peaks
1. Sucrose, 2. Cellobiose, 3. Maltose, 4. Lactose, 5. Rhamnose, 6. Ribose
7. Mannose, 8. Arabinose, 9. Galactose, 10. Xylose, 11. Glucose
Fig. 1.17.1 Flow Diagram of the System Fig. 1.17.2 Chromatogram of a Standard Mixture of 11 Saccharides
(200 μmol/L each, except sucrose at 2 mmol/L, 10 μL injected)
26
H O
1.17 Analysis of Saccharides Using Post-Column

Derivatization System (2) - LC
QHigh-Sensitivity Analysis Q Effect of Cell Temperature Control
The RF-20Axs offers unprecedented levels of sensitivity; It is generally known that the f luorescence intensity
a water Raman S/N ratio is at least 2000. This allows the drops as the temperature rises; because the molecular
reducing sugar analysis system to detect saccharides with collisions increase in frequency with increase in the
much higher sensitivity. Fig. 1.17.3 shows a chromatogram temperature, therefore molecules lose their potential
obtained f rom analysis of a standard solution of energy. In other words, a f luctuation of the ambient
saccharides (2 μmol/L each, except sucrose at 20 μmol/L, (detection) temperature changes the uorescence intensity
10 μL injected). In this case, glucose is clearly detected of some compounds, and this negatively influences the
even when injected at the amount of 20 pmol (3.6 ng). accuracy of analysis. The RF-20A XS has a temperature-
controlled cell as a standard feature, ensuring the high
reliability of analysis that is not affected by a temperature
fluctuation. Fig. 1.17.4 shows a comparison of the peak
mV
intensities at cell temperatures of 25 °C and 30 °C (using
0.6 Q Peaks the same saccharide solution and analytical conditions as
1. Sucrose 7. Mannose in Fig. 1.17.2). The comparison of two chromatogram at
0.5 2. Cellobiose 8. Arabinose cell temperatures of 25 °C and 30 °C reveals a decrease
3. Maltose 9. Galactose in peak intensity over 10 % for every compound at the
0.4 4. Lactose 10. Xylose higher cell temperature. By maintaining a constant cell
5. Rhamnose 11. Glucose
10
temperature, peak intensity and detection sensitivity will
0.3 6. Ribose
11 not be compromised if the room temperature changes
8 9 during the sequence run.
0.2 6 7
5
0.1
4
1 2 3
0.0
0.0 25.0 50.0 75.0 min
Fig. 1.17.3 Chromatogram of a Standard Mixture of 11 Saccharides

(2 μmol/L each, except sucrose at 20 μmol/L, 10 μL injected)
mV
Peak Height
Cell Temperature
QPeaks
6 Ý& Ý& Ý&Ý&
6 Same as Fig. 1.17.2 Ý&
Ý& Sucrose 4546 4043 88.9
Cellobiose 4082 3639 89.1

1 10
5 Maltose 2966 2645 89.2
2
4 Lactose 3175 2830 89.1
4 Rhamnose 4232 3717 87.8
3 8
9 Ribose 5852 5118 87.5
7
Mannose 2201 1961 89.1
2 11
Arabinose 2820 2445 86.7
Galactose 2489 2184 87.7
Xylose 4263 3759 88.2
0 Glucose 1623 1454 89.6

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 min
Fig. 1.17.4 Effect of Cell Temperature on Peak Intensity
27
1.18 Analysis of Ethyl Į -D-Glucoside in Japanese Sake - LC
Q Explanation mV
Japanese sake typically contains ethyl Ơ -D-glucoside 200

Q Peaks
(Ơ -EG), a substance believed to promote beautiful skin, 1. Ethyl њ -D-Glucoside
2. Fructose
and it is now receiving attention for its effectiveness 150
3. Glucose
4. Sucrose
1
in improving dry skin conditions even when taken in a 5. Maltose
2
drink. Here we present an example of analysis of ethyl
Ơ -D-glucoside and other saccharides in sake using 100 3 4
hydrophilic interaction liquid chromatography (HILIC). 5
50
QAnalysis of Standard Solution

Fig. 1.18.1 shows the st r uct ural for mula of ethyl 0
Ơ -D-glucoside (Ơ -EG). Ơ -EG is a highly polar compound, 0.0 5.0 10.0 min
so by using hydrophilic interaction liquid chromatography

(HILIC) Ơ -EG and other saccharides can be analyzed Fig. 1.18.2 Chromatogram of a Standard Mixture of Ethyl Ơ -D-Glucoside
at the same time. Since Ơ -EG shows almost no UV and 4 Saccharides (20 g/L each, 10 μL injected)
absorption, common with other saccharides, in addition
to the refractive index detector used for this analysis, an QAnalysis of Sake (Alcoholic Beverage from Rice)
evaporative light-scattering detector can also be used. Fig. 1.18.3 and Fig. 1.18.4 show chromatograms of
Fig. 1.18.2 shows the chromatogram obtained from commercial sake. In both cases, simultaneous analysis
analysis of a standard solution of ethyl Ơ -D-glucoside and was conducted for Ơ -EG and glucose, the primary
other saccharides (fructose, glucose, sucrose, and maltose, saccharide ingredient of sake. After filtering the sake
each at 20 g/L). through a 0.45 μm membrane lter, 10 μL was injected for
each analysis.
mV
HO
QPeaks
1. Ethyl њ -D-Glucoside
2 2. Glucose
O 75
OH 50
1
HO OC2H5
25
OH
0
Fig. 1.18.1 Structure of Ethyl Ơ -D-Glucoside 0.0 5.0 10.0 min
Fig. 1.18.3 Chromatogram of Sake A (10 μL injected)
QAnalytical Conditions mV
QPeaks
Column : Asahipak NH2P - 50 4E 1. Ethyl њ -D-Glucoside
2. Glucose
PP/ðPP,' 2
50
Mobile Phase : Water / Acetonitrile = 30/70
25
1
Detection : Refractive Index Detector
0
0.0 5.0 10.0 min
Fig. 1.18.4 Chromatogram of Sake B (10 μL injected)
28
H O
1.19 Analysis of Alliin in Garlic - LC

The active ingredient alliin in garlic quickly changes Column : Shim-pack Amino-Na
to allicin by the action of the enzyme alliinase. As the PP/ðPP,'
ef cacy of these substances has been elucidated, they are Mobile Phase : A: 0.1 mol/L Sodium Citrate
often marketed as health food products. Since alliin is a %XIIHU6ROXWLRQS+
type of amino acid, it can be analyzed by the post-column B: 0.2 mol/L Sodium Hydroxide
derivatization method with fluorescence detection using $TXHRXV6ROXWLRQ
OPA (o-phthalaldehyde) as the reaction reagent. Here Step Gradient Elution Method
we introduce the analysis of alliine in garlic using the ²PLQ A 100 %
Shimadzu amino acid analysis system. ²PLQ B 100 %
²PLQ A 100 %
NH2
Mobile Phase Flowrate : 0.4 mL/min
CH CH2 S CH2 CH CH2
COOH O Reaction Reagent : $PLQR$FLG5HDJHQW.LW6ROXWLRQ%
Fig. 1.19.1 Structure of Alliin Reaction Reagent Flowrate : 0.4 mL/min
Reaction Temp. : 60 °C
Garlic 700 mg(grinded) Detection : Fluorescence Detector
or
Processed Foods 100 mg Ex: 350 nm Em: 450 nm
5 %Trichloroacetic acid aq.
50 mL
Mixing
Q Peak
1. Alliin
Filtration 1
HPLC 10 ѥL Inj.
Fig. 1.19.2 Sample Preparation
Q Peak
1. Alliin
0 10 20 30 min
1
Fig. 1.19.4 Analysis of Processed Garlic (tablet)
0 10 20 30 min
Fig. 1.19.3 Analysis of Garlic
29
1.20 High Speed Analysis of Catechins in Green Tea (1) - LC

Along with the health consciousness boom that has been Column : HALO®&PP/ðPP,'ƫP
ongoing in recent years, green tea and green tea drinks Mobile Phase : A: 3KRVSKRULF$FLGLQ
have been receiving heightened attention. :DWHU7HWUDK\GURIXUDQ YY
Here we introduce an example of high-speed analysis of B: Acetonitrile / Tetrahydrofuran = 990
14 catechins in green tea, including methyl catechins, YY
which have been reported to display anti-allergic action. Gradient Elution Method
The analytical system consisted of the Prominence Time Program : %PLQ¤PLQ
¤PLQ
UFLC XR ultra-fast, high-resolution LC system with
a high-speed, high-resolution column. In addition, Column Temp. : 40 °C
detection was also conducted using the LCMS-2020, and Injection Volume : ƫ/
these results were compared to those obtained using UV Detection : SPD-M20A at 230 nm
detection. UV Cell : Semi-micro cell
QAnalysis of Standard Solution mAU

Fig. 1.20.1 shows the structures of the catechins analyzed 1 2
here, including methyl catechins, gallic acid and 400 3
caffeine. Fig. 1.20.2 shows a chromatogram of a standard
mixture of these14 substances (100 mg/L each), which
5
were analyzed all together with a high-speed and high- 6
resolution HALO ® C18 column (particle size 2.7 μm) 200
4
7 8 10
from AMT Ltd. 9
11 12 13 14
1. Gallic Acid (GA) 2. Gallocatechin (GC) 3. Epigallocatechin (EGC)

OH OH
COOH OH OH
0
HO O HO O
OH OH
0.00 0.50 1.00 1.50 2.00 2.50
HO OH OH
OH min
OH OH OH Peaks
4. Caffeine 5. Catechin (C) 6. Epicatechin (EC) 1. GA, 2. GC, 3. EGC, 4. Caffeine, 5. C, 6. EC, 7. EGCG, 8. GCG,
OH
O OH
9. EGCG4”Me, 10. EGCG3”Me, 11. ECG, 12. CG, 13. ECG4”Me, 14. ECG3”Me
H3C
HO O
N CH3 OH
N HO O
OH
N N O
OH
OH
OH Fig. 1.20.2 Chromatogram of a Standard Mixture of Catechins,
Gallic Acid and Caffeine (100 mg/L, 2 μL injected)
CH3 OH
7. Epigallocatechin Gallate 8. Gallocatechin Gallate 9. Epigallocatechin 3-(4”-O -

(EGCG) OH (GCG) methyl) gallate
OH
OH OH
OH (EGCG4”Me)
OH
HO O
OH HO O HO O
OH OH
O
O O
OH OH
OH OH OH OMe
O
O O
OH
OH OH
OH
OH OH
10. Epigallocatechin 3-(3”-O - 11. Epicatechin Gallate 12. Catechin Gallate (CG)
methyl) gallate (ECG)
(EGCG3”Me) OH
OH
OH
HO O
OH OH
HO O
OH
HO O
OH O
O OH OH
O
OH OH O
OH OH
O
O OH
OMe OH OH
OH OH
13. Epicatechin 3-(4”-O - 14. Epicatechin 3-(3”-O -

methyl) gallate methyl) gallate
(ECG4”Me) (ECG3”Me)
OH
OH OH
OH
HO O
HO O
O
O
OH OH
OH OMe
O
O
OH OMe
OH OH
Fig. 1.20.1 Structures of Catechins, Gallic Acid and Caffeine
30
H O
1.20 High Speed Analysis of Catechins in Green Tea (2) - LC
QAnalysis of Green Tea

Fig. 1.20.3 and Fig. 1.20.4 show the chromatograms of commercial green teas A and B, respectively. The green tea
leaves (3 g per sample) were infused in 100 mL of hot water, diluted by a factor of 10 with puri ed water and the diluted
infusion was passed through a membrane lter (pore size 0.22 μm). The methyl catechins EGCG3” Me and ECG3” Me
were con rmed to be present at different concentrations, respectively, in green teas A and B.
mAU mAU
3 3
7
4
7
100 4
100
6 6
2
2
(1)
11 11
5 10 14 (1) 10
5 14
0 0
0.00 0.50 1.00 1.50 2.00 2.50 min 0.00 0.50 1.00 1.50 2.00 2.50 min
Peaks Peaks
(1. GA), 2. GC, 3. EGC, 4. Caffeine, 5. C, 6. EC, 7. EGCG, 8. GCG, (1. GA), 2. GC, 3. EGC, 4. Caffeine, 5. C, 6. EC, 7. EGCG, (8. GCG),
(9. EGCG4”Me), (10. EGCG3”Me), 11. ECG, (12. CG), (13. ECG4”Me), 14. ECG3”Me (9. EGCG4”Me), 10. EGCG3”Me, 11. ECG, (12. CG), (13. ECG4”Me), 14. ECG3”Me
Fig. 1.20.3 Chromatogram of Green Tea A Fig. 1.20.4 Chromatogram of Green Tea B
QAnalysis of Green Tea by LC-MS QAnalytical Conditions

Fig. 1.20.5 shows the MS chromatogram of green tea B QLC Conditions
analyzed using the LCMS-2020. The acid added to the Column : HALO®&PP/ðPP,'ƫP
mobile phase for the LC-MS measurement was changed Mobile Phase : A: 0.4 % Formic Acid in Water /
7HWUDK\GURIXUDQ YY
to the volatile formic acid, and it was also added to the
B: 0.4 % Formic Acid in
tetrahydrofuran in Solvent B. (Since a different mobile Tetrahydrofuran / Acetonitrile =
phase than that used with UV detection was used in the YY
LC-MS method, the order of elution of peak 4 (C) and Gradient Elution Method
peak 5 (caffeine) was observed to be reversed. In addition, Time Program : %PLQ¤PLQ
GA, which was observed in Fig. 1.20.4, was not con rmed ¤PLQ
in the LC-MS analysis results of Fig. 1.20.5, suggesting Flowrate : 2.0 mL/min
that is may be a compound other than GA. Column Temp. : 40 °C
(× 100,000) QMS Conditions (LCMS-2020)
3 Probe Voltage : N9(6,3RVLWLYH0RGH
5.0
6 7
N9(6,1HJDWLYH0RGH
2 10 11 14 Nebulizing Gas Flow : 1.5 L/min.
4 TIC (Negative) Drying Gas Flow : 20 L/min.
5 TIC (Positive) DL Temp. : 250 °C
m/z 441
m/z 457 DL/Q-array Voltage : Using default values
m/z 305 Monitoring Ions : m/z1HJDWLYHIRU(&*DQG&*
m/z 289 m/z1HJDWLYHIRU(*&*DQG*&*
m/z 195
m/z 169 m/z1HJDWLYHIRU*&DQG(*&
m/z 471 m/z1HJDWLYHIRU&DQG(&
0.0 m/z 455
0.00 0.50 1.00 1.50 2.00 2.50 min
m/z3RVLWLYHIRU&DIIHLQH
m/z1HJDWLYHIRU*$
Peaks
(1. GA), 2. GC, 3. EGC, 4. C, 5. Caffeine, 6. EC, 7. EGCG, (8. GCG), m/z1HJDWLYHIRU(*&*µ0H
(9. EGCG4”Me), 10. EGCG3”Me, 11. ECG, (12. CG), (13. ECG4”Me), 14. ECG3”Me
DQG(*&*µ0H
Fig. 1.20.5 MS Chromatograms of Green Tea B m/z1HJDWLYHIRU(&*µ0HDQG
(&*µ0H
31
1.21 High Speed Analysis of Resveratrol in Wine - LC

Resveratrol, a type of polyphenol, is a phytoalexin that is Column : Shim-pack XR-ODS Ⅲ
synthesized by plants when they are exposed to stress due PP/ðPP,'ƫP
to disease and pests. In addition to providing antioxidative Mobile Phase : A: 0.2 % Formic Acid - Water
effects, resveratrol is reported to offer other health bene ts B: 0.2 % Formic Acid - Acetonitrile
including support for increased longevity. For these Gradient Elution Method
reasons, this substance is actively being researched, and Time Program : %PLQ ¤PLQ
recently, resveratrol is being added to food products and ¤ PLQ ¤ 100 % (2.51-
to cosmetics. Here we introduce an example of ultra fast PLQ¤PLQ
analysis of resveratrol in red wine using the Nexera ultra Flowrate : 0.7 mL/min
high performance liquid chromatograph and the Prominence Column Temp. : 60 °C
RF-20Axs high-sensitivity uorescence detector. Injection Volume : ƫ/
Detection : RF-20Axs Ex. at 300 nm, Em. at 386 nm
QAnalysis of Standard Solution Cell Temp. : 20 °C
Fig. 1.21.1 shows the structural formulas of cis- and trans-
resveratrol. Fluorescence detection can be used for both QAnalysis of Red Wine
forms. Fig. 1.21.2 shows the results of measurement of a Fig. 1.21.3 shows the sample preparation procedure,
standard mixture of trans- and cis-resveratrol* (25 mg/L and Fig. 1.21.4 shows the chromatogram obtained from
each in 50 % methanol). The system pressure during this analysis of red wine prepared using this procedure.
analysis was 75 MPa (10,900 psi) at maximum, while the
Sample 5 mL
pressure tolerance of the Shim-pack XR-ODS Ⅲ column
Adjusted to pH2
(2.2 μm particle size) is rated at 100 MPa (14,500 psi). by adding 0.1 mol/L HCl
* The cis- form of the resveratrol standard contains a small amount of the
Diethylether 5 mL
trans- isomer.
Mixing 5 min
HO
H
Aqueous Organic Phase
trans - Resveratrol (lower)
HO H
OH
(upper)
H Evaporate to dryness
H
50 % Methanol 1 mL
OH cis - Resveratrol
Filtration (0.2 μm)
HO OH
Fig. 1.21.1 Structures of cis- and trans-Resveratrol 1 μL injection

mV
mV
Q Peaks
200 1. trans-Resveratrol
2. cis-Resveratrol 50
175
40
150
30
125 1
20 1
100 2
2
10
75
0
50 Sample 1 2
-10
25
-20
0 Standard (5 mg/L)
-30
-25
0.0 1.0 2.0 min
0.0 1.0 2.0 min
Q Peaks
1. trans-Resveratrol 2. cis-Resveratrol
Fig. 1.21.2 Chromatogram of a Standard Mixture of cis- and
trans-Resveratrol (25 mg/L each, 1 μL injected) Fig. 1.21.4 Chromatogram of Red Wine
32
H O
1.22 High Speed Analysis of Phenolic Acids (1) - LC

Phenolic acid exists in higher plants as esters, ethers or in Column : Shim-pack XR-ODSⅡ
its free state, and is a compound that has been receiving PP/ðPP,'ƫP
increased attention in recent years due to its antioxidant Mobile Phase : A: 50 mmol/L Ammonium Formate
effects. HPLC is often used for quantitative analysis of %XIIHUS+
phenolic acid in processed foods containing fruits and fruit B: Methanol
materials, but the analysis is often quite time-consuming Gradient Elution Method
because a relatively long column and gradient elution Time Program %PLQ¤PLQ
are required to achieve separation of the contaminants ¤PLQP/PL[HU
in actual samples. Here we present an example of high Flowrate : 0.9 mL/min
speed, high resolution analysis of phenolic acids using Column Temp. : 40 °C
the Shimadzu Prominence UFLCXR ultra high-speed LC Injection Volume : ƫ/
system with the SPD-M20A photodiode array detector. Detection : 63'0$0D[SORWQP
UV Cell : Semi-micro cell
QSimultaneous Analysis of 11 Phenolic Acids
and Benzoic Acid
Fig. 1.22.1 shows a chromatogram of a standard mixture
of 11 phenolic acids and benzoic acid (50 mg/L each),
analyzed using a Shim-pack XR-ODS Ⅱhigh speed, high
resolution column. For comparison, data acquired using a
conventional Shim-pack VP-ODS column are also shown.
mAU
Shim-pack VP-ODS (250 mm L. = 4.6 mm I.D., 4.6 ѥm)
200 Injection Volume: 10 ѥL, UV Cell: Conventional cell
100
0
0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0 min
mAU
Shim-pack XR-ODSⅡ(100 mm L. × 3.0 mm I.D., 2.2 ѥm)
4
250
1
2
200
6
150 9
3 5
100
7 10
8 11
12
50
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min
■ Peaks
1. Gallic acid, 2. Protocatechuic acid, 3. Chlorogenic acid, 4. p -Hydroxybenzoic acid, 5. Vanillic acid, 6. Caffeic acid, 7. Syringic acid
8. Salicylic acid, 9. p -Coumaric acid, 10. Ferulic acid, 11. Benzoic acid, 12. Sinapic acid
Fig. 1.22.1 Chromatograms of a Standard Mixture of 11 Phenolic Acids and Benzoic Acid (50 mg/L each)
33
1.22 High Speed Analysis of Phenolic Acids (2) - LC
QAnalysis of Chlorogenic Acid ■ Peak

mAU
Here we present a high speed analysis of chlorogenic acid 1. Chlorogenic acid
in commercially available fruit juices. 500 1
400
HOOC OH
OH 300
O
Berry Juice
200
HO O OH
100 Filtration (0.2 +m)
OH
0
Fig. 1.22.2 Structure of Chlorogenic Acid 0.0 0.5 1.0 min HPLC
mAU
Column : Shim-pack XR-ODS
PP/ðPP,'ƫP 60 1
Mobile Phase : A: 50 mmol/L Ammonium Acetate 50

%XIIHUS+
40
B: Methanol
30
Gradient Elution Method Peach Juice 1 mL
Time Program : %PLQ¤PLQ 20 Water 4 mL
¤PLQ 10 Filtration (0.2 +m)

Flowrate : 0.9 mL/min 0
Column Temp. : 40 °C 0.0 0.5 1.0 min HPLC

Detection : 63'0$QP Fig. 1.22.3 Chromatograms of Berry Juice (upper) and Peach Juice (lower)
QAnalysis of Ellagic Acid
Ellagic acid is found in many plants, in which much of
it exists in the form of tannin combined with gallic acid. ■ Peak
mAU
Here we analyzed free ellagic acid in processed food. 1. Ellagic acid
7.5 Jam 1 g
O
1
O OH 5.0
Homogenization
Methanol 4 mL
HO OH 2.5
Ultrasonic Extraction
(30 min)
0.0
HO O
Filtration (0.2 +m)
O 0.0 0.5 1.0 min
Fig. 1.22.4 Structure of Ellagic Acid HPLC
mAU
Column : Shim-pack XR-Phenyl 25

PP/ðPP,'ƫP 20
Mobile Phase : $PPRO/2[DOLF$FLGDT 15
%0HWKDQRO$FHWRQLWRULOH YY Red Wine
10
B Conc. 35 %
5 1
Flowrate : 1.0 mL/min Filtration (0.2 +m)
Column Temp. : 40 °C 0
Injection Volume : ƫ/ 0.0 0.5 1.0 min HPLC
Detection : 63'0$QP

UV Cell : Semi-micro cell Fig. 1.22.5 Chromatograms of Raspberry Jam (upper) and Red Wine (lower)
34
H O
1.23 High Speed Analysis of Į -Acids and ȕ -Acids in Hops (1) - LC

Hops are one of the basic ingredients in beer and contain Column : HALO®&PP/ðPP,'ƫP
Ơ -acids (humulones) and ơ -acids (lupulones). During Mobile Phase : A::DWHU0HWKDQRO3KRVSKRULF$FLG
the brewing process the Ơ -acids are converted through Triethylamine = 300 mL/700 mL/19.6 g/15.1 g
isomerization into iso-Ơ -acids (isohumulons), which are B: Methanol
largely responsible for the bitterness of beer. ơ -acids, on Gradient Elution Method
the other hand, do not produce much bitterness during the Time Program : %PLQńPLQ
boiling stage of beer production, but they are said to play Flowrate : 1.1 mL/min
a role in "balancing" the bitter avor during fermentation Column Temp. : 50 °C
and storage. Injection Volume : ƫ/
HPLC is generally used for the analysis of Ơ -acids Detection : SPD-20AV at 330 nm
and ơ -acids, but this analysis typically takes as long Flow Cell : Semi-micro cell
as 30 minutes. Here we introduce an example of high-
speed analysis of Ơ -acids and ơ -acids in hops using the * Composition of "International Calibration Extract 2"
Prominence UFLC XR ultra fast, high resolution LC - Cohumulone 14.45 %
system with a high-speed, high-resolution column. - Humulone + Adhumulone 34.94 %
- Colupulone 12.92 %
QAnalysis of Standard Solution - Lupulone + Adlupulone 12.02 %
We selected 6 target analytes in hops, consisting of
3 Ơ -acids (humulone, cohumulone, and adhumulone)
and 3 ơ -acids (lupulone, colupulone, and adlupulone). mAU
250
The structural formulas of these substances are shown 2
in Fig. 1.23.1. A standard solution was prepared by 225

Q Peaks
dissolving 0.1 g of the standard extract "International 200 1. Cohumulone
Calibration Extract 2" (American Society of Brewing 175
2. Humulone
3. Adhumulone
Chemists)* in methanol, after which the volume was 4. Colupulone
5. Lupulone
adjusted to 100 mL, and the resultant solution was ltered 150 1
6. Adlupulone
through a 0.22 μm membrane lter. Fig. 1.23.2 shows the 125
results of measurement of a 4 μL injection of this standard 100

solution. For the high-speed, high-resolution column, we
used the Advanced Materials Technology HALO ® C18 75
3 4
column (particle size 2.7 μm). The system pressure in this 50 5
analysis was about 56 MPa at maximum. 25 6
њ -Acids -25
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min
Humulone R=CH2CH (CH3)2
Cohumulone R=CH (CH3)2
Adhumulone R=CH (CH3)CH2CH3 Fig. 1.23.2 Chromatogram of a Standard Mixture of Ơ -Acids and ơ -Acid
ћ -Acids
Lupulone R=CH2CH (CH3)2
Colupulone R=CH (CH3)2
Adlupulone R=CH (CH3)CH2CH3
Fig. 1.23.1 Structures of Ơ -Acids and ơ -Acids
35
1.23 High Speed Analysis of Į -Acids and ȕ -Acids in Hops (2) - LC
QAnalysis of Hop Pellets

Fig. 1.23.3 shows the results of analysis of a commercially
available hop pellets. Sample preparation was conducted Sample 10 g
according to the procedure1) of Fig. 1.23.4. Even in
the case of an actual sample, excellent separation was
obtained just as with the standard solution (Fig. 1.23.2). Grind
2.5 g
Toluene (HPLC grade) 25 mL

mAU
35.0
Shake (30 min)
32.5 2
Q Peaks
30.0
1. Cohumulone
5
27.5 2. Humulone
Centrifuge (1000 rpm, 10 min)
3. Adhumulone 4
25.0
4. Colupulone
22.5 5. Lupulone
20.0 6. Adlupulone
Supernatant Residue
17.5
5 mL
15.0
12.5 1
Evaporate
10.0
7.5
6 Methanol (HPLC grade) 25 mL
3
5.0
Filter
2.5
0.0
-2.5
HPLC 4 μL
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min
Fig. 1.23.3 Chromatogram of Hop Pellets Fig. 1.23.4 Sample Preparation
[Reference]
1) Brewery Convention of Japan [Analysis Committee] Edition, Methods of Analysis of BCOJ (Revised edition), Brewing Society of Japan (2004)
36
H O
1.24 High Speed Analysis of Iso-Į-Acids and Į-Acids in Beer (1) - LC

Iso-Ơ -acids (iso-humulones) are compou nds that Column : HALO®&PP/ðPP,'ƫP
contribute to the bitter taste of beers and are generated Mobile Phase : 3KRVSKRULF$FLGPPRO/
through the isomerization of Ơ -acids (humulones) during ('7$Ã1DDT$FHWRQLWULOH YY
the brewing process. Here we introduce an example of Flowrate : 1.4 mL/min
analysis of iso-Ơ -acids and Ơ -acids using the ultra-high Column Temp. : 30 °C
speed, high resolution Prominence UFLCXR system with Injection Volume : ƫ/
a high-speed, highresolution column. Detection : 63'$9DWQPPLQQP
PLQ
QAnalysis of Standard Solution Flow Cell : Semi-micro cell
We selected 6 target analytes, consisting of 3 iso-Ơ -acids
(isohumulone, isocohumulone, and isoadhumulone, all * Composition of “International Calibration Standard I2”
trans isomers) and 3 Ơ -acids (humulone, cohumulone, Isohumulone + isocohumulone + isoadhumulone: 64.3 % (all
and adhumulone). The structural formulas of the iso- trans isomers)
Ơ -acids (cis and trans isomers) and Ơ -acids are shown
* Composition of “International Calibration Extract 2”
in Fig. 1.24.1. A standard solution was prepared by - Cohumulone: 14.45 %
dissolving 0.2 g of the standard extract “International - Humulone + Adhumulone: 34.94 %
Calibration Standard I2” and “International Calibration - Colupulone: 12.92 %
Ext r a ct 2” ( bot h A me r ica n Societ y of Brew i ng - Lupulone + Adlupulone: 12.02 %
Chemists)* in 50 mL of methanol. A 1/100 dilution of the *** Since the ơ -acids (colupulone, lupulone, and adlupulone)
all elute after 13 minutes under these analytical conditions
stock solution (using methanol) was filtered through a and were not of interest in this note, an acetonitrile rinse
0.22 μm membrane lter. Fig. 1.24.2 shows the results of step was added when conducting analysis of the standard
measurement of a 4 μL injection of this standard solution. solution to ush the ơ -acids from the column.
For the high-speed, high-resolution column, we used
the AMT HALO ® C18 column (particle size 2.7 μm).
The maximum system pressure during analysis was mAU
approximately 56 MPa (8100 psi). 10
9
1
Q Peaks
O O 8 1. trans-Isocohumulone
trans-Iso-њ -Acids 2. trans-Isohumulone
R 7
H 3. trans-Isoadhumulone
trans-Isohumulone R=CH2CH(CH3)2 4. Cohumulone
HO 6 2
O OH trans-Isocohumulone R=CH(CH3)2 5. Humulone
5 6. Adhumulone
trans-Isoadhumulone R=CH(CH3)CH2CH3
4
3
O O
2 5
cis-Iso-њ -Acids
3 4
R 1
H cis-Isohumulone R=CH2CH(CH3)2
6
HO 0
O OH cis-Isocohumulone R=CH(CH3)2
cis-Isoadhumulone R=CH(CH3)CH2CH3 -1
0.0 2.5 5.0 7.5 10.0 min
OH O Fig. 1.24.2 Chromatogram of Standard Mixture of Iso-Ơ -Acids

њ -Acids and Ơ -Acids
R Humulone R=CH2CH(CH3)2
HO O Cohumulone R=CH(CH3)2
HO
Adhumulone R=CH(CH3)CH2CH3
Fig. 1.24.1 Structures of Iso-Ơ -Acids and Ơ -Acids
37
1.24 High Speed Analysis of Iso-Į-Acids and Į-Acids in Beer (2) - LC
QAnalysis of Beer
Fig. 1.24.3 shows the result of analysis of a commercial
beer. Sample preparation was conducted according to
the procedure1) shown in Fig. 1.24.4. Peaks a, b, and c in
the chromatogram are presumed 2) to be the cis isomers
of isocohumulone, isohumulone, and isoadhumulone,
respectively.
mAU
30.0
Sample 20 mL
Q Peaks
27.5
1. trans-Isocohumulone
a
2. trans-Isohumulone
25.0 Degas
3. trans-Isoadhumulone
22.5 4. Cohumulone
b 5. Humulone 10 mL
20.0 6. Adhumulone
6 mol/L Hydrochloric Acid 0.5 mL
17.5 a. cis-Isocohumulone
15.0 1
( b. cis-Isohumulone
c. cis-Isoadhumulone ) Shake (30 min)
2,2,4-Trimethylpentane
(Spectrometry Grade) 20 mL
12.5
10.0 2 (After 15 min)

7.5
c
5.0
Organic Layer Aqueous Layer
3
2.5
4 5 6 12 mL
0.0
-2.5 Evaporate
0.0 2.5 5.0 7.5 10.0 min
Methanol (HPLC Grade) 1 mL
Fig. 1.24.3 Chromatogram of Beer Sample Filtrate
HPLC 4 μL
[References]
1) Brewery Convention of Japan [Analysis Committee] Edition, Revised BCOJ Beer Analysis Method, Brewing Society of Japan (2004)
2) B. Jaskula, K. Goiris1, G. De Rouck, G. Aerts, L. De Cooman : J. Inst. Brew., 113(4), 381-390 (2007)(see pdf)
38
H O
1.25 Analysis of Lycopene and ȕ-Carotene in Tomato - LC

Lycopene is a type of carotenoid, and is found in large Column : Shim-pack VP-ODS
quantities as a red pigment in red tomatoes, etc. The PP/ðPP,'
antioxidative effects of lycopene are said to be 100 times Mobile Phase : $FHWRQLWULOH(WKDQRO YY
stronger than that of vitamin E and more than twice Flowrate : 1.0 mL/min
stronger than that of ơ -carotene, and lycopene is receiving Column Temp. : 50 °C
attention for its effects to prevent lifestyle diseases such Injection Volume : ƫ/
as cancer and arteriosclerosis, and to slow aging. Though Detection : SPD-M20A 450 nm
lycopene and ơ -carotene have similar structures, they can Slit width: 8 nm, Band width: 8 nm
be easily separated by reversed phase chromatography Cell Temp. : 50 °C
using a non-aqueous mobile phase.
CH3 CH3 CH3 H3C H 3C CH3 CH3 CH3 H3C

H3C
CH3
H3C CH3
CH3 CH3 CH3 CH3 CH3 CH3 CH3
Lycopene ћ -Carotene
Fig. 1.25.1 Structure of Lycopene and ơ -Carotene
Tomato 5 g
Chloroform 40 mL
Homogenize, 1 min. Standard
Tomato
Shake, 5 min.
Aqueous(upper) Organic phase (lower layer)
waste Evaporate to dryness

Chloroform 5 mL
5 μL Injection
Fig. 1.25.2 Sample Preparation Fig. 1.25.4 Spectra of Lycopene
mAU
100
1
mAU
100
75
1: Lycopene 80
2: ћ -Carotene
60
40
50
20
0
200 min
25 300 15
2 400 10
500 5
0 0
600
0 5 10 15 20 nm
min
Fig. 1.25.3 Chromatogram of Tomato Fig. 1.25.5 3-D Chromatogram of Tomato
39
1.26 Determination of Cyanidin-3-Glucoside in Black Soybeans - LC

Anthocyanins, essential factor to x the color tone of owers Column : 6KLPSDFN932'6PP/ðPP,'
and fruits, are the generic name of the glycosides which have Guard Column 6KLPSDFN*932'6PP/ðPP,'
anthocyanidins as aglycones. The color tone of anthocyanins Mobile Phase : $PPRO/6RGLXP3KRVSKDWH%XIIHU
varies greatly according to the pH. It is known that many S+
types of anthocyanins exist widely throughout higher plants, B: Acetonitrile
and recently they are receiving much attention due to their
Initial B.Conc = 5 %
antioxidative properties. Among these, cyanidin-3-glucoside,
the glucose glycoside of cyanidin, is said to account for more 7LPHPLQ %&RQF
than 90 % of the anthocyanidins present in black soybeans. 10.00 30
Here we introduce an analysis of cyanidin-3-glucoside using 15.00 100
the Prominence SPD-M20A photodiode array detector. 20.00 100
20.01 5
OH 35.00 STOP
OH Flowrate : 1.0 mL/min
+ Column Temp. : 40 °C
HO O
Detection : 63'0$DWQP6OLWZLGWKQP
O
mAU
OH
CH2OH 50
O
OH Standard
40
HO
OH Sample
30
Fig. 1.26.1 Structure of Cyanidin-3-Glucoside
QAnalysis of Black Soybeans 20
Fig. 1.26.2 shows the chromatogram of commercial black

soybean extract solution obtained at two wavelengths. 10
After pulverizing the black soybeans, 5 mL of methanol

containing 1 % hydrochloric acid was added to 1 g of the 0
ground powder. Cyanidin-3-Glucoside was extracted using 200 300 400 500 600 nm
ultrasonication, and centrifugation and ltering to the extract
were performed. And then, mobile phase A was added to
Fig. 1.26.3 Spectra of Cyanidin-3-Glucoside in Black SoybeanExtract
make a 10-fold dilution. Fig. 1.26.3 shows the spectra of the and Standard Compound
cyanidin-3-glucoside in the black soybean extract solution
and the standard solution. A 3-D plot is shown in Fig. 1.26.4.
Q Peak
35 1. Cyanidin-3-Glucoside
30
25
20
mAU
15
10
280 nm
5
1
520 nm
0
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0

min
Cyanidin-3-Glucoside
Fig. 1.26.2 Chromatograms of Black Soybean Extract (10 μL Inj.) Fig. 1.26.4 3-D Plot of Black Soybean Extract
40
H O
1.27 Analysis of Capsaicinoids in Spices - LC

When absorbed by the body, capsaicins, the spice element Column : 6KLPSDFN932'6P/ðPP,'
found in chili pepper, has the effect of stimulating adrenaline Mobile Phase : $FHWLF$FLGDT$FHWRQLWULOH YY
secretion and causing perspiration. Since ancient times, Flowrate : 1.2 mL/min
capsaicins have been known for their anti-bacterial, stomachic, Column Temp. : 40 °C
and body-warming properties. Furthermore, capsaicins are very Injection Volume : ƫ/
stable elements that maintain their spiciness even when used Detection : SPD-20A at 280 nm
in a variety of cooking methods. The unit used to indicate the RF-10AXL Ex at 280 nm, Em at 325 nm
spiciness of a spice is known as the “Scoville Scale”. This scale mAU
is indicated in magnitudes based on the amount of time until a 1.00
UV Q Peaks
taster no longer feels the spiciness of a spice extract dissolved in 1. Capsaicin
0.75
sugar water. In recent years, to gain more objective indicators, 2. Dihydrocapsaicin
quantitative methods that use HPLC to analyze the capsaicinoids 0.50

1
in a spice also are in use. This issue introduces examples of the

analysis of capsaicinoids in spice using HPLC. 0.25 2
0.00
O
CH3 0 5 10 15
N min
H CH3 mV
HO
OCH3 Fluorescnce Q Peaks

Capsaicin 300 1. Capsaicin
O 2. Dihydrocapsaicin
CH3
N 200 1
H CH3
HO
OCH3 Dihydrocapsaicin 100 2

O CH3
N CH3 0
HO
H 0 5 10 15
min
OCH3 Nordihydrocapsaicin
Fig. 1.27.3 Chromatogram of Red Pepper
Fig. 1.27.1 Structure of Capsaicinoids
mAU
1.00
UV 1 Q Peaks
QAnalysis of Spice 1. Capsaicin
0.75
Fig. 1.27.2 shows the sample preparation steps used on the 2. Dihydrocapsaicin
commercial spices and Figs. 1.27.3, Figs. 1.27.4 show the results 0.50 2
of the analysis. With the actual samples, because impurity
substances are eluted out after the dihydrocapsaicin, in order 0.25
to remove those substances from the column we recommend
cleansing the column by f lowing the mobile phase with 0.00
increased level of acetonitrile before each analysis. 0 5 10 15
* The peak indicated by the arrow in the chromatogram is assumed to min
be nordihydrocapsaicin. mV
Fluorescnce 1 Q Peaks
300 1. Capsaicin
Sample(Fig. 1.27.3: 0.1 g, Fig. 1.27.4: 2.0 g) 2. Dihydrocapsaicin
Ethanol 10 mL 200
2
([WUDFWLRQLQ:DWHUEDWKÝ& 1 hour) 100
Filtration
0
0 5 10 15
+3/&ѥL inj. min
Fig. 1.27.2 Sample Preparation Fig. 1.27.4 Chromatogram of Pepper Sauce
41
1.28 High Speed Analysis of Isothiocyanates and Sinigrin - LC

The isothiocyanates contained in cruciferous vegetables, Column : 6KLPSDFN;52'6PP/ðPP,'ƫP
such as Japanese horseradish and must ard, have Mobile Phase : A : PPRO/,PLGD]ROH3KRVSKDWH
been receiving attention for their antibacterial and Buffer [pH 2.6]
antioxidative properties. It is known that these pungent B : Acetonitrile
components usually exist as glycosides in plant tissue, and Gradient Elution Method
Time Program : %PLPńPLP
when they are hydrolyzed by myrosinase, they take part in ń 50 % PLPńPLP
a transfer reaction, and are released as isothiocyanates. Flowrate : 1.2 mL/min
A n example of the si mult aneous analysis of the Column Temp. : 40 °C
isothiocyanates in Japanese horseradish and sinigrin, a Injection Volume : ƫ/
representative glycoside, performed using the Prominence Detection : 63'0$ DW QP PLQ DQG
UFLC ultra fast LC system and the Shim-pack XR-ODS QPDIWHUPLQ
high-performance column, which is used in high-speed,
high-resolution applications, is presented here.
mAU
80 1
70 2
QSimultaneous Determination of Isothiocyanates 60
3
and Sinigrin in Japanese Horseradish 50
40 4
The simultaneous analysis of isothiocyanates and the 30
56
representative glycoside sinigrin (Fig. 1.28.1) in powdered 20

10
Japanese horseradish was performed using a Shim-pack 0
XR-ODS column (50 mm) under high-speed separation -10
conditions. Fig. 1.28.2 shows an example of the analysis -20
of standard solution. When water is added to the sinigrin 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 min
in Japanese horseradish, hydrolysis occurs, and mainly Q Peaks

1. Sinigrin (90 mg/L) 4. n-Propyl Isothiocyanate (60 mg/L)
allyl isothiocyanate is released. On comparing the results 2. Ethyl Isothiocyanate (60 mg/L) 5. 2-Phenylethyl Isothiocyanate (30 mg/L)
3. Allyl Isothiocyanate (60 mg/L) 6. Phenyl Isothiocyanate (120 mg/L)
obtained immediately after adding water to commercially
available dried, powdered horseradish and those obtained Fig. 1.28.2 Chromatogram of a Standard Mixture of Isothiocyanates
after adding water, kneading the mixture, and waiting 5 and Sinigrin
minutes (Fig. 1.28.3-upper chromatogram: immediately
mAU
after; lower chromatogram: after waiting 5 minutes), there 40 2
are certain changes in peak intensities of sinigrin and allyl 30
isothiocyanate. 20
1
10
0
-10
R-N=C=S
CH2CH=CH2 R: 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 min
CH2OH CH3CH2- Ethyl Isothiocyanate
S C NOSO3K
CH2=CHCH2- Allyl Isothiocyanate
H O mAU
CH3CH2CH2- n-Propyl Isothiocyanate
H 50 2
OH H
CH2CH2 2-Phenylethyl Isothiocyanate 40
HO H 30
H HO Phenyl Isothiocyanate 20 1
10
Sinigrin Isothiocyanates
0
-10
min
Fig. 1.28.1 Structures of Isothiocyanates and Sinigrin 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0
Q Peaks
1. Sinigrin
2. Allyl Isothiocyanate
Fig. 1.28.3 Chromatogram of Jpanese Horseradish

Upper: Immediately after adding water to the sample
Lower: 5 minutes after adding water to the sample
42
H O
+LJK6SHHG$QDO\VLVRI,VRÀDYRQHVLQ6R\- LC

Soy iso avones are a group of compounds that are present Column : 6KLPSDFN;52'6PP/ðPP,'ƫP
in large quantity in soybeans, and especially in the soy Mobile Phase : A : 0.1 % Formic Acid -Water
germ. Recently, these compounds are receiving much B : Acetonitrile
attention due to the similarity of their activity with the Gradient Elution Method
female hormone estrogen. Here we introduce an example Time Program : %PLQńPLQ
of the simultaneous analysis of seven isoflavones using ńPLQńPLQ
the high-performance “Shim-pack XR-ODS” column. Flowrate : 1.5 mL/min
Soy food products (soybean flour, miso, soymilk) were Column Temp. : 40 °C
prepared according to the procedure shown in Fig. 1.29.2, Injection Volume : ƫ/
and high-speed analysis was conducted as shown in Detection : SPD-20A at 254 nm
Fig. 1.29.3 through 5.
CH2OH CH2OCOCH2COOH CH2OCOCH2COOH

O O O
O O O O O O HO O
HO HO HO
OH OH OH
OH OH OH
O O OH O OH O
OH OH OH OH
Daidzin Malonyldaidzin Malonylgenistin Genistein
CH2OH CH2OCOCH2COOH
O O
O O O O HO O
HO HO
OH OH
OH OH
H3CO
OH O O O
OH OH OH
Genistin Malonylglycitin Daidzein
Fig. 1.29.1 Structural Formulas of Seven Iso avones
Sample 0.25 g mAU
2
Water / Ethanol=3/7(v/v)12.5 mL
7
Stir(30 min)
20
1
Centrifuge(3,000 rpm, 15 min)
5
6
Supernatant 3
(4)
0
Filtration (0.45 ѥm) 0.00 0.50 1.00 1.50 2.00 2.50 min
Q Peaks
1. Daidzin, 2. Genistin, 3. Malonyldaidzin, (4. Malonylglycitin),
4 ѥL Inject into HPLC
5. Malonylgenistin, 6. Daidzein, 7. Genistein
Fig. 1.29.2 Sample Preparation Fig. 1.29.4 Chromatogram of Miso (Soybean Paste)
mAU mAU
40
200
5
2
100 3
20 5
1 7 2
1 3
4 6
4 (6) (7)
0 0
0.00 0.50 1.00 1.50 2.00 2.50 min 0.00 0.50 1.00 1.50 2.00 2.50 min
Q Peaks Q Peaks
1. Daidzin, 2. Genistin, 3. Malonyldaidzin, 4. Malonylglycitin, 1. Daidzin, 2. Genistin, 3. Malonyldaidzin, 4. Malonylglycitin,
5. Malonylgenistin, 6. Daidzein, 7. Genistein 5. Malonylgenistin, (6. Daidzein), (7. Genistein)
Fig. 1.29.3 Chromatogram of Soybean Flour Fig. 1.29.5 Chromatogram of Soymilk
43
1.30 Analysis of Flavonoids in Ginkgo Biloba Extract (1) - LC

Flavonoids are a kind of polyphenol, and the name also Column : 6KLPSDFN932'6PP/ðPP,'
refers to a class of plant metabolites. Recently, there Mobile Phase : A 3KRVSKRULF$FLGDT
has been much research on flavonoids with regard to B : Acetonitrile
their physiological activity, and in particular, their anti- Gradient Elution Method
oxidative effects have been reported. Ginkgo biloba Time Program : %PLQń90 % (12.01-
leaves are said to contain as many as 20 types of PLQńPLQ
flavonoids, and among these are quercetin, kaempferol Flowrate : 1.5 mL/min
and isorhamnetin, 3 types that are present in large Column Temp. : 60 °C
quantities. Here we introduce an example of the analysis Injection Volume : ƫ/
of these 3 avonoids present in ginkgo biloba leaves using Detection : SPD-M20A at 370 nm
the SPD-M20A photodiode array detector.
mAU
QAnalysis of Standard Solution 250
Fig. 1.30.1 shows the structures of the 3 f lavonoids 225
(quercetin, kaempferol and isorhamnetin) analyzed here. 200
Fig. 1.30.2 shows the UV-VIS absorption spectrum of 175
quercetin, indicating that all of these compounds display 150
maximum absorption in the vicinities of 250-260 nm
125
and 370 nm. Fig. 1.30.3 shows a chromatogram of the 3
100
avonoids in a standard solution at 370 nm. For detection,
the SPD-M20A photodiode array detector was used. 75
50
25
0
R2
200 250 300 350 400 nm
OH
Fig. 1.30.2 UV-VIS Spectrum of Quercetin
HO
mAU
OR1 175
QPeaks
OH O 1. Quercetin (40 mg/L)
150 2. Kaempferol (40 mg/L)
1 3. Isorhamnetin (10 mg/L)
125
Compound R1 R2
Kaempferol H H 100
2
Quercetin H OH
75
Isorhamnetin H OCH 3
50
25 3
Fig. 1.30.1 Structures of 3 Flavonoids
0
0.0 2.5 5.0 7.5 10.0 min
Fig. 1.30.3 Chromatogram of a Standard Mixture of 3 Flavonoids
44
H O
1.30 Analysis of Flavonoids in Ginkgo Biloba Extract (2) - LC
QAnalysis of Ginkgo Biloba Extract Supplement

We conducted analysis of a commercially available
ginkgo biloba leaf extract (tablet) after preparing the
sample according to the procedure shown in Fig. 1.30.4.
Fig. 1.30.5 shows the obtained chromatogram. Fig. 1.30.6
shows the respective spectra of the f lavonoids in the
sample overlaid with the corresponding standard spectra.
Tablet Sample mAU
Milling 450 Q Peaks

2 mL Methanol 1. Quercetin
400 1
Sonicate 3 min 2. Kaempferol
350 3. Isorhamnetin
2 mL HCl
Sonicate 10 min 300
2 mL Methanol 250
Centrifuge (14000 rpm) 10 min 200 2
150
Supernatant
100
Filtration (0.45ƫm) 50 3
0
Heat (70 ÝC) 25 min -50
0.0 2.5 5.0 7.5 10.0 min
Inject to HPLC 10 ƫL
Fig. 1.30.4 Sample Preparation Fig. 1.30.5 Chromatogram of Ginkgo Biloba Dietary Supplement
mAU mAU mAU

700 350 60
600 Quercetin 300
Kaempferol 50
Isorhamnetin
500 250 40
400 200
30
300 150
20
200 100
100 50 10
0 0 0
200 250 300 350 400 nm 200 250 300 350 400 nm 200 250 300 350 400 nm
Standard, Sample
Fig. 1.30.6 UV-VIS Spectra of 3 Flavonoids in Ginkgo Biloba Dietary Supplement
[Reference]
United States Pharmacopeia (USP32-NF27)
45
1.31 Analysis of Terpenoids in Ginkgo Biloba - LC

Ginkgo biloba extract contains avonoids and terpenoids that Column : 6KLPSDFN)&2'6PP/ðPP,'
have been reported to be effective for improving poor blood Mobile Phase : A: Water
circulation in the brain as well as poor peripheral blood vessel B: Methanol
circulation. This ginkgo biloba extract is used as a health dietary Gradient Elution Method
supplement in Japan and the United States. Here we present an Time Program : %PLQńPLQń 80 %
example of analysis of terpenoids in ginkgo biloba extract using PLQńPLQ
the ELSD-LT Ⅱevaporative light scattering detector.
QAnalysis of Standard Solution Column Temp. : 50 °C
Terpenoids that are known to be present in large quantities Detection : ELSD-LT Ⅱ
in gin kgo biloba include bilobalide, gin kgolide A, Temperature : 40 °C
ginkgolide B and ginkgolide C (Fig. 1.31.1). Because these Gain :6
compounds have no chromophores, use of the evaporative Nebulizer Gas : N2
light scattering detector together with reversed-phase
Gas Pressure : 350 kPa
gradient elution is an effective means of analysis. Fig. 1.31.2
shows a chromatogram obtained from analysis of a standard QAnalysis of Dietary Ginkgo Biloba Supplement
solution of these four terpenoids (200 mg/L each, methanol). Analysis was conducted after performing sample preparation of a
commercially available dietary ginkgo biloba supplement according to
Bilobalide Ginkgolide
the procedure shown in Fig. 1.31.3. Fig. 1.31.4 shows the chromatogram.
Tablet Sample
Milling
Methanol
Sonicate (20 min)
Centrifuge (14000 rpm) 10 min
Compound R1 R2
Ginkgolide A H H
Supernatant
Ginkgolide B OH H Filter (0.45 ѥm)

Ginkgolide C OH OH Dilute
Methanol
Fig. 1.31.1 Structures of 4 Terpenoids Inject to HPLC 10 ѥL

mV
mV
25.0 50
3
Q Peaks 45
1. Bilobalide
20.0 2. Ginkgolide C Q Peaks
40
3. Ginkgolide A 1. Bilobalide
1 4. Ginkgolide B 2. Ginkgolide C
2 35 3. Ginkgolide A
4. Ginkgolide B
15.0
4 30
25
10.0
20
15 1
5.0 3
10
2
4
0.0 5
0.0 5.0 10.0 15.0 20.0 min
0
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 min
Fig. 1.31.2 Chromatogram of a Standard Mixture of 4 Terpenoids
in Ginkgo Biloba (200 mg/L each, 10 μL injected) Fig. 1.31.4 Chromatogram of Dietary Ginkgo Biloba Supplement
46
H O
1.32 Analysis of Ginkgolic Acids in Ginkgo Biloba Extract (1) - LC

Ginkgo leaf extract (ginkgo biloba extract), which contains Column : 6KLPSDFN&/&&PP/ðPP,'
active ingredients extracted from ginkgo biloba leaves, is Mobile Phase : A: 3KRVSKRULF$FLG:DWHU
reported to be effective in improving cerebral and peripheral B: 3KRVSKRULF$FLG$FHWRQLWULOH
blood circulation de ciencies. Gradient Elution Method
It is used in Japan and the United States in the form of a dietary
supplement, and in Germany, France, and other European Time Program : %PLQńPLQ
countries as a prescription medication. However, alkylphenols ńPLQ
which are present in the ginkgolic acids contained in ginkgo Flowrate : 1.0 mL/min
leaves are known to cause allergic reactions. For this reason, Column Temp. : 35 °C
the United States Pharmacopeia (USP) has established an upper Injection Volume : ƫ/
limit for ginkgolic acid content in ginkgo biloba extract. Here we Detection : SPD-M20A at 311 nm
introduce an example of analysis of gingkolic acids contained in
ginkgo biloba leaves.

The ginkgolic acids in ginkgo biloba leaves that were
analyzed include ginkgolic acid C13:0 (hereafter, GA
C13:0), GA C15:0, GA C15:1, and GA C17:1. Fig. 1.32.1
shows the structural formula of these 4 substances. Due
to the high hydrophobicity of these ginkgolic acids, the
Shim-pack CLC-C8 in which the silica gel is modi ed with
an octyl group (C8) was used, and chromatography was
conducted using gradient elution. For detection, the SPD-
M20A photodiode array detector was used. Fig. 1.32.2
shows the spectrum of GA C17:1, and Fig. 1.32.3 shows a
chromatogram of a standard mixture of 4 ginkgolic acids.
Fig. 1.32.2 UV Spectrum of Ginkgolic Acid C17:1
Fig. 1.32.1 Structures of Ginkgolic Acids
Fig. 1.32.3 Chromatogram of a Standard Mixture of 4 Ginkgolic Acids
47
1.32 Analysis of Ginkgolic Acids in Ginkgo Biloba Extract (2) - LC
QAnalysis of Ginkgo Biloba Extract Supplement

Analysis of the ginkgo biloba extract was conducted was barely detected in this supplement, the chromatogram
after performing sample pretreatment of the commercial also shows the results of analysis of the prepared sample
supplement containing the extract as shown in Fig. 1.32.4. solution spiked with ginkgolic acid standard.
Fig. 1.32.5 shows the chromatogram. Since ginkgolic acid
mAU
QPeaks
1. GA C13:0 (Spiked at 0.0625 mg/L)
2 4
1 3
Fig. 1.32.4 Sample Preparation Fig. 1.32.5 Chromatogram of Ginkgo Biloba Extract Supplement
(Upper: Spiked, Lower: Not Spiked)
[Reference]
United States Pharmacopeia (USP32-NF27)
48
H O
1.33 High Speed Analysis of Gingerol and Shogaol in Ginger (1) - LC

Ginger is not only used as a spice, but has also been used Column : 6KLPSDFN;52'6PP/ðPP,'ƫP
in traditional Chinese herbal medicine since ancient Mobile Phase : A: Water
times. Investigation into the ef cacy of ginger has been B : Acetonitrile
attracting a lot of attention in recent years, and the use of Gradient Elution Method
ginger in health foods has seen great increases. Here we Time Program : B 30 ńPLQń 90 %
introduce an example of the analysis of 6-gingerol and PLQ ńPLQ
6-shogaol, constituents of ginger, using the Prominence ńPLQ
U FLC ult ra fast LC system with the SPD -M20A
photodiode array detector.
Detection : 63'0$DWQPVOLWZLGWKQP
Fig. 1.33.1 shows the str uctures of 6-gingerol and
6-shogaol. Homologs of both gingerol and shogaol exist,
and in the case of gingerol, these include 6-gingerol,
mAU
8-gingerol and 10-gingerol, with 6-gingerol the most
abundant found in ginger. Gingerol is converted to 70 QPeaks
Q Peaks
shogaol during the dehydration reaction caused by 1.
1. 6-Gingerol
6-Gingerol
2.
2. 6-Shogaol
6-Shogaol
heating, but the content levels of these constituents vary 60
depending on the type of ginger. Fig. 1.33.2 shows the 1 2
chromatogram obtained from analysis of a 6-gingerol 50
and 6-shogaol standard solution (each 100 mg/L, in
methanol solution) using the Shim-pack XR-ODS high- 40
speed, high-resolution column. It should be noted that a
column rinsing procedure was added to these conditions 30
for analysis of the actual sample.
20
2 2+ 10
+&2
*LQJHURO
0
+2
2 0.0 1.0 2.0 min

+&2
6KRJDRO
+2 Fig. 1.33.2 Chromatogram of a Standard Mixture of 6-Gingerol
and 6-Shogaol (100 mg/L, 2 μL injected)
Fig. 1.33.1 Structures of 6-Gingerol and 6-Shogaol
49
1.33 High Speed Analysis of Gingerol and Shogaol in Ginger (2) - LC
QAnalysis of Ginger Extract

After sample preparation according to the procedure of mAU
225
Fig. 1.33.3, analysis of ginger was conducted. Fig. 1.33.4 Q
QPeaks
Peaks
200
shows the resulting chromatogram. Fig. 1.33.5 shows the 1.
1. 6-Gingerol
6-Gingerol
175
overlaid spectra of 6-gingerol obtained from analysis of a 2.
2. 6-Shogaol
6-Shogaol
1
6-gingerol standard sample and of a ginger extract. 150
125
100
Ginger 1 g 75
Methanol 3 mL 50
2
Homogenize 25
0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 min
Residue
Supernatant Methanol 2 mL
Fig. 1.33.4 Chromatogram of Ginger Extract
Inject into HPLC 2 ѥL

mAU
1500
Standard
1250
1000
750
Ginger
Extract
500
250
200.0 225.0 250.0 275.0 300.0 325.0 nm
Fig. 1.33.5 Spectra of 6-Gingerol
50
H O
1.34 High Speed Analysis of Flavanones in Citrus Juice (1) - LC

Here we introduce an example of high-speed analysis Column : 6KLPSDFN;52'6PP/ðPP,'ƫP
of 6 avanones. The LCMS-2010EV mass spectrometer Mobile Phase : A: 10 mmol/L Ammonium Formate Buffer
was added to the Prominence U FLC/SPD -M20A S+ $FHWRQLWULOH YY
con guration, and the analysis of minor components were B: 10 mmol/L Ammonium Formate Buffer
veri ed using UV spectra as well as mass spectra. S+$FHWRQLWULOH YY
Time Program : %PLQńPLQ ń
QAnalysis of 6 Flavanones PLQńPLQ
Flowrate : 0.4 mL/min with Semi-micro mixer
Fig. 1.34.1 shows the structures of the 6 f lavanones
analyzed here. Fig. 1.34.2 shows the results of simultaneous Injection Volume : ƫ/
analysis of a standard mixture of the 6 avanones using the Detection : SPD-M20A at 285 nm
SPD-M20A. Flow Cell : Semi-micro cell
Fig. 1.34.1 Structures of the Flavanones
Fig. 1.34.2 Chromatogram of a Standard Mixture of 6 Flavanones

(100 mg/L each, 2 μL injected)
51
1.34 High Speed Analysis of Flavanones in Citrus Juice (2) - LC
QAnalysis of Grapefruit Juice QLC/MS Analytical Conditions

Fig. 1.34.4 and Fig. 1.34.5 show the results of analysis of Probe Voltage : N9(6,1HJDWLYH0RGH
commercial 100 % grapefruit juice using the SPD-M20A Nebulizing Gas Flow : 1.5 L/min
and LCMS-2010EV. Sample preparation was conducted Drying Gas Pressure : 0.1 MPa
using the procedure described in Fig. 1.34.3, and 2 μL CDL, Q-array Voltages : Using default values
CDL Temp. : 250 °C
was injected. From the chromatogram, it is evident Block Heater Temp. : 200 °C
that hesperidin and neohesperidin are included along Scan Range : m/z 100 - 650
with narirutin and naringin. However, the qualitative
information is further supplemented by comparing the Sample 3 mL
UV spectra and mass spectra. In addition, since the m/z 60 % Acetonitrile 3 mL
507 ion in the hesperidin mass spectrum is absent from
standard solution mass spectrum, it is presumed to be Mixing
an unknown compound. Even in this type of situation,
monitoring of the m/z 609 ion can provide very accurate Centrifugation
Filtration HPLC
quantitation. (10,000 rpm = 5 min)
Fig. 1.34.3 Preparation of Citrus Juice Sample

mAU (×100) mAU
500 3000
Q Peaks 450 2750
1. Narirutin 2
1.4 400 Narirutin 2500 Naringin
2. Naringin 2250
350 Standard
1 2000
3. Hesperidin 300 Grapefruit Juice 1750
1.2 4. Neohesperidin 250 1500
200 1250
150 1000
1.0 750
100
500
50 250
0 0
0.8
200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 nm 200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 nm
mAU mAU
0.6 70
60
60
50
Hesperidin Neohesperidin
50
0.4 3 40
40
30 30
0.2 4 20 20
10 10
0.0 0 0
0.0 1.0 2.0 3.0 4.0 5.0 min 200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 nm 200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 nm
Fig. 1.34.4 UV Chromatogram and Spectra of Flavanones in Grapefruit Juice (2 μL injected)
Inten. (×1,000,000)
Q Peaks 2 Inten. (×100,000) Inten. (×100,000)
1. Narirutin [M-H]- 579 [M-H]- 579
4.00 2. Naringin 2.0 Narirutin Naringin
3. Hesperidin 5.0
1 3 1.5
4. Neohesperidin
3.50
4 TIC 1.0
2.5
3.00 0.5
2 271 557 557
0.0 0.0
2.50 300 400 500 600 m/z 300 400 500 600 m/z
1
2.00 Inten. (×10,000) Inten. (×10,000)
m/z 579 507 1.0 609
2.0 Hesperidin Neohesperidin [M-H]-
1.50
4
1.5 [M-H]-
0.7
1.00 609 0.5
3 1.0
0.50 0.5 600 0.2 552

397 433 459 501
0.0 0.0
0.00 m/z 609
300 400 500 600 m/z 300 400 500 600 m/z
0.0 1.0 2.0 3.0 4.0 5.0 min
Fig. 1.34.5 Mass Chromatograms and Mass Spectra of Flavanones in Grapefruit Juice using Scan Mode (2 μL injected)
52
H O
1.35 High Speed Analysis of Ginsenosides in Ginseng (1) - LC

Ginseng is a widely used herbal medicine with a Column : 6\QHUJLƫP3RODU53c
number of reported health benefits including stress PP/ðPP,'ƫP
reduction, building resistance to disease, and promoting Mobile Phase : A: Water
concentration and memory function. Compounds called B: Acetonitrile
ginsenosides are believed to be the active constituents Gradient Elution Method
behind ginseng's efficacy. Analysis of ginsenosides by Time Program : % PLQ ń PLQ ń
HPLC has traditionally been a relatively time-consuming PLQńPLQ
process due to the time required for separation of these Flowrate : 0.6 mL/min
structurally similar analytes as well as their separation Column Temp. : 35 °C
from complex contaminants. Here we introduce an Injection Volume : ƫ/
example of the analysis of ginsenosides in ginseng using Detection : SPD-20A at 203 nm
the ultra fast LC system “Prominence UFLC” with the Flow Cell : Semi-micro cell
Phenomenex Synergi 2.5 μm Polar-RP high-speed, high-
resolution column. mAU
20.0
QPeaks
QAnalysis of Standard Solution 17.5
1. Ginsenoside Rg1
2. Ginsenoside Re
The structural formulas of the 5 ginsenosides that are 3. Ginsenoside Rb1
4. Ginsenoside Rc
15.0
the subject of determination in this analysis are shown 5. Ginsenoside Rd
in Fig. 1.35.1. Here, separation of the ginsenosides Rg1 12.5

and Re in particular was conducted ef ciently using the
10.0
high-speed, high-resolution Phenomenex Synergi 2.5 μm 1
Polar-RP (particle diameter 2.5 μm) column. Fig. 1.35.2 7.5

2 3 4 5
shows the results of analysis of a solution (60 % methanol
5.0
aqueous solution) of the 5 ginsenosides, each present at
50 mg/L in the 2 μL sample. 2.5
0.0
R3 0.0 2.5 5.0 7.5 min

OH
Fig. 1.35.2 Chromatogram of a Standard Mixture of 5 Ginsenosides
(50 mg/L each)
R1
R2
Compound R1 R2 R3
Ginsenoside Rg1 OH O-Glc O-Glc
Ginsenoside Re OH O-Glc(2-1)Rha O-Glc
Ginsenoside Rb1 O-Glc(2-1)Glc H O-Glc(6-1)Glc
Ginsenoside Rc O-Glc(2-1)Glc H O-Glc(6-1)Araf
Ginsenoside Rd O-Glc(2-1)Glc H O-Glc
Glc : ћ -D-glucose
Rha : њ -L-rhamnose
Araf : њ -L-arabinose (furanose)
Fig. 1.35.1 Structures of Ginsenosides
53
1.35 High Speed Analysis of Ginsenosides in Ginseng (2) - LC
QAnalysis of Powdered Ginseng

Fig. 1.35.3 shows the preparation procedure for herbal (SPE) is incorporated in the sample preparation procedure
medicines as described in the Japanese Pharmacopeia. of Fig. 1.35.3, using a reversed phase sorbent cartridge
Fig. 1.35.4 shows the results of analysis of commercial (Phenomenex “strata-X”), and the results of that analysis
ginseng powder, using a 2 μL injection of the sample are shown in Fig. 1.35.6. Compared to the results of
prepared using the process shown in Fig. 1.35.3. Fig. 1.35.5 Fig. 1.35.4, it is clear that the high-polarity contaminants
shows the procedure in which solid phase extraction are effectively removed during the SPE step.
Sample 1.0 g
60 % methanol (aq.) (30 mL)

Sonicate (15 min)
Sample Preparation 1 strata-X (60 mg/3 mL)
2 mL Aliquot Condition with methanol (2 mL)
Supernatant ① Residue ①
Dilute with water to 10 mL Condition with water (2 mL)
60 % methanol (aq.) (15 mL)
Sonicate (15 min)
Load the sample
Wash with 10 % methanol (aq.) (2 mL)
Supernatant ② Residue ②
Elute with methanol (2 mL)
Supernatants ① + ②
Inject to HPLC
Adjust to 50 mL using 60 % methanol (aq.)
Inject to HPLC
Fig. 1.35.3 Sample Preparation 1 Fig. 1.35.5 Sample Preparation 2
mAU mAU
20.0 20.0
QPeaks QPeaks
1.Ginsenoside Rg1 1.Ginsenoside Rg1
17.5 2.Ginsenoside Re
17.5 2.Ginsenoside Re
3.Ginsenoside Rb1 3.Ginsenoside Rb1
15.0 4.Ginsenoside Rc 15.0 4.Ginsenoside Rc
5.Ginsenoside Rd 1 5.Ginsenoside Rd
12.5 12.5
10.0 10.0 1
3 3
7.5 2 7.5
5.0 4 5.0
4
5 2
5
2.5 2.5
0.0 0.0
0.0 2.5 5.0 7.5 min 0.0 2.5 5.0 7.5 min
Fig. 1.35.4 Chromatogram of Powdered Ginseng Fig. 1.35.6 Chromatogram of Powdered Ginseng
(Using Sample Preparation 1) (Using Sample Preparation 2)
[Reference]
The 15th Revision of the Japanese Pharmacopeia (Society of Japanese Pharmacopeia)
54
H O
1.36 High Speed Analysis of Lutein and Zeaxanthin (1) - LC

Lutein and zeaxanthin are types of carotenoids, and Column : 6KLPSDFN;52'6PP/ðPP,'ƫP
are constituents of marigold pigment, a food additive Shim-pack VP2'6PP/ðPP,'ƫP
which also occurs naturally in various foods. Recent Mobile Phase : A :Methanol / Tetrahydrofuran / Water
studies have suggested that these substances may be YYY
effective in preventing cataracts and age-related macular B :Tetrahydrofuran
degeneration syndrome (AMD). Gradient Elution Method
Time Program : [XR-ODS]
Here we introduce an example of analysis of lutein and %PLQń 100 % (4.51-
zeaxanthin in marigold supplement extract using the PLQńPLQ
ultra fast LC “Prominence UFLC” with the SPD-M20A [VP-ODS]
photodiode array detector. %PLQń100 % (15.51-
PLQń 0 % PLQ
QAnalysis of Standard Solution Flowrate : P/PLQ;52'6P/PLQ932'6
Fig. 1.36.1 shows the structures of lutein and zeaxanthin, Column Temp. : 50 °C
which reveals that these 2 compounds are structurally Injection Volume : ƫ/;52'6, ƫ/932'6
similar, and in fact, str uctural isomers. Complete Detection : SPD-20A at 450 nm
separation of such substances generally requires the use Flow Cell : 6HPLPLFURFHOO;52'6
&RQYHQWLRQDOFHOO932'6
of reversed phase chromatography. In order to achieve
more efficient separation through improved selectivity,
we added tetrahydrofuran to the water / methanol mobile mAU
Shim-pack VP-ODS (150 mm L. ×4.6 mm I.D.)
phase. In addition, use of the Shim-pack XR-ODS high 22.5 2

20.0
speed, high resolution column (particle diameter 2.2 μm) 17.5
1
allows the analysis time to be shortened to about 1/4 the 15.0

time achieved with the conventational Shim-pack VP- 12.5
10.0
ODS (particle diameter 4.6 μm), while maintaining the 7.5
same resolution. 5.0
2.5
0.0
CH3
0.0 2.5 5.0 7.5 10.0 12.5 15.0 min
OH
CH3 CH3 CH3
H 3C
CH3
CH3 CH3 CH3
HO
CH3
Lutein Shim-pack XR-ODS (75 mm L. ×3.0 mm I.D.)
mAU
CH3
OH 22.5
CH3 CH3 CH3 20.0
H 3C 2
17.5
1
CH3 15.0
CH3 CH3 CH3
HO 12.5
CH3
Zeaxanthin 10.0
7.5
5.0
Fig. 1.36.1 Structures of Lutein and Zeaxanthin 2.5
0.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 min
QPeaks
1. Zeaxanthin (10 mg/L) 2. Lutein (10 mg/L)
Fig. 1.36.2 Chromatograms of Lutein and Zeaxanthin Standard Solution
55
1.36 High Speed Analysis of Lutein and Zeaxanthin (2) - LC
QAnalysis of Dietary Supplement mAU Lutein

Fig. 1.36.3 shows the analysis results of commercial
marigold extract contained in a dietary supplement 100
(capsule form). Fig. 1.36.4 shows the sample preparation
75
procedure. Fig. 1.36.5 shows overlay spectra of standard
and sample solutions of lutein and zeaxanthin in a dietary 50
supplement, respectively. In addition, Fig. 1.36.6 shows a
3-D plot of the dietary supplement. 25
0
mAU
150 300 400 500 600
nm
Q Peaks Zeaxanthin
125 1. Zeaxanthin 6
2. Lutein
2
5
100
4
75 3
2
50
1
25
0
1
0 300 400 500 600

nm
-25
0.0 1.0 2.0 3.0 4.0 5.0
Fig. 1.36.5 Spectra of Lutein and Zeaxanthin
min
Fig. 1.36.3 Chromatogram of Dietary Supplement
4 capsules
Tetrahydrofuran 10 mL
Sample solution 1 mL
3 % Pyrogallol-ethanolic solution 10 mL
1 % NaCl 0.5 mL
60 % KOH 1.0 mL
Saponification (30 min, Ý&
Cooling (5 min) 35
30
Ethyl acetate / Hexane (1:9) 15 mL 200 25
1 % NaCl 22.5 mL 250 Lutein 20
Shaking (5 min)
300 15
350 10 Intenisty
Centrifugation (mAU)
400 Zeaxanthin 5
Wavelength 450 0
Aqueous (Lower layer) (nm) 4,000
500
Supernatant (Upper layer) ① Ethyl acetate / Hexane (1:9) 15 mL 3,000
550
Mixing (5 min) ×2 2,000 Time
(min)
1,000
Centrifugation
Supernatant ② Residue
Fig. 1.36.6 3-D Plot of Dietary Supplement

Supernatant ① + ②
Evaporation to dryness
Tetrahydrofuran 10 mL
2 μL Injection to HPLC

56
H O
1.37 Determination of Coenzyme Q10 in Food - LC

In Japan, coenzyme Q10 has traditionally been used as Column : 6KLPSDFN)&2'6PP/ðPP,'
a pharmaceutical for improving myocardial metabolism. Mobile Phase : 0HWKDQRO(WKDQRO YY
In accordance with revisions to the Food and Medicine Flowrate : 1.5 mL/min
Differentiation List (Pharmaceuticals and Food Safety Column Temp. : 40 °C
Bureau, Ministry of Health, Labour and Welfare, Japan) Injection Volume : ƫ/
in 2001, coenzyme Q10 was moved to the food section Detection : SPD-20A at 275 nm
of the list. It is now the focus of attention as a food Slit Width : 8 nm
supplement. According to the Japanese Pharmacopoeia, Cell Temp. : 40 °C
which lists coenzyme Q10 under the pharmacological
name, “Ubidecarenone”, the recommended analysis QPeak
1
method for coenzyme Q10 is the HPLC method. Here we 1. Coenzyme Q10
introduce an analysis of coenzyme Q10 in commercially
available food products using the Prominence Photodiode
Array UV-Vis detector SPD-M20A.
O
H3CO CH3
H3CO H
O CH3 10
Fig. 1.37.1 Structure of Coenzyme Q10

0.0 2.5 5.0 7.5 min
QAnalysis of Food Sample Fig. 1.37.2 Chromatogram of Food Sample (Capsule)

Fig. 1.37.2 shows the resulting chromatogram, using a
photodiode array detector, of a food sample (capsule) Sample
Coenzyme Q10 Standard
containing coenzyme Q10. The sample was dissolved*
in ethanol (10 g/L) and the solution was ltered through
a membrane filter (0.45 μm) before injection (5 μL).
Fig. 1.37.3 shows comparison of the spectra of coenzyme
Q10 in the standard solution and that of corresponding
peak in the sample solution. We can see that the spectra
closely match. Using a photodiode array detector makes
it easy to obtain qualitative information from the UV
absorption spectrum.
*A high concentration sample was used in this measurement.
275
However, dilution by a factor of 100 is recommended for

routine analytical in order to reduce load on the column.
200.0 225.0 250.0 275.0 300.0 325.0 350.0 nm
Fig. 1.37.3 UV Spectra of Coenzyme Q10
57
2. Food Additives
2.1 Analysis of Food Preservatives (1) - GC/MS
Q Explanation
Food preservatives are used to prevent adverse changes (×10 6)
3.5
and rotting of foods by suppressing the proliferation 3.0
of microorganisms; they are used in accordance with 2.5

2.0
the properties of the food to which they will be added. 1.5
1.0
Many of the analytical methods used for analyzing food 0.5 TIC
preservatives employ extraction of the compounds of 0.0
(×10 5) %
interest by the steam distillation technique, followed 1.5 100
97
Sorbic acid
67
by quantitative analysis using high performance liquid 1.0 50
112 O
chromatography (HPLC). Gas chromatography can 0

OH
also be used for measurement of many compounds 0.5 50 100 150 200
97
used as food preservatives, including benzoic acid, 0.0

112
(×105) %
sor bic a cid , met hyl p -hyd rox yb e n z oat e ( PH BA 100
105 Benzoic acid
122 HO O
77
methyl), ethyl p-hyd roxybenzoate (PHBA ethyl), 1.0
50 51
propyl p-hydroxybenzoate (PHBA propyl), and butyl 0.5 0
p-hydroxybenzoate (PHBA butyl), and therefore, gas 50 100 150 200
105
chromatography / mass spectrometery (GC/MS) is an 122
effective analysis technique for verifying the detection (×10 6)

2.5
%
100
121 PHBA methyl
O O
results obtained by HPLC. Here we introduce an example 2.0

50 152
of GC/MS analysis of six types of food preservatives. 1.5 65 93
OH
1.0 0
50 100 150 200
0.5 121
152
QAnalytical Conditions (×10 6)

3.0
%
100
121 PHBA ethyl
O O
2.5
Instrument : GCMS-QP2010 Plus
2.0
50
93 138 166
-GC- 1.5
0
65
OH
Column : 5W[06PðPP,'GI ƫP 1.0 50 100 150 200 121
138
0.5
Injection Temp. : &&PLQ&PLQ 0.0
166
Carrier Gas : He, 45.0 cm/sec (×10 6)

3.0
%
100
121 PHBA propyl
O O
Carrier Gas Mode : Constant Linear Velocity Mode 138
50
Injection Temp. : 250 °C 2.0
65 93
180 OH
Injection Method : Split Injection 1.0
0
50 100 150 200 121
Split Ratio : 1:10 138

180
Injection Volume : ƫ/ 0.0

(×10 6) % 121 PHBA butyl
3.0 100 138 O O
-MS- 2.0
50
65 93 OH
I.F. Temp. : 260 °C 0
194
1.0 50 100 150 200
I.S. Temp. : 230 °C 121
138
Ionization Method: EI 0.0

194
5.0 7.5 10.0

Scan Range : m/z 40-300
Scan Interval : 0.3 sec
Fig. 2.1.1 Chromatograms of 6 Preservatives
58
Food Additives
2.1 Analysis of Food Preservatives (2) - GC/MS
QResults Liquid Condiment

Standard Samples In Japan, the use of PHBA esters is permitted in liquid
Each of six preservatives was used to prepare a 10 mg/L condiments such as soy sauce. A liquid condiment sample
standard solution. The resulting chromatograms are shown spiked with 5 μg/g of PHBA esters was also analyzed as
part of this study. For the pretreatment, the sample was
in Fig. 2.1.1. The uppermost data is the TIC chromatogram,
diluted 10 to 1 with ethanol and centrifuged for 5 minutes at
a nd t he r e m a i n i ng c h r o m a t og r a m s a r e t he m a s s 1000 rpm. The precipitate was removed, and the supernatant
chromatograms corresponding to the characteristic m/z of solution was analyzed without any further pretreatment.
each of the substances; the mass spectrum of each substance The analytical results are shown in Fig. 2.1.3. The mass
is also shown on the corresponding mass chromatogram. chromatograms of the added PHBA esters demonstrate that
Although sensitivities for benzoic acid and sorbic acid were they were clearly detected.
lower than those for the PHBA esters, the peaks for benzoic Many chromatographic peaks were observed in the TIC
acid and sorbic acid were clearly detected. chromatograms of both the soft drink and the liquid
condiment. Although the compounds of interest are
Soft Drinks difficult to observe in the TIC, the target substances are
Many commercially available soft drinks contain sodium clearly detected in the corresponding mass chromatograms.
Furthermore, mass spectra of the preservatives were
benzoate as a preservative, and one such soft drink in
easily obtained using spectrum subtraction. These results
which sodium benzoate was indicated as a preservative demonstrate that the preservative could be detected not only
ingredient was analyzed as part of this study. The in the standard sample but in the actual samples as well
pretreatment consisted only of diluting the sample 50 to using a simple pretreatment procedure. Clearly the results
1 with ethanol; the diluted sample was analyzed directly. of this study show that analysis by GC/MS is effective as a
The analytical results indicate the detection of benzoic con rmation technique for these substances.
acid in the mass chromatogram (Fig. 2.1.2).
(×10 7)
5.0
(×10 6)
2.5
5.0
4.0 TIC
3.0 0.0
%
2.0 (×10 5) 100 121 PHBA methyl
1.0 3.0
TIC 50 72 152
93
0.0 2.0
(×10 5) 0
% 50 100 150 200
100 105 1.0 121
3.0 122 Benzoic acid
77 152
50 51
2.0 (×10 5) %
100 121 PHBA ethyl
1.0 0
50 100 150 200
105 3.0 50
122 138
65 93 166
3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 2.0
0
50 100 150 200 121
1.0 138
166
Fig. 2.1.2 Result of Soft Drink Analysis (×10 5)

% 121
100 PHBA propyl
75 138
3.0 50
25 65 93 180
2.0 0
50 100 150 200 121
1.0 138
180
(×10 5) % 121 PHBA butyl

100 138
3.0 50
65 93 139 159
2.0 194
0
50 100 150 200 121
1.0 138
194
5.0 7.5 10.0
Fig. 2.1.3 Chromatograms of Soy Sauce Spiked with 4 kind of

Preservatives
59
2.2 High Speed Analysis of Benzoic Acid and Sorbic Acid - LC

Benzoic acid (as sodium benzoate), sorbic acid (as Column : Shim-pack XR-ODS
potassium sorbate), and sodium dehydroacetate, etc. are PP/ðPP,'ƫP
used as preservatives in food with the aim of preventing Mobile Phase : PPRO/ 6RGLXP &LWUDWH %XIIHU
food poisoning and the proliferation of microorganisms. S+$FHWRQLWULOH YY
Determination of these compound levels in food is Flowrate : 1.0 mL/min
generally conducted using HPLC. Here we introduce an Column Temp. : 40 °C
example of high speed analysis of benzoic acid, sorbic Injection Vol. : ƫ/
acid, and dehydroacetic acid using the Prominence UFLC Detection : SPD-20A at 230 nm
ultra fast LC system with the Shim-pack XR-ODS high-
speed, high-resolution column.
QAnalysis of a Soft Drink QAnalysis of Pickle Juice

Fig. 2.2.1 shows the results of analysis of a commercial Fig. 2.2.2 shows the results of analysis of pickle juice.
soft drink. A 10-fold dilution of the soft drink was After centrifuging the pickle juice, the supernatant was
prepared using distilled water, and after filtering it collected and diluted 10 times using distilled water,
through a membrane lter (0.2 μm pore diameter), 4 μL ltered through a membrane lter (0.2 μm pore diameter),
were injected. and 4 μL were injected.
mAU mAU
55
70 Q Peak Q Peak
1 1. Benzoic Acid 50 1. Sorbic Acid
60 45
40
50 1
35
40 30
25
30 20
15
20
10
10 5
0
0
-5
0.0 0.5 1.0 1.5 2.0 2.5 min 0.0 0.5 1.0 1.5 2.0 2.5 min
Fig. 2.2.1 Chromatogram of a Soft Drink Fig. 2.2.2 Chromatogram of Pickle Juice
60
Food Additives
2.3 Analysis of Natamycin in Cheese - LC

Natamycin is a polyene macrolide antibiotic that Column : Shim-pack VP-ODS
specifically inhibits the growth of mold and yeast, and PP/ðPP,'
was speci ed as a food additive (surface treatment agent :
Mobile Phase Methanol/Water/Acetic Acid = YYY
for cheese) to the “Japanese Food Additive Analysis Flowrate : 1.0 mL/min
Methods” revised on November 28, 20051), and the HPLC Column Temp. : 40 °C
natamycin analytical method was added 2) . Here we Detection : SPD-20A at 304 nm
introduce an example of analysis of natamycin in cheese
using HPLC. mAU
1.0
Q Peak
0.9
1. Natamycin
H OH
H 0.8
HOOC H OH H 0.7
O
H
0.6
O
H OH 0.5
H
CH3 O 0.4
H O
H O O 0.3
NH2 HO
1
HO H CH3 0.2
H H
H
0.1
Fig. 2.3.1 Structure of Natamycin 0.0
QAnalysis of Natamycin in Cheese 0.0 2.5 5.0 7.5 10.0 12.5 min
Two types of commercial cheese samples were prepared Fig. 2.3.3 Chromatogram of Cheese A (spiked with natamycin)
according to the procedure shown in Fig. 2.3.2, and
analysis was conducted. Each of these samples were mAU
spiked with an appropriate amount of natamycin standard 1.0
to achieve a concentration of 0.5 mg/kg. Fig. 2.3.3 and Q Peak

0.9
1. Natamycin
Fig. 2.3.4 show the respective analytical results.
0.8
0.7
Sample 10 g
0.6
Methanol 50 mL
0.5
Homogenize (5 min)
0.4
Water 50 mL
Natamycin (500 mg/L), 10 ѥL 0.3 1
&RROLQJÝ&KRXU 0.2
0.1
Filtration
0.0
HPLC 5 ѥL injection 0.0 2.5 5.0 7.5 10.0 12.5 min
Fig. 2.3.2 Sample Preparation Fig. 2.3.4 Chromatogram of Cheese B (spiked with natamycin)
[References]
1) “Ministerial Ordinance Revising Part of the Enforcement Regulations of the Food Sanitation Law, and Partial Revision of Standards for Foods
and Food Additives” (July 28, 2005 Ministry of Health, Labour and Welfare, Food Safety Supplement No. 1128002, Japan)
2) “Food Additive Analysis Methods” (July 28, 2005 Ministry of Health, Labour and Welfare, Food Safety Supplement No. 1128001, Japan)
61
2.4 Analysis of Food Antioxidants (1) - GC/MS

Food antioxidants serve to prevent the oxidation of food Instrument : GCMS-QP2010 Plus
ingredients by becoming oxidized themselves instead. BHT -GC-
(dibutylhydroxytoluene) and BHA (butylhydroxyanisol), Column : 5W[06PðPP,'GI ƫP
which had become widely used as food antioxidants, are Column Temp. : &&PLQ&PLQ
almost never used now because of concern related to their Carrier Gas : +HFPVHFN3D
reported carcinogenicity. Instead, vitamin C and vitamin Carrier Gas Mode : Constant Pressure Mode
E have recently become widely used. The use of some Injection Temp. : 250 °C
food additives is not permitted in Japan, but many such Injection Method : Split Injection
substances are permitted in some countries. For example, the Split Ratio : 1:5
antioxidant TBHQ (t-butylhydroquinone) is not permitted Injection Volume : ƫ/
to be used in Japan; however, since it may be used in many
other countries, there is a possibility of it being included -MS-
in food imports to Japan. Here we introduce the results of I.F. Temp. : 260 °C
analysis of the antioxidants BHA, BHT and TBHQ using I.S. Temp. : 230 °C
a gas chromatograph mass spectrometry (GC/MS). The Ionization Method : EI
antioxidants were added to a butter sample, and then extracted Scan Range : m/z 40-300
using a simple pretreatment process. In this analysis, the Scan Interval : 0.3 sec
backflush technique was employed to prevent high-boiling
point substances in the actual sample from being introduced
into the detector. (×106)
QResults and Discussion 2.0

Analysis of Standard Sample 1.5
A sample solution consisting of 10 mg/L each of BHA, BHT
and TBHQ in acetone was analyzed. The results are shown 1.0
in Fig. 2.4.1. The TIC chromatogram is shown at the top, and 0.5
the mass spectra of the respective constituent peaks and mass
chromatograms at the characteristic m/z values are shown below. (×105)
Each constituent peak was clearly detected. 3.0
Recovery Analysis of Spiked Components in 2.0

Actual Sample
1.0
Recovery analysis was conducted for substances added to an
actual sample of commercially available butter. After dissolving
100 mg of butter in 1 mL acetone, centrifugal separation (×106)
1.00
(1000 rpm, 5 minutes) was conducted to precipitate the extract,
and supernatant was collected and analyzed. For recovery testing, 0.75
BHA, BHT and TBHQ were added to the butter sample to attain
a nal concentration of 5 mg/kg each (concentrations of BHA, 0.50
BHT and TBHQ in sample following pretreatment were 0.5 mg/L, 0.25
respectively). The resulting chromatogram is shown in Fig. 2.4.2.
As in Fig. 2.4.1, the TIC chromatogram is shown at the top, and (×105)
the mass spectra of the respective constituent peaks and mass 1.5
chromatograms at the characteristic m/z values are shown
below. In the actual sample as well, peak detection could be 1.0
conducted selectively for the target constituents of the mass
chromatograms at the characteristic m/z values. Mass spectra 0.5
similar to those obtained using the standard solution were also
obtained. The analysis was conducted 3 times to determine 0.0
3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0
the repeatability of the quantitation values. The repeatability
results and recoveries based on the average quantitation value
with n=3 are shown in Table 2.4.1. Excellent repeatability and Fig. 2.4.1 Chromatograms of BHA, BHT and TBHQ (10 mg/L)
recovery were obtained for the actual sample.
62
Food Additives
2.4 Analysis of Food Antioxidants (2) - GC/MS
Table 2.4.1 Results of Butter Sample Analysis maintenance, since the high-boiling point substances are
Quantitation Values (mg/kg) RSD Average Recovery not introduced into the detector. In addition, high-boiling
1 2 3 Average (%) Rate (%) point substances may not be completely discharged
BHT 6.4 6.2 5.8 6.1 4.7 123 from the column during the analysis. Residual high-
BHA 4.7 4.6 4.6 4.6 1.3 93 boiling point substances in the column can cause such
TBHQ 5.9 5.9 5.3 5.7 6.4 114 problems as reduced sensitivity and poor repeatability,
so discharging them via the injection port provides an
ef cient means of removing them from the column while
(×105) extending the life of the column. The back ush settings
3.0
were implemented via time program, so that following
2.5
elution of the target components (in this analysis, at 9
2.0
minutes), the split vent pressure was raised from 20 kPa
1.5
to 200 kPa, and at the same time the column pressure was
1.0
reduced to 20 kPa. This program can be set automatically
0.5
using the backflush software utility. Fig. 2.4.3 shows a
(×104)
1.00 comparison of TIC chromatograms of butter analysis
with and without the use of the back ush technique. The
0.75
zoomed chromatograms in Fig. 2.4.3 display only the
0.50 data acquired up to the completion of elution of BHA,
0.25
BHT and TBHQ. Similar chromatograms were obtained
when backf lush was used and when it was not used.
(×104) When backflushing was conducted, however, no peaks
3.0
were detected after the 9 minutes corresponding to the
time setting. Implementing the backf lush prevented
2.0
introduction of the high-boiling point substances into
1.0
the detector, and provided a means of discharging them
ef ciently from the column.
(×103)
4.0
(×106) TIC
3.0 5.0
2.0 4.0
1.0 3.0
0.0 2.0 Without backflush

3.0 4.0 5.0 6.0 7.0 8.0 9.0
1.0
With backflush
0.0
5.0 10.0 15.0 20.0 25.0 30.0
Fig. 2.4.2 Chromatograms of Butter Sample (5 mg/L) 6
(×10 ) TIC Frame Magnified
6.0
&DSLOODU\%DFNÀXVK6\VWHP 5.0
In this investigation, act ual sample analysis was 4.0 Without backflush
conducted using a simple sample preparation process 3.0
(m i n i m a l s a mple cle a nup wa s p e r for me d a f t e r 2.0
extraction). Therefore, most of the high-boiling point 1.0 With backflush
substances would typically be introduced into the GC/ 0.0

3.0 4.0 5.0 6.0 7.0 8.0 9.0
MS.Backf lushing reverses the f low of carrier gas in
the column to discharge high-boiling point substances Fig. 2.4.3 Comparison of Butter Sample Analysis with/without Back ush
from the split vent of the injection port. One of the
benefits of backf lushing is the reduced frequency of
63
2.5 High Speed Analysis of Ascorbic Acid and Erythorbic Acid - LC

As an analytical method for determination of the food Column : Phenomenex LUNA NH2
antioxidants ascorbic acid and erythorbic acid, the (50 mm /ðPP,'ƫP
hydrophilic interaction liquid chromatography (HILIC) Mobile Phase : PPRO/7ULHWKDQRODPLQH3KRVSKDWH
mode, a type of normal-phase chromatography using an %XIIHUS+$FHWRQLWULOH YY
amino column, is used in combination with an acetonitrile Flowrate : 0.8 mL/min
/ aqueous mobile phase. Here we introduce an example Column Temp. : 40 °C
of high-speed, high-resolution analysis of ascorbic acid Injection vol. : ƫ/
and erythorbic acid using the Prominence UFLC ultra Detection : SPD-20AV at 240 nm
fast LC system with the high-speed analysis HILIC mode
Phenomenex LUNA NH2 column (particle diameter 3 μm). QAnalysis of Soft Drinks
Figs. 2.5.3 and Fig. 2.5.4 show the analysis of commercial
QAnalysis of Standard Solution soft drinks. Soft drink A (vitamin-added soft drink) was
Fig. 2.5.1 shows the structural formulas of ascorbic acid diluted 10 times with distilled water, and further diluted
and erythorbic acid. Fig. 2.5.2 shows the analysis of a 10 times with an acetonitrile / water (8/2, v/v) solution.
standard mixture of ascorbic acid and erythorbic acid Soft drink B (green tea beverage) was diluted 5 times
(20 mg/L each). The standard solution was prepared using with distilled water, and further diluted 5 times with an
an acetonitrile / water (8/2, v/v) solution, and 2 μL of acetonitrile solution. Each of these solutions was then
ltered through a membrane lter (pore diameter 0.2 μm),
this solution were immediately injected. When using the
and 2 μL of each were injected for analysis.
Phenomenex LUNA NH2 column (particle diameter 3 μm),
the analysis can be completed within 1.5 minutes using a
50 mm column while maintaining excellent resolution. mAU
30.0
Q Peak
CH2OH CH2OH 25.0 2. Ascorbic acid
2
HO CH HC OH 20.0
O O
O O
15.0
HO OH HO OH 10.0
Erythorbic acid Ascorbic acid 5.0
Fig. 2.5.1 Structural Formulas 0.0

0.00 0.50 1.00 1.50 min
mAU
1 Fig. 2.5.3 Chromatogram of Soft Drink A (2 μL injected)
Q Peaks
25.0 1. Erythorbic acid
2
2. Ascorbic acid mAU
30.0
20.0
Q Peak
25.0
15.0 2. Ascorbic acid
20.0
10.0
2
15.0
5.0
10.0
0.0
0.00 0.50 1.00 1.50 min 5.0
Fig. 2.5.2 Chromatogram of a Standard Mixture of Ascorbic Acid 0.0
and Erythorbic Acid (20 mg/L each, 2 μL injected) 0.00 0.50 1.00 1.50 min
Fig. 2.5.4 Chromatogram of Soft Drink B (2 μL injected)

64
Food Additives
2.6 High Speed Analysis of Tocopherols - LC

Tocopherols (Vitamin E) consist of a class of nutrients that Column : Shim-pack XR-SIL
are not only used as food additives such as antioxidants PP/ðPP,'ƫP
and nutritional supplements, but are also included in 6KLPSDFN&/&6,/0
natural ingredients. Tocopherols are known to exist in PP/ðPP,'ƫP
various forms, including the ơ -, Ƣ -, and ƣ - isomers, not Mobile Phase : +H[DQH3URSDQRO YY
to mention Ơ -tocopherol (Fig. 2.6.1). HPLC analysis of Flowrate : P/PLQ;56,/
tocopherol isomers is typically conducted using normal P/PLQ&/&6,/0
phase chromatography combined with f luorescence Column Temp. : 30 °C
detection. Here we conducted ultra fast analysis using the Injection Volume : ƫ/;56,/ƫ/&/&6,/0
Shim-pack XR-SIL high-speed, high-resolution analytical Detection : RF-20AXS Ex. at 298 nm, Em. at 325 nm
column (particle diameter 2.2 μm) together with the RF- Cell Temp. : 25 °C
Flow Cell : 6HPLPLFURFHOO;56,/
20A XS detector. Fig. 2.6.2 shows an example of analysis
&RQYHQWLRQDOFHOO&/66,/0
of a standard solution of 4 tocopherols (2 mg/L each)
using the Shim-pack CLC-SIL (M) conventional column
(particle diameter 5 μm) and the Shim-pack XR-SIL. The
Shim-pack XR-SIL shortened the analysis time to less Shim-pack CLC-SIL(M)
1
than one-fourth. 4
2 3
HO
H H 0.0 2.0 4.0 6.0 8.0 10.0 12.0 min

O
1 Shim-pack XR-SIL
Fig. 2.6.1 Structure of Ơ -Tocopherol 4

3
2
0.0 0.5 1.0 1.5 2.0 2.5 min

Q Peaks
1. њ -Tocopherol, 2. ћ -Tocopherol, 3. ќ -Tocopherol, 4. ѝ -Tocopherol
Fig. 2.6.2 Chromatograms of Standard Mixture of Tocopherols

(2 mg/L each)
65
2.7 Simultaneous Determination of Sweeteners in Food (1) - LC

Several different low calorie sweeteners have been used Column : 3KHQRPHQH[/XQD3)3
in a variety of foods and beverages in recent years. These PP/ðPP,'ƫP
sweeteners present a distinct taste that set them apart Mobile Phase : A: 10 mmol/L Ammonium Formate
from sucrose, glucose, fructose, etc., and the overall %XIIHUS+
taste can be manipulated by mixing multiple sweeteners. B: Acetonitrile
Here we introduce an example of simultaneous analysis Gradient Elution Method
of 6 sweeteners using the ELSD-LTⅡ Evaporative Light Time Program : B 5 % ńPLQ
Scattering Detector. ńPLQ
ńPLQ
QAnalysis of Standard Solution Flowrate : 1.0 mL/min
Fig. 2.7.1 shows the st r uct ural for mulas of the 6 Column Temp. : 40 °C
sweeteners analyzed for this experiment. Because the Injection Volume : ƫ/
polarity varies greatly among these, analysis must Detection : ELSD-LTⅡ
Temperature : 40 °C
be conducted by gradient elution. Moreover, because
Gain :6
sucralose displays little UV absorption, simultaneous
Nebulizer : GasN2
analysis was attempted using an evaporative light Gas Pressure : 350 kPa
scat ter ing detector (ELSD). Fig. 2.7.2 shows the
chromatogram of the standard mixture. The 6 compounds
were effectively separated without the use of an ion- mV
pair reagent by using the Phenomenex Luna PFP (2) Peaks
40
(particle size: 5 μm) column, which incorporates a 1. Acesulfame K
2. Saccharin
penta uorophenyl solid phase as the analytical column. 3. Sucralose
4. Aspartame
It was con rmed that glucose and sucrose were eluted as 5. Glycyrrhizic acid
unretained peaks. 6. Neotame 6
30
O O
4
H3C O
S
NH O
N K
S
O O O 3
Saccharin Acesulfame K
20 2
O
OH
H
Cl OH O
H 5
Cl
O
HO
O O
10
OH
O
HO Cl COOH
H H
1
O
OH
OH
OH
COOH O
O
OH
Sucralose
OH
OH
Glycyrrhizic acid 0
O OCH3 O OCH3
0.0 5.0 10.0 15.0 min
O O
HOOC N HOOC N
H H
H3C NH NH2 Fig. 2.7.2 Chromatogram of a Standard Mixture of 6 Sweeteners
H 3C
CH3
(100 mg/L each, 10μL injected)
Neotame Aspartame
Fig. 2.7.1 Structures of 6 Sweeteners
66
Food Additives
2.7 Simultaneous Determination of Sweeteners in Food (2) - LC
QAnalysis of Soft Drink

Fig. 2.7.3 to Fig. 2.7.5 show examples of analysis of uV
1000
commercial soft drinks. Using 100 mmol/L ammonium Peak
3. Sucralose
formate buffer solution, the low-sugar coffee and sports
drink were diluted 5-fold and 2-fold, respectively, and 800
after filtering through a 0.2 μm membrane filter for
aqueous solutions, each sample was injected at a volume 600
of 10 μL for analysis. For the carbonated beverage, after
the carbon dioxide was removed by ultrasonic treatment,
400
the same buffer solution was used to prepare a 5-fold
dilution. Then, after ltering through a 0.2 μm membrane 3
lter for aqueous solutions, 10 μL was injected. 200
0.0 5.0 10.0 15.0 min
Fig. 2.7.4 Chromatogram of Sports Drink (10 μL injected)
uV uV
Peaks Peaks
10000 1. Acesulfame K 10000 1. Acesulfame K
3. Sucralose 4
4. Aspartame
8000 8000
6000 6000
4000 4000
2000 2000
3
1
1
0 0
0.0 5.0 10.0 15.0 min 0.0 5.0 10.0 15.0 min
Fig. 2.7.3 Chromatogram of Coffee Drink (10 μL injected) Fig. 2.7.5 Chromatogram of Carbonated Beverage (10 μL injected)
67
2.8 Analysis of Cyclodextrins in Food (1) - LC

Cyclodextrins are cyclic oligosaccharides consisting of linked Column : Asahipak NH2P-50 4E
D-glucose units in a ring-shaped structure, and it is known PP/ðPP,'
that various compounds can be enclosed within that cyclic Mobile Phase : 5 mmol/L Ammonium Acetate /
structure. Properties such as water solubility and stability of $FHWRQLWULOH YY
the “guest molecules” incorporated into the “host” cyclodextrin Flowrate : 1.0 mL/min
can become greatly modi ed. For this reason they are widely Column Temp. : 40 °C
used in the food, pharmaceutical, chemical and other fields.
Injection Vol. : ƫ/
Here we introduce an example of hydrophilic interaction liquid
Detection : ELSD-LTⅡ
chromatography (HILIC) in which cyclodextrins in a food
product are separated, and then detected using the ELSD-LT Ⅱ
Gain :6
Evaporative Light-Scattering Detector.
Nebulizer Gas : N2
In the food industry, Ơ -cyclodextrin, ơ -cyclodextrin and
Ƣ-cyclodextrin are commonly used as food additives. These mV
100
differ according to the number of linked D-glucose units in Q Peaks
the structure, with the Ơ type containing 6 units, the ơ type 1. њ -Cyclodextrin
7 units, and the Ƣ type 8 units of linked D-glucose. Fig. 2.8.1 75 1 2. ќ -Cyclodextrin
3. ћ -Cyclodextrin
shows the structure of Ơ -cyclodextrin. Here, separation
of the cyclodextrins by hydrophilic interaction liquid
50
chromatography (HILIC) was conducted using a polyamine
column as the stationary phase, and a mixture of acetonitrile 2
3
and water as the mobile phase. Detection was conducted 25
using an evaporative light-scattering detector (ELSD).
Although detection is also possible using a differential
refractive index detector, the ELSD is often very effective in 0
analysis of foods, which typically contain many impurities, 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min
because of the applicability of gradient elution methods with
the ELSD. Fig. 2.8.2 shows the analysis results of a standard
mixture of the 3 cyclodextrins (500 mg/L each). Fig. 2.8.2 Chromatogram of a Standard Mixture of 3 Cyclodextrins
(500 mg/L each)
OH
O
OH O O
HO OH O
OH
HO OH
O OH O
HO
OH
O HO O
HO OH
O HO
HO OH
O O OH
O
HO
Fig. 2.8.1 Structure of Ơ -Cyclodextrin
68
Food Additives
2.8 Analysis of Cyclodextrins in Food (2) - LC
QAnalysis of Food Samples QAnalytical Conditions

Analyses were conducted for cyclodextrin food additives Column : $VDKLSDN1+3(PP/ðPP,'
using a functional drink (isotonic drink), green tea, and Mobile Phase : A : 5 mmol/L Ammonium Acetate
Japanese horseradish paste as samples. The hydrophilic B : Acetonitrile
impurities contained in these food products were strongly Gradient Elution Method
retained on the column speci ed in the original analytical Time Program : %PLQń PLQ
conditions, so new analytical conditions, including the ń PLQ
post-analysis column washing process indicated in this Flowrate : 1.0 mL/min
page, were used instead. Fig. 2.8.3 shows the respective Column Temp. : 40 °C
sample pretreatment procedures, and Figs. 2.8.4-6 show Injection Vol. : ƫ/
the chromatograms obtained for each sample. Detection : ELSD-LT Ⅱ
Gain :6
1) Functional Drink
Nebulizer Gas : N2
Sample 2 mL
Water/Acetonitrile = 5/5 (v/v) 8 mL mV
Stir 10 min 60
QPeak
1. ќ -Cyclodextrin
Filtration (0.45 ѥm) 50
40
Inject to HPLC 10 ѥL
30
20
2) Instant Green Tea 1
10
Sample 0.5 g
Water 20 mL 0
Stir 10 min 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min
Centrifuge (3000 rpm) 10 min Fig. 2.8.4 Chromatogram of Functional Drink
Supernatant 5 mL mV
Acetonitrile 5 mL 20
■ Peak
Filtration (0.45 ѥm) 1.ћ -Cyclodextrin
15
10
1
3) Japanese Horseradish Paste 5

Sample 1.5 g
Water 50 mL
0
Stir 10 min
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min
Fig. 2.8.5 Chromatogram of Instant Green Tea
Supernatant 5 mL
Acetonitrile 5 mL
mV
Filtration (0.45 ѥm) 150
■ Peak
125 1. ћ -Cyclodextrin
100
75
Fig. 2.8.3 Preparation of Samples 50
25
1
0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min
Fig. 2.8.6 Chromatogram of Japanese Horseradish Paste
69
2.9 Analysis of Sugar Alcohols in Food (1) - LC

Sugar alcohols are widely used in the food industry as Column : 8QLVRQ8.$PLQRPP/ðPP,'ƫP
low-calorie sweeteners that cause almost no tooth decay. Mobile Phase : A : Water
Here we introduce an example of hydrophilic interaction B : Acetonitrile
liquid chromatography (HILIC) in which 8 sugar alcohols Gradient Elution Method
are separated by gradient elution, and then detected using Time Program : %PLQńPLQń 40 %
the ELSD-LTⅡ Evaporative Light Scattering Detector. PLQńPLQ
QAnalysis of Standard Solution Detection : ELSD-LTⅡ
Sugar alcohols are chain polyhydric alcohols obtained Temperature : 40 °C
through reduction of the aldose or ketose carbonyl group. Gain :6
Fig. 2.9.1 shows the structural formulas of 2 typical Nebulizer Gas : N2
sugar alcohols, sorbitol and xylitol. Sugar alcohols, as Gas Pressure : 350 kPa
with sugars in general, are substances that have no UV
chromophore and while the differential refractive index
detector (RID) is generally applicable for detection, it is mV
110
not very suitable for multi-compound analysis because Peaks
100
it cannot be applied in gradient elution analysis. As 90
1. Erythritol
2. Xylitol
5. Inositol
6. Maltitol
7
an alternative, we used an evaporative light scattering 80
3. Sorbitol
4. Mannitol
7. Lactitol
8. Palatinol
5 6
detector (ELSD) for simultaneous analysis of 8 sugar 70 2

1 34
alcohols by gradient elution. An aminosilica column was 60
used as the analytical column, and the sugar alcohols were 50
eluted by hydrophilic interaction liquid chromatography 40 8*
30
(HILIC) using an acetonitrile / water mobile phase. Since
20
the highly hydrophilic impurities that are common in
10
foods are strongly retained on the column, analysis was 0
conducted with a column-washing procedure added to the -10
conditions. Fig. 2.9.2 shows the chromatogram obtained 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0
min
following injection of 10 μL of a standard mixture of 8
sugar alcohols (each 250 mg/L). Fig. 2.9.2 Chromatogram of a Standard Mixture of 8 Sugar Alcohols
(250 mg/L, 10 μL injected)
HO HO
*Since Palatinol is a mixture of 1-o-Ơ -D-glucopyranosyl-D-
H H glucitol and 1-o-Ơ -D-glucopyranosyl-D-mannitol, two peaks
OH are eluted.
HO
H OH H OH
Sorbitol
HO H HO H
HO OH
H OH
Xylitol
Fig. 2.9.1 Structures of Sorbitol and Xylitol
70
Food Additives
2.9 Analysis of Sugar Alcohols in Food (2) - LC
QAnalysis of Food Samples

mV
A functional beverage, a sugarless candy and a sugarless 450
mint tablet were analyzed as food products to which 400
1 Peaks
1. Erythritol
sugar alcohols had been added. Fig. 2.9.3 shows the 350 2. Glucose
sample preparation procedure. Erythritol was detected in
300
the functional beverage (Fig. 2.9.4), xylitol, sorbitol and
250
maltitol in the sugarless candy (Fig. 2.9.5), and sorbitol
was detected in the sugarless mint tablet (Fig. 2.9.6). 200
150
100
50
1) Functional Beverage 2
Sample 1 mL 0
Water / Acetonitrile = 5/5 (v/v) 9 mL 0.0 5.0 10.0 15.0 20.0 25.0 min
Stir
Filtrate (0.45 ѥm) Fig. 2.9.4 Chromatogram of Functional Beverage
mV
2) Sugarless Candy 3
800 Peaks
Sample 1.0 g 1. Xylitol
Water 20 mL 700 2. Sorbitol
3. Maltitol
Stir 20 min 600
Centrifuge (3000 rpm) 10 min 500

400
Supernatant 1 mL 1
Water / Acetonitrile = 5/5 (v/v) 19 mL 300
Filtrate (0.45 ѥm) 200
Inject to HPLC 10 mL 100

2
0
3) Sugarless Mint Tablet 0.0 5.0 10.0 15.0 20.0 25.0 min
Sample 0.6 g
Water 20 mL
Fig. 2.9.5 Chromatogram of Sugarless Candy
Stir 20 min

mV
Supernatant 1 mL 500
Water / Acetonitrile = 5/5 (v/v) 19 mL 1
Peak
Filtrate (0.45 ѥm) 450 1. Sorbitol
400
Inject to HPLC 10 ѥL) 350
300
250
Fig. 2.9.3 Sample Preparation 200
150
100
50
0
0.0 5.0 10.0 15.0 20.0 25.0 min
Fig. 2.9.6 Chromatogram of Sugarless Mint Tablet
71
2.10+LJK6SHHG$QDO\VLVRI$UWL¿FLDO&RORULQJV - LC

Here we present an example of ultra-high-speed analysis Column : 3KHQRPHQH[.LQHWH[ƫP&c
of artificial colorings using the Nexera UHPLC (Ultra PP/ðPP,'ƫP
High Performance Liquid Chromatography) System Mobile Phase : A: 10 mmol/L Ammonium Acetate
together with the Phenomenex Kinetex C18 high-speed, B: 10 mmol/L Ammonium Acetate /
high-resolution analytical column. $FHWRQLWULOH YY
Time Program : %PLQńPLQ
Q$QDO\VLVRI$UWL¿FLDO&RORULQJV ńPLQ
We conducted simultaneous analysis of 12 kinds of tar 0L[HUƫ/
synthetic dyes. The Phenomenex Kinetex-C18 (particle Flowrate : 2.5 mL/min
size 2.6 μm) was used for the separation. This is a Core- Column Temp. : 40 °C
Shell column consisting of a 1.9 μm solid core coated Injection Volume : ƫ/
with a bonded multilayer (0.35 μm thick) of porous silica Detection : SPD-M20A Max Plot 400-700 nm
gel micro particles. Monitoring was conducted using the Flow Cell : Semi-micro cell
maximum absorbance (MAX plot) from 400 to 700 nm
with the SPD-M20A photodiode array detector. Fig. 2.10.1 Table 2.10.1 Repeatability of 12 Arti cial Colorings (n=6)
shows the chromatogram of a standard mixture of the 12
arti cial colorings (each at 50 mg/L in aqueous solution),
Peak No. Retention Time %RSD Peak Area %RSD
in addition to a contour plot. The 12 dyes were separated 1 0.103 0.117
within 1 minute. In addition, excellent repeatability of 2 0.069 0.161
retention time and peak area were obtained for all of 3 0.047 0.289
the components, using 6 consecutive injections (1-μL 4 0.068 0.219
injections), as shown in Table 2.10.1. 5 0.091 0.116
6 0.057 0.203
7 0.066 0.170
mAU 8 0.056 0.206
9 0.021 0.073
10
300 78 11 10 0.034 0.117
11 0.032 0.140
200 9 12 12 0.037 0.148
5 6 mAU
100 1 23 4
750
0.0
500
0.00 0.25 0.50 0.75 1.00 min
nm
300 250
400
500
0.0
600 0.00 0.25 0.50 0.75 1.00 min
700
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 min
■ Peaks
1. Y4 (Tartrazine), 2. R2 (Amaranth), 3. B2 (Indigo Carmine),
4. R102 (New Coccine), 5. Y5 (Sunset Yellow FCF),
6. R40 (Allura Red AC), 7. G3 (Fast Green FCF),
8. B1 (Brilliant Blue FCF), 9. R3 (Erythrosine), 10. R106 (Acid Red),
11. R104 (Phloxine), 12. R105 (Rose Bengale)
Fig. 2.10.1 Chromatogram and Contour Plot of a Standard

Mixture of 12 Arti cial Colorings (50 mg/L each)
72
Food Additives
+LJK6SHHG$QDO\VLVRI$UWL¿FLDO&RORULQJV- LC
Q$QDO\VLVRI$UWL¿FLDO&RORULQJVLQ)RRG
Pickle juice and liquid extract of candy were measured using the analytical conditions shown on the previous page.
The retention times and spectral patterns of the detected peaks matched those of the standard substances.
mAU mAU
40 mAU mAU 40 mAU
30 ■ Peaks 30 ■ Peak
1.0 Peak 4 Peak 10
4. R102
Peak 3
30 30 20 3. B2
20 10. R106
0.5 10 3
10 10
20 0.0 20
0 0
300 500 nm 300 500 nm 300 500 nm
10 10
4
0 0
0.00 0.25 0.50 0.75 1.00 min 0.00 0.25 0.50 0.75 1.00 min
Fig. 2.10.2 Chromatograms and Spectra of Arti cial Colorings in Food (Left: Pickle, Right: Candy)
Q$QDO\VLVRI$UWL¿FLDO&RORULQJV mAU
150
We conducted analysis of 21 artificial substances, 13 16 18 20
+ 10 11
Max.Plot
100 14 15 17 7 8
19 9 21
consisting of those listed on the previous page together 50 1 23 4 5 6 12
with 9 additional dye substances. Based on investigation 0

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 min
of the gradient analysis conditions (Table 2.10.2), mAU
simultaneous analysis was completed within 2 minutes. 30 1 5 450 nm
13 20
Fig. 2.10.3 shows the MAX plot and the chromatograms 20
19
obtained using 3 different wavelengths. Chromatographic 10
0
peaks were identified for yellow dyes at 450 nm, red 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 min
dyes at 520 nm, and blue-green dyes at 620 nm. Thus, mAU
50 6 9
simultaneous quantitation can be achieved by selecting 2 4
16 10 11
520 nm
25 15
the appropriate detection wavelength for each substance 14 17 12
using the photodiode ar ray detector. In the MAX 0

0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 min
plot, peak 20 (Orange Ⅱ ) and peak 21 (Patent Blue V)
mAU
are overlapping, however, as shown in Fig. 2.10.4, at 100 7 8 21 620 nm
450 nm and 620 nm, almost none of the other substances 18
50 3
are present, allowing quantitation at each separate
wavelength. 0
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 min
■ Peaks
Table 2.10.2 Analytical Conditions 1. Y4 (Tartrazine), 2. R2 (Amaranth), 3. B2 (Indigo Carmine),
4. R102 (New Coccine), 5. Y5 (Sunset Yellow FCF),
Time Program : %PLQńPLQ 6. R40 (Allura Red AC), 7. G3 (Fast Green FCF),
8. B1 (Brilliant Blue FCF), 9. R3 (Erythrosine), 10. R106 (Acid Red),
ńPLQ 11. R104 (Phloxine), 12. R105 (Rose Bengale)
Detection : SPD-M20A 13. Orange G, 14. Black PN, 15. Red 2G, 16. Fast Red E,
17. Azo Rubine, 18. Green S, 19. Quinoline Yellow S,
Max Plot 400-700 nm, 450 nm, 520 nm, 20. OrangeⅡ, 21. Patent Blue V
620 nm
Fig. 2.10.3 Multi-Chromatogram of a Standard Mixture of 21
*The other conditions are the same as speci ed on the previous page. Arti cial Colorings (25 mg/L each)
mAU
500
Peak 20 Peak 21
400
300
200
100
0
300 400 500 600 nm
Fig. 2.10.4 Spectra of Peak 20 and Peak 21

73
2.11 Analysis of Curcumin in Turmeric - LC

Analysis of herbal medicines by HPLC generally requires Column : Shim-pack XR-ODS
separation of the impurities and active constituents, so PP/ðPP,'ƫP
the time to required to complete an analysis becomes Mobile Phase : A: 2 %$FHWLF$FLGDT
relatively long. Here we introduce examples of high- B : Acetonitrile
speed analysis of herbal medicines using the “Prominence A / B = 55/45 (v/v
UFLC” ultra-fast LC system with the “Shim-pack XR- Flowrate : 1.0 mL/min
ODS” high-speed, high-resolution column. Column Temp. : 40 °C
QAnalysis of Curcumin in Turmeric Detection : SPD-20AV at 425 nm
Curcumin, which is present in turmeric, is used not Flow Cell : Semi-micro Cell
only as an arti cial yellow coloring agent, but it is also
effective for enhancing liver function and promoting bile
secretion. The sample preparation procedure is shown
in Fig. 2.11.1. Fig. 2.11.2 shows an analysis example of mAU
300
curcumin in turmeric. Q Peak
1. Curcumin
250
Sample 0.5 g
Methanol 5 mL 200
1
Ultrasonic Extraction (30 min)
Centrifuge (3000 rpm, 5 min) 150
Supernatant Residue
100
Filtration
Inject to HPLC 50

0
0 1 2 3 min
Fig. 2.11.2 Chromatogram of Turmeric
74
Food Additives
2.12 Analysis of Impurities in Curcumin (1) - LC/MS

Here we present an example of analysis of a curcumin Column : Shim-pack XR-ODS
using photodiode array (PDA) detection and mass PP/ðPP,'ƫP8)/&
spectrometry (MS). Shim-pack VP-ODS
PP/ðPP,'ƫP+3/&
QAnalysis of Curcumin Standard Solutions Mobile Phase : 0.1 % Formic Acid / Acetonitrile = 60/40
Curcumin is a type of polyphenol present in turmeric, and Flowrate : P/PLQ8)/&P/PLQ+3/&
is used as a food dye. Fig. 2.12.1 shows the structrures Column Temp. : 40 °C
of curcumin in addition to two similar compounds Injection volume : ƫ/
(curcuminoids). The measurement samples consisted Detection
of commercial curcumin standards of different purity, PDA
referred to as grade A and grade B. The standard solutions SPD-M20A : 425 nm
were prepared by dissolving the standards in methanol MS
and adjusting the respective concentrations to 1 mg/mL. Probe Voltage : N9(6,1HJDWLYH0RGH
PDA detection was conducted at a wavelength of 425 nm. Nebulizing Gas Flow : 1.5 L/min
MS analysis was conducted using electrospray ionization, Drying Gas Pressure : 03D8)/&03D+3/&
and the deprotonated molecules were detected. Grade A CDL Temp. : 250 °C
showed a relatively low purity of curcumin, and more Block Heater Temp. : 200 °C
than 20 minutes was required to complete the analysis CDL, Q-array Voltages : Using default values
using a 5 μm particle diameter column (Fig. 2.12.2 Scan Range : m/z 150 - 500
(a), (b)), while this analysis time was shortened by 7
minutes using the Prominence UFLC (Fig. 2.12.2 (c), P$8

,QWHQ
(d)). The area percentages in the PDA chromatogram D E

were 1.11 % for bisdemethoxycurcumin, 11.46 % for

demethoxycurcumin, and 87.19 % for curcumin. Other
than these compounds, minute peaks were also noticeable

at tR=1.34 min (ƪ max 338 nm, m/z 337), tR=1.48 min (ƪ
max 349 nm, m/z 367), and tR=2.77 min (ƪ max 429 nm,
P$8
PLQ
,QWHQ
PLQ
m/z 383). As for the highpurity grade B sample, neither

F G
bisdemethoxycurcumin nor demethoxycurcumin were

detected (Fig. 2.12.2 (e), (f)).

H PLQ PLQ
O O
P$8 ,QWHQ
(1)
H I
HO OH

H

O O
(2)

HO OH
OCH3
H PLQ PLQ
O O
Q3HDNV
(3) %LVGHPHWKR[\FXUFXPLQ'HPHWKR[\FXUFXPLQ&XUFXPLQ
HO OH
OCH3 OCH3 Fig. 2.12.2 PDA Chromatograms, Total Ion Chromatograms
(TIC) of Curcumin Standard Solutions
(a) PDA chromatogram of curcumin (grade A) by HPLC,
Fig. 2.12.1 Structures of Curcuminoids (b) TIC of curcumin (grade A) by HPLC-MS
(1) Bisdemethoxycurcumin (MW 308), (c) PDA chromatogram of curcumin (grade A) by UFLC
(2) Demethoxycurcumin (MW 338), (d) TIC of curcumin (grade A) by UFLC-MS
(3) Curcumin (MW 368) (e) PDA chromatogram of curcumin (grade B) by UFLC
(f) TIC of curcumin (grade B) by UFLC-MS
75
2.12 Analysis of Impurities in Curcumin (2) - LC/MS
Q PDA Data
Fig. 2.12.3 shows the UV spectra of curcuminoids. All of them clearly possess a maximum absorption wavelength near
420 nm. When the peak purity is calculated from the grade A data, an impurity was detected at 5.35 minutes (Fig. 2.12.4 (2)).
mAU mAU mAU
17.5 150 1000
(1) 417 (2) 421 (3) 426
900
15.0 125
800
12.5 700
100
10.0 600
75 500
7.5
400
50
5.0 246 300
240 263
200
2.5 25
100
600
0.0 0 0
200 300 400 500 nm 200 300 400 500 nm 200 300 400 500 nm
Fig. 2.12.3 UV Spectra of Curcuminoids ((1) Bisdemethoxycurcumin, (2) Demethoxycurcumin, (3) Curcumin)
mAU mAU mAU

0.85 15.0
Purity Curves Purity Curves Purity Curves
130 0.9 Zero Line 900
0.80 Zero Line Zero Line
14.0 0.6
peak peak peak
0.75
(1) 13.0 (2) 120 0.8 (3) 800
0.70 0.5
12.0 110
0.65 0.7 700
0.60 11.0 0.4 100
0.55 10.0 90 0.6 600
0.50 9.0 0.3 80
0.45 0.5 500
8.0 70
0.40 0.2
0.35 7.0 60 0.4 400
0.30 6.0 0.1
50 0.3
0.25 5.0 300
40
0.20 4.0 0.0 0.2
0.15 3.0 30 200
0.10 -0.1 20 0.1
0.05 2.0 100
0.00 1.0 -0.2 10 0.0
0.0 0 0
4.2 4.3 4.4 4.5 min 5.00 5.25 min 5.75 6.00 6.25 6.50 min
Fig. 2.12.4 Purity Curves of Curcuminoids ((1) Bisdemethoxycurcumin, (2) Demethoxycurcumin, (3) Curcumin)
QAnother Impurity
Focusing on the peak (compound X) at 5.35 minutes in the in the vicinity of 370 nm, and the m/z 369 negative ion was
mass chromatograms of Fig. 2.12.4 (2), the UV spectrum detected. Therefore, the molecular weight of compound X is
and mass spectrum were obtained for this peak, as shown presumed to 370. Thus, by using both PDA and MS in UFLC
in the Fig. 2.12.5. The maximum absorption wavelength is detection, greater ef ciency is believed to be possible.
Inten. (×1,000,000) (×1,000,000)
3.50
3.25 (a) 10.0 (b)
3
3.00 7.5 373
2.75 QPeaks
2.50 1. Bisdemethoxycurcumin,2 5.0
2. Demethoxycurcumin,
2.25 2.5 255 282
3. Curcumin
2.00 X
1 0.0
1.75 TIC
1.50 200 300 400 500 nm
Inten. (×10,000)
1.25 m/z 307 6.0
(c) 369
1.00 5.0
m/z 337
0.75 4.0
0.50 m/z 369 3.0
0.25 2.0
0.00 m/z 367
1.0
169 397
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 min 0.0
200 300 400 m/z
Fig. 2.12.5 Mass chromatograms of curcumin standard solution (grade A, (a)) and UV spectrum (b), mass spectrum (c) of compound X
76
Food Additives
2.13 Analysis of Sudan Dyes in Foods - LC

Sudan dyes are oil-soluble red synthetic dyes that are Column : Shim-pack VP-ODS
used in industrial products. These dyes are forbidden PP/ðPP,'
to be added to food products in Japan, Europe and the Mobile Phase : YY )RUPLF$FLG DT
United States, however, since SudanⅠ was detected in $FHWRQLWULOH YY
imported cayenne in Europe in May 2003, the presence Flowrate : 1.0 mL/min
of SudanⅠ in foods have been reported in Europe and Column Temp. : 40 °C
other places. Monitoring inspections for Sudan dyes are Injection Vol. : ƫ/
being reinforced in Japan, as well. Sudanese dyes include Detection : SPD-20AV at 480 nm
SudanⅠ , Sudan Ⅱ , Sudan Ⅲ , and Sudan Ⅳ . Sudan Ⅳ
has also been found in Europe and other places. Here we
introduce simultaneous HPLC analysis of seven dyes, QAnalysis of Curry Powder
including SudanⅠ- Ⅳ , Sudan Orange G, Sudan Red G and Fig. 2.13.3 shows the chromatogram of commercial
Sudan Red 7B. curry powder spiked with standard samples of Sudan
Dyes. Seven types of Sudan dyes were added to the
curry powder to make 100 μg/g for each dye, and after
pretreatment as shown in Fig. 2.13.2, 10 μL of the sample
Fig. 2.13.1 shows the chromatogram of a 10 μL injection of
solution was injected into the HPLC.
a standard mixture of Sudan dyes (each concentration was
100 mg/L, which was obtained by 10-fold dilution of 1 g/L
of acetone solution with ethanol.). 6DPSOHJ67'
* Since Sudan Orange G was con rmed as two isolated peaks, they are
ĸ(WKDQROP/
indicated as Sudan Orange G-a and Sudan Orange G-b respectively. 6KDNHPLQ
&HQWULIXJHPLQ
6XSHUQDWDQW 5HVLGXH
ĸ(WKDQROP/
6KDNHPLQ
P$8 Q3HDNV &HQWULIXJHPLQ

6XGDQ2UDQJH*D
6XSHUQDWDQW5HVLGXH
6XGDQ2UDQJH*E
6XGDQ5HG* )LOOXSWRP/ZLWKHWKDQRO
6XGDQⅠ
6XGDQⅡ )LOWHUHG

6XGDQⅢ ,QMHFWWR+3/&
6XGDQ5HG%
6XGDQⅣ
Fig. 2.13.2 Sample Pretreatment
PLQ
Fig. 2.13.1 Chromatogram of a Standard Mixture of Sudan Dyes

P$8
(100 mg/L each, 10 μL inj.) Q3HDNV
6XGDQ2UDQJH*D

6XGDQ2UDQJH*E
6XGDQ5HG*
6XGDQⅠ

&XUU\SRZGHU 6XGDQⅡ
6XGDQG\HV
6XGDQⅢ
6XGDQ5HG%
&XUU\SRZGHU
6XGDQⅣ
PLQ
Fig. 2.13.3 Chromatogram of Curry Powder Spiked with Sudan

Dye Standards (each added at 100 μg/g)
77
2.14 Analysis of Sudan Dyes and Para Red - LC/MS

Sudan dyes are red or orange synthetic pigments used to color Column : Shim-pack FC-ODS
various dyeing oils and waxes, etc. Sudan dyes are not permitted (2.0 mm ,'ðPP
in food in many countries, including Japan, the EU and the Mobile Phase : Water containing 0.1 % Formic
United States, due to concerns that they may be carcinogenic.
Acid / Acetonitrile = 15/85
However, there are some countries where these dyes are still
being used for coloring of such spices as chili powders and Flowrate : 0.2 mL/min
paprika. Since SudanⅠ was rst detected in cayenne products Column Temp. : 40 °C
imported into the EU in May 2003, there have been many Injection Volume : ƫ/
reported cases of its detection, and this has led to strengthened Probe Voltage : N9(6,3RVLWLYH0RGH
inspection of such products when they are imported into Japan.
The Ministry of Health, Labour and Welfare Administration of CDL Temp. : 250 °C
Food Safety Publication No.0501008, “Test Methods for Sudan BH Temp. : 200 °C
Dyes and Para Red in Foods” (hereafter referred to as “of cial Nebulizing Gas Flow : 1.5 L/min
methods”), dated May 1, 2006, specifies the use of HPLC as Drying Gas Pressure : 0.10 MPa
the quantitation method, and LC/MS as one of the con rmation CDL Voltage : Using default value
test methods. Here we introduce an example of simultaneous
quantitative analysis of Sudan dyes and Para Red using LC/ Q-array DC Voltage : Using default value
MS. Fig. 2.14.1 shows the structural formulas and mass spectra Q-array RF Voltage : Using default value
for SudanⅠ-Ⅳ and Para Red. Fig. 2.14.2 shows the SIM Scan Range : m/z 90-500
chromatograms obtained for SudanⅠ- Ⅳ and Para Red added to SIM Monitoring Ions : m/z 294, 249, 277, 353, 381
commercial chili powder.
Inten.(×1,000,000) Inten.(×1,000,000) Inten.(×1,000,000)

5.0
5.0 249 HO
277 HO 353
N 5.0 H3 C N N HO
N N N N
CH3 N
2.5 SudanⅠ SudanⅡ 2.5 Sudan Ⅲ

C16H12N2O 2.5 C18H16N2O C22H16N4O
ExactMass: 248.09 278 Exact Mass: 276.13 Exact Mass:352.13
232 156
0.0 0.0 0.0
100 200 300 400 m/z 100 200 300 400 m/z 100 200 300 400 m/z
Inten.(×1,000,000) Inten.(×1,000,000)
381 294
-
O HO
H3 C N HO 1.0 N+ N
N N O N
CH3 N
2.5
SudanⅣ
C24H20N4O 0.5 Para Red
ExactMass:380.16 C16H11N3O3
383 ExactMass:293.08
105 249 335
0.0 0.0
100 200 300 400 m/z 100 200 300 400 m/z
Fig. 2.14.1 Positive ESI Mass Spectra of Sudan Dyes and Para Red
Para Red SudanⅠ SudanⅡ Sudan Ⅲ SudanⅣ

(10,000) (100,000) (100,000) (10,000) (10,000)
1.25 3.5
4.0
1.00 5.0 3.0
1.00
3.0 4.0 2.5
0.75
0.75
3.0 2.0
2.0 0.50 0.50 1.5
2.0
1.0
1.0 0.25 0.25
1.0
0.5
0.00
2.5 5.0 3.2 5.0 7.2 7.5 10.0 12.5 15.0 22.5 25.0
Fig. 2.14.2 SIM Chromatograms of Chili Powder Extract with 5 μg/mL Sudan Dyes and Para Red (Black), Chili Powder Extract (Red),
and Sudan Dyes and Para Red (Blue) at 500 ng/mL each
78
Food Additives
2.15 Analysis of Essential Oil - GC

Here, direct GC analysis examples of peppermint oil and Column : ULBON HR-20M
spearmint oil used as avorings are introduced. PðPP,'GI ƫP
Column Temp. : 60 °C -3 °C/min - 220 °C
Injector Temp. : 250 °C
Detector Temp. : 250 &),'
Carrier Gas : +HP/PLQ
Injection Method : Split Injection
Split Ratio : 1:15
■ Peaks ■ Peaks
1. Limonene 1. Limonene
2. 1,8-Cineole 2. 1,8-Cineole
3. trans-Sabinenehydrate 3. Menthone
4. L-Menthone
5. Menthoturan 4. Terpinene-4-ol
6. D-Isomenthone 5. ћ -Caryophyllene
7. Neo-Menthol 6. Dihydrocarvone
8. Terpinene-4-ol 7. Carvone
9. ћ -Caryophyllene
12 3 4 5 6 78 9 10 10. Menthol 12 3 4 5 6 7
12
16
20
24
28
32
36
40
44
48
52
56
12
16
20
24
28
32
36
40
44
48
52
56
0
4
8
0
4
8
Fig. 2.15.1 Analysis of Peppermint Oil Fig. 2.15.2 Analysis of Spearmint Oil
79
2.16 Flavor Analysis by Fast - GC/MS (1) - GC/MS
Q Explanation
Fast-GC/MS is an effective way to improve laboratory acquisition technology. Shimadzu GCMS systems offer
productivit y by shor tening analysis cycle times. the high-performance levels necessary to satisfy these
Fast-GC/MS methods require a system with a high- requirements. Here we show the results from analyzing
performance AFC controller that is compatible with lavender oil using conventional methods and Fast-GC/MS
narrow bore capillary columns and high-speed data methods.
Conventional-GC/MS
Instrument : GCMS-QP2010 Ultra
Column : 5W[06P/ðPP,'ƫP
Glass Insert : Split insert with deactivated glass wool
31
[GC] [MS]
Injection Temp. : 250 °C Interface Temp. : 250 °C
Column Temp. : &PLQ&PLQ& Ion Source Temp. : 200 °C
PLQ Measurement Mode : Scan
Injection Mode : Split Mass Range : m/z 40 - 400
Control Mode : /LQHDUYHORFLW\FPVHF Emission Current : ƫ$
Split Ratio : 1:100 KLJKVHQVLWLYLW\
Fast-GC/MS
Instrument : GCMS-QP2010 Ultra
Column : 5W[P/ðPP,'ƫP
Glass Insert : Split insert with deactivated glass wool
31
[GC] [MS]
Column Temp. : &PLQ&PLQ& Ion Source Temp. : 200 °C
&PLQ&PLQ Measurement Mode : Scan
Injection Mode : Split Mass Range : m/z 40 - 400
Control Mode : /LQHDUYHORFLW\FPVHF Emission Current : ƫ$
Split Ratio : 1:1800 KLJKVHQVLWLYLW\
80
Food Additives
2.16 Flavor Analysis by Fast - GC/MS (2) - GC/MS
QResults
Fast-GC/MS provided separation patterns similar to results from conventional methods, but analysis times were 1/7 of
conventional analysis times.
(×1,000,000)
7
10
9
TIC
13
1
5.0
3
4.0
3.0
6
12
2
2.0
5
11
15
4
1.0
14
16
8
19
18
17
7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 min
(×1,000,000)
10
9
TIC
7
13
3.0
1
2.5
3
2.0
6
12
1.5
2
1.0
5
11
15
14
4
0.5
16
19
17
18
8
2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 min
Fig. 2.16.1 Total Ion Current Chromatograms Obtained Using Conventional Method (upper) and Fast-GC/MS Method (lower)
ID Compound Name ID Compound Name ID Compound Name

1 alpha-Pinene 8 gamma-Terpinene 14 Bornyl acetate
2 Camphene 9 Linalool 15 Neryl acetate
3 beta-Pinene 10 Camphor 16 (Z)-beta-Farnesene
4 Myrcene 11 Isoborneol 17 Germacrene D
5 Cymene 12 alpha-Terpineol 18 beta-Bisabolene
6 Limonene 13 Linalyl acetate 19 Caryophyllene oxide
7 Eucalyptol
81
2.17 Analysis of Benzoyl Peroxide in Foods - LC

I n Japa n, t he food addit ive ben zoyl peroxide is Column : Shim-pack VP-ODS
permitted to be used as a processing agent only in PP/ðPP,'
flour, and its concentration is limited to 0.30 g/kg or Mobile Phase : :DWHU$FHWRQLWULOH YY
less in the “Specifications and Standards of Foods and Flowrate : 1.0 mL/min
Food Additives” published by the Japanese Ministry Column Temp. : 40 °C
of Health, Labour and Welfare. In the Food Safety Detection : UV235 nm
Standards Noti cation No. 0513003 (May 13, 2004), the
benzoyl peroxide analysis method was changed from gas
chromatography to HPLC. Here we introduce an example QAnalysis of Flour
of benzoyl peroxide analysis based on the method Fig. 2.17.2 shows the sample preparation procedure for
speci ed in the Food Safety Standards Noti cation No. analyzing benzoyl peroxide in our. Fig. 2.17.3 shows the
0513003. chromatograms obtained by analyzing a sample of our
produced in Japan (after preparation), and a sample spiked
with 1.0 mg/L benzoyl peroxide (equivalent to 5.0 mg/kg
in our).
Flour 10 g
5.0 g/L STD 10 ѥL
Add 50 mL Acetonitrile
O O
C O O C Agitation for 15 min by a stirring bar
Filtration (pore size 0.45 ѥm)
20 ѥL inj.
Fig. 2.17.1 Structure of Benzoyl Peroxide Fig. 2.17.2 Sample Preparation
mAU
1 Q Peak
1.Benzoyl Peroxide
5.0
4.0
3.0
2.0
1.0
0 5 10 15 20 25 30 35 40 45
min
Fig. 2.17.3 Chromatogram of Flour sample - Upper: Spiked with 1.0 mg/L Benzoyl Peroxide, Lower: Not spiked (20 μL inj. each)
82
3. Aromas and Odors
3.1 Analysis of Aroma Compounds in Peach Juice (1) - GC/MS
Q Explanation
The OPTIC-4 is a multimode injection system with a variety of adsorbent materials used in the MonoTrap, it
thermal desorption function that enables performing can efficiently concentrate many compounds, including
thermal desor ption by placing an adsorbent in the polar compounds, enabling ultra-high-sensitivity analysis.
injection port liner cup. In combination with the new This was used as a monolithic material sorptive extraction
MonoTrap adsorbent, the system enables analyzing aroma (MMSE) method for GC/MS pretreatment. In this case,
components with high sensitivity. the MonoTrap RGC18TD (for thermal desorption), a
The MonoTrap is a completely new trapping tool with a hybrid graphite carbon and octadecyl group type sorbent,
proprietary monolithic porous high-purity silica structure was used to concentrate aroma components in peach
that concentrates trace components using the adsorptive juice, which were analyzed using a high-polarity capillary
capacity provided by its large surface area. Due to a wide column.
Q Experimental
Collection
30 mL of commercial peach juice and MonoTrap RGC18
TD were placed in a 40 mL vial and stirred for one hour at
room temperature for sampling.
Analysis
0RQR7UDS5*&7' 6SHFLDOL]HG/LQHU
The MonoTrap material was removed from the vial, &DW1R &DW1R
its surface lightly rinsed with water, and placed in a 0DQXIDFWXUHGE\*/6FLHQFHV,QF 0DQXIDFWXUHGE\*/6FLHQFHV,QF
specialized liner, then placed in the OPTIC-4 inlet for

thermal desorption.
Instruments
Injection (TD) : 237,&$7$6*/,QWHUQDWLRQDO%9(LQGKRYHQWKH1HWKHUODQGV
GC-MS : *&06438OWUD6KLPDG]X
Column : InertCap Pure-WAX (0.25 mm × 30 m, df ƫP*/6FLHQFHV-DSDQ
[Injector] [MS]
Thermal Desorption Temp. : & &VHF Interface Temp. : 250 ºC
200 ºC Ion Source Temp. : 200 ºC
Carrier Gas : He Solvent Elution Time : 0.5 min
Column Flowrate : 1.0 mL/min Data Sampling Time : 1.0 - 60 min
Split Flowrate : 5:50 Measurement Mode : TIC
Cryofocus Temp. : Trap -160 ºC Mass Range : m/z 30-600
Introduction 250 ºC Detector Voltage : N9DEVROXWHYDOXH
[GC]
Column Temp. : &PLQ&PLQ&PLQ
83
3.1 Analysis of Aroma Compounds in Peach Juice (2) - GC/MS
( 10,000,000) ( 10,000,000)
TIC TIC
3.0
30 29
32
3.0
16
29
33
4
2.5
8
2.5
5
6
38
10
8
9
4
1
2
33
38
2.0 2.0
16
10
9
15
28
19
30
1.5 1.5
5
2
18
20
25
24
13
1
1.0 1.0
12
15
37
31
22
27
25
20 19
28
11
37
23 22
35
0.5 0.5
31
34
36
21
24
14
23
36
35
12
26
18
3
17
27
26
13
21
0.0 0.0
5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 5.0 10.0 15.020.0 25.0 30.0 35.0 40.0 45.0
Time (min) Time (min)
MMSE Method SBSE Method (conventional method)
Fig. 3.1.1 Comparison of Chromatograms Obtained Using the MMSE (with MonoTrap) and Conventional (SBSE) Methods
1. Ethyl acetate 11. 2-Isopropyl-4-methylthiazole 20. Benzyl acetate 29. delta-Undecalactone

2. Ethyl butanoate 12. Octyl acetate 21. cis-Geraniol 30. delta-Decalactone
3. Butyl acetate 13. Benzaldehyde 22. beta-Damascenone 31. Eudesm-7 (11) -en-4-ol
4. Isoamyl acetate 0HWKyl-4-propyl-1,3-oxathiane 0HWKyl-2- (1-methyl-1- 32. Ethyl caproate
5. D-Limonene p0HQWKDQRQH sulfanylethyl) cyclohexanone 33. delta-Undecalactone
6. Isobutyl isovalerate 16. beta-Linalool trans-Geraniol 34. n-Decanoic acid
7. 2-Hexenal 17. 0HWKylbutanoic acid 25. gamma-Butylbutyrolactone 35. delta-Hexylvalerolactone
8. Hexyl acetate 18. gamma-Caprolactone 26. beta-Ionone 36. gamma-Dodecalactone
9. 3-Hexenyl acetate 19. Terpineol 27. gamma-n-Amylbutyrolactone 37. delta-Dodecalactone
10. 2-Hexenyl acetate 28. Triacetin 38. Nootkatone
In Fig. 3.1.2, the peak area values obtained for each

component by the SBSE method are indicated as "1" to
show how MMSE sensitivity compares to SBSE. Because
the MonoTrap RGC18 TD material is a hybrid of graphite
carbon and octadecyl groups, it is more effective in
collecting polar compounds than the SBSE method, which
uses agitator paddles coated with PDMS. Therefore, the
MonoTrap material enabled highly sensitive analysis. It
SBSE Method
is especially useful for sulfur compounds and lactones, (red)
such as gamma-caprolactone (18) and 2-Isopropyl-4-

methylthiazole (11).
Lactones are the components in peaches, other fruits, dairy
products, etc., that give them a sweet smell. 2-Isopropyl-4-
methylthiazole is used as a fruit and vegetable avoring. Fig. 3.1.2 Sensitivity Comparison Between MMSE and Conventional
The monolith manufacturing technology with sol-gel method is new (SBSE) Methods
technology developed in Japan by Dr. Soga and Dr. Nakanishi at Kyoto
University.
H. Minakuchi, K. Nakanishi, N. Soga, N. Ishizuka, N.Tanaka, Anal.
Chem. 1996, 68, 3498-3501.
Nakanishi, K., Pore structure control of silica gels based on phase
separation. J. Porous Materials, 4 (1997) 67
QSummary
The MMSE method using the MonoTrap monolithic which had previously been an issue when analyzing aroma
adsorbent enabled the use of a simple procedure to highly components, was presumably due to the ef cient thermal
concentrate trace aroma components. The high sensitivity desorption provided by the OPTIC-4 multimode inlet.
is not only due to the high concentration levels, but also The combination of concentration by the MMSE method
due to the improved adsorption ef ciency of the graphite and the OPTIC-4 multimode inlet provides an excellent
carbon contained in the MonoTrap’s base silica structure alternative to SBSE and other typical concentrating
and to the use of a high-polarity deactivated column. In methods for detecting or screening low-concentration
addition, the improved sensitivity for polar compounds, compounds in gaseous form.
84
Aromas and Odors
3.2 Analysis of Aroma Compounds in Cheese (1) - GC/MS
Q Explanation
Volatile compounds, including aroma compounds, carbon (graphite carbon for Mono Trap TD) and the
in Parmesan and Blue cheese were analyzed using octadecyl functional group. GC-MS equipped with the
adsorption and thermal desorption GC/MS (TD-GC/ OPTIC-4 multi-purpose injector was used for thermal
MS). MonoTrap was used as adsorbant. It is a state-of- desorption. The OPTIC-4 allows direct introduction
the-art silica monolithic and hybrid adsorbent having a of desorbed gas into a capillary column without re-
large surface area and properties based on silica, activated adsorption.
Q Experimental
Sample Preparation
MonoTrap RGC18 TD is conditioned and packed into an
ampoule (Fig. 3.2.1) before shipment and therefore showed
an extremely low blank. It was used without conditioning.
Ten grams of each cheese sample were weighed and placed
into a vial (40 mL). MonoTrap was placed over the sample in Fig. 3.2.1 MonoTrap Packed into an Ampoule for TD (left)
MonoTrap RGC18 TD (right)
the vial using the MonoTrap holder. The vials were capped
and agitated for 3 hours at 600 °C.
TD-GC/MS Analysis
MonoTrap RGC18 TD was removed and placed into the
OPTIC-4 liner.
Instruments
Injection Unit : OPTIC-4
GC-MS : GCMS-QP2010 Ultra
Auto-Sampler : AOC-5000 Plus LINEX system
Column : InertCap Pure-WAX (60 m × 0.25 mm I.D.,
df ƫP*/6FLHQFHV,QF
Fig. 3.2.2 OPTIC-4 (left) and GCMS-QP2010 Ultra equipped
[OPTIC-4]
with OPTIC-4 and AOC-5000 Plus (right). Liner can
Desorb Temp. : 200 ºC be automatically exchanged using the AOC-5000 Plus.
Desorb Time : 5 min
Carrier Gas : He
Column Flow : 1.0 mL/min
Injection Mode : Splitless
Cryo Trapping : -150 ºC
Injection Temp. : 250 ºC
[MS]
Interface Temp. : 230 ºC
Ion Source Temp. : 200 ºC
Acquisition Mode : Scan
Mass Range : m/z 29-600
[GC]
Column Temp. : &PLQ&PLQ&
85
3.2 Analysis of Aroma Compounds in Cheese (2) - GC/MS
QResults and Discussion

Fig. 3.2.3 and Fig. 3.2.4 show total ion current chromatograms Sulfur compounds, such as dimethyl disulfide and dimethyl
of Parmesan and Blue cheese, respectively. The detected sulfone, were extracted and detected. It is known to be dif cult
compounds were identi ed using a mass spectral library search. to detect sulfur compounds using conventional TD system.

7,&

PLQ
Fig. 3.2.3 Total Ion Current Chromatogram of Parmesan Cheese
1. Methanethiol, 2. Ethyl acetate, 3. 2-Butanone, 4. 2-Methylbutanal, 5. 3-Methylbutanal, 6. 1-Propanol, 7. Toluene,

8. Dimethyl disulfide, 9. Hexanal, 10. 2-Pentenal, 11. 3-Penten-2-one, 12. 2-Heptanone, 13. D-Limonene, 14. Acetoin,
15. Acetol, 16. Dimethylpyrazine, 17. Dimethylpyrazine, 18. Dimethylpyrazine, 19. 2-Nonanone, 20. 2, 5-Dimethyl-3-ethylpyrazine,
21. Benzaldehyde, 22. Isobutyric acid, 23. 2-Undecanone, 24. Butanoic acid, 25. 2-Furanmethanol, 26. Acetamide,
27. 2-Tetradecanol, 28. 2-Tridecanone, 29. Hexanoic acid, 30. Dimethyl sulfone, 31. ѝ -Octalactone, 32. 2-Pentadecanone,
33. Octanoic acid, 34. ѝ -Decalactone, 35. Decanoic acid

7,&

PLQ
Fig. 3.2.4 Total Ion Current Chromatogram of Blue Cheese
1. Acetaldehyde, 2. Butanal, 3. Ethyl acetate, 4. Isovaleraldehyde, 5. 2-Pentanone, 6. Ethyl butyrate, 7. 2-Hexanone,

8. Isobutyl alcohol, 9. 2-Heptanone, 10. Methylhexanoate, 11. Ethylhexanoate, 12. 1-Pentanol, 13. 2-Heptanol, 14. 2-Nonanone,
15. Ethyloctanoate, 16. 2-Decanone, 17. 2-Nonanol, 18. Methyldecanoate, 19. 2-Undecanone, 20. Butanoic acid,
21. Ethyldecanoate, 22. 3-Methylbutanote, 23. ќ -Caprolactone, 24. 2-Undecanol, 25. 2-Tridecanone, 26. Hexanoic acid,
27. 2-Pentadecanone, 28. Octanoic acid, 29. Decanoic acid, 30. Dodecanoic acid
QSummary
Sulfur compounds in the cheese were easily and simply detected using MonoTrap and TD-GC/MS (OPTIC-4 and GC-MS).
86
Aromas and Odors
3.3 Higher Sensitivity Analysis of 2-Methoxy-3-Isobutylpyrazine

(MIBP) in Wine (1) - GC/MS/MS
Q Explanation
2-Methoxy-3-isobutylpyrazine (MIBP) is an aromatic concentration and selective separation and detection are
substance with the fragrance of bell peppers. It is found in essential to analysis.
sauvignon blanc (a type of grape used for white wine) and The trace amounts of MIBP in wine were selectively
cabernet sauvignon (a type of grape used for red wine), detected by utilizing the MonoTrap® silica monolithic
and gives the wines a favorable aroma. MIBP, which has a absorbent for collection and concentration, and a GC/
signi cant impact on the avor of wine, has an extremely MS/MS (GCMS-TQ8030) in positive chemical ionization
low threshold value in sensory tests, on the order of (PCI) mode. The MRM acquisition mode was used to
a few ng/L. Since wine contains many components, monitor speci c transitions for the compound of interest.
Q Experimental
A standard MIBP solution was added to commercially- and then the gaseous phase MIBP was collected using
available sauvignon blanc (produced in Chile in 2012) MonoTrap® RGPS TDNote (GL Sciences, P/N: 1050-
and cabernet sauvignon (produced in Chile in 2012) at 74202). After collection, the MonoTrap® RGPS TD was
different concentrations (0 ng/L, 1 ng/L, 5 ng/L, 10 ng/L, measured using the following analytical conditions.
and 20 ng/L). The samples were heated for 1 hour at 50 °C,
Autosampler : AOC-5000 Plus
OPTIC Liner Auto Exchange System : CDC + LINEX
Multipurpose Injection Port : OPTIC-4
GC-MS : GCMS-TQ8030
Column : InertCap 17MS (Length: 30 m, 0.25 mm I.D., df = 0.25 ƫP
*/6FLHQFHV31
[OPTIC-4] [GC]
Initial Temp. : 35 ºC Column Temp. : &PLQ-&PLQ-&PLQ
Ramp Rate : 10 ºC/sec [MS]
Hold Temp. : 250 ºC Ion Source Temp. : 200 ºC
Hold Time : 300 sec Interface Temp. : 250 ºC
Column Flow1 : 5 mL/min Ionization Method : 3RVLWLYHFKHPLFDOLRQL]DWLRQ3&,
Column Flow Time : 320 sec Reagent Gas : ,VREXWDQHN3D
Column Flow2 : 1.5 mL/min Acquisition Mode : MRM
Split Flow1 : 5 mL/min Event Time : 0.3 sec
Split Flow Time : 320 sec Monitor Ion and Collision : m/z!9
Split Flow2 : 50 mL/min Energy (CE) m/z!9
QResults
Fig. 3.3.1 shows the calibrations curves for sauvignon in the wines. Table 3.3.1 shows the results of quantitatively
blanc and cabernet sauvignon via the standard addition analyzing each wine 3 times via the standard
method. Favorable results were obtained for the addition method and the repeatability. The respective
correlation coef cient (R) between area and concentration concentrations of MIBP in the wines were 5.4 ng/L for the
for each MIBP-spiked sample, with 0.9999 for the sauvignon blanc and 12.1 ng/L for the cabernet sauvignon.
sauvignon blanc and 0.9998 for the cabernet sauvignon. In addition, favorable results of 3 % RSD were obtained
Fig. 3.3.2 shows the MRM chromatograms for the MIBP for the repeatability.
87
3.3 Higher Sensitivity Analysis of 2-Methoxy-3-Isobutylpyrazine
(MIBP) in Wine (2) - GC/MS/MS
Area Area
35000
R=0.9999 R=0.9998
10000 30000
25000
7500
20000
5000 15000
10000
2500
5000
0 0
0.0 2.5 5.0 7.5 ⃰ᗘ
Conc. 0.0 5.0 10.0 15.0 ⃰ᗘ
Conc.
Fig. 3.3.1 Calibration Curves for the Wines via the Standard Addition Method
Left: Sauvignon Blanc (Concentrations of 0 ng/L, 1 ng/L, 5 ng/L, and 10 ng/L)
Right: Cabernet Sauvignon (Concentrations of 0 ng/L, 5 ng/L, 10 ng/L, and 20 ng/L)
(×1,000) (×1,000)
167.10>124.10 167.10>124.10
6.0 167.10>135.10
2.5 167.10>135.10
5.0
2.0
4.0
1.5
3.0
1.0
2.0
0.5 1.0
14.00 14.25 14.00 14.25
Fig. 3.3.2 MRM Chromatograms for the MIBP in the Wines (Left: Sauvignon Blanc, Right: Cabernet Sauvignon)
Table 3.3.1 Quantitative Results for the MIBP in the Wines via the Standard Addition Method
(Concentration Units: ng/L), and the Repeatability (n=3)
Wine Type 1 2 3 Average Standard Deviation C.V. (%)

Sauvignon Blanc 5.5 5.3 5.3 5.4 0.1 2.47
Cabernet Sauvignon 11.8 12.3 12.1 12.1 0.2 1.91
QConclusion
The trace quantities of MIBP in the wines were collected and concentrated by the MonoTrap® RGPS TD, and then
selectively detected by utilizing the GC-MS/MS in PCI mode with MRM acquisition mode. It was thus possible to detect
MIBP at the ng/L level with high sensitivity.
88
4. Residual Pesticides
4.1 Analysis of Organophosphorus Pesticides in Baby
Foods (1) - GC/MS/MS
Q Introduction The selectivity of the MRM mode is illustrated in the
Contamination of food products with pesticides is a chromatograms of three OP pesticides shown in Fig. 4.1.2.
growing global concern, particularly for baby foods. GC/ In Fig. 4.1.2, the top chromatograms are the extracted ion
MS/MS operated in the Multiple Reaction Monitoring chromatograms (mass chromatograms) from the full-scan
(MRM) mode is a technique of choice for analysis of (Q3 scan) data, and the bottom chromatograms are the
trace contaminants in complex matrices because of its corresponding chromatograms from the MRM mode. The
improved sensitivity and selectivity compared to single MRM operation mode allows detection and quantitation
quadrupole GCMS. of trace concentrations of analytes in complex matrices
such as food extracts, virtually eliminating background
interference.
Q Experimental
Samples were prepared using the QuEChERS (Quick Calibration Results and Assessment of Precision
Easy Cheap Ef fect ive Rugged a nd Safe) sa mple Seven calibration standards were prepared in the blended
preparation method1). Analyses were conducted using peas extract over the range of 0.5-200 ng/mL (ppb).
a Shimadzu GCMS-TQ8030 GC/MS/MS operated in Response factors were calculated and relative standard
the MRM mode using conditions detailed Shimadzu deviation (RSD) determined by the GCMSsolution
Application News GCMS-13042). A 7-point calibration software. The precision of the calibration was evaluated
curve was prepared for 24 OP-pesticides using the matrix- using the RSD of the response factors and the correlation
matched internal standard procedure, and a sample of coef cient (r) for each of the analytes. In general, the %
organic blended peas to minimize contribution of analytes RSD was 25 % and correlation coef cient values for the
from the sample matrix. multi-point calibration were > 0.999.
Example calibration curves are shown in Fig. 4.1.3;
chromatograms correspond to the 1 ppb standard.
QResults and Discussion
GCMSMS Operation in the MRM Mode
Operation of the GCMS-TQ8030 in the MRM mode
provides excellent sensitivity and selectivity for analysis
of trace level contaminants in complex matrices.
Relationship between the responses of the pesticides of
interest relative to the matrix background is shown in
Fig. 4.1.1.
Black trace: Blended peas extract Scan mode
Pink trace: Pesticide Standard (100 ppb) MRM mode
Fig. 4.1.1 Total Ion Chromatograms of Blended Peas Extract and Pesticides Standard (100 ppb)
89
4.1 Analysis of Organophosphorus Pesticides in Baby
Fig. 4.1.2 Extracted Ion and MRM Chromatograms for Selected Pesticides
Fig. 4.1.3A Ronnel Calibration Fig. 4.1.3B Stirophos Calibration
Eight replicate injections of the 1 ng/mL and 10 ng/mL QConclusion

standards were analyzed to assess the precision of the Detection of the organophosphorus pesticides was
analytical method and the accuracy of measurement near demonstrated at low ng/mL (ppb) levels in baby food
the low end of the calibration range. The mean measured extract; linear calibration was demonstrated from 0.5-
concentrations and % RSD for the 1 ng/mL replicate 200 ng/mL. Precision and accuracy were demonstrated
analyses were 0.75-1.25 ng/mL and < 20 %; mean by replicate analyses of matrix spiked aliquots at 1 and
concentrations and % RSD for the 10 ng/mL replicate 10 ng/mL. Calibration was conducted in the blended peas
analyses were 8.0-11.0 ng/mL and < 10 %, respectively. 2) QuEChERS extract, and provided accurate, repeatable
results for the sample matrix. A Shimadzu GCMS-
TQ8030 system operated in the MRM mode was shown to
be a rapid, sensitive, and selective technique for analysis
of organophosphorus pesticides in baby foods.
[References]
1) AOAC Of cial Method 2007.01, Pesticide Residues in Foods by Acetonitrile Extraction and Partitioning with Magnesium Sulfate (2007)
2) Shimadzu Application News GCMS-1304
90
Residual Pesticides
4.2 Analysis of Pesticides in Baby Foods (1) - GC/MS/MS
Q Introduction Calibration and Assessment of Precision

Contamination of food products with pesticides is a with Variable MS Resolution
growing global concern, particularly for baby foods. GC/ One of the purposes of this study was to evaluate
MS/MS operated in the Multiple Reaction Monitoring calibration linearity, analytical precision, and qualitative
(MRM) mode is a technique of choice for analysis of speci city in the MRM mode with variable resolution on
trace contaminants in complex matrices because of its Q1 and Q3. Variable resolution settings can be selected for
improved sensitivity and selectivity compared to single the MRM transitions for a given analyte. As resolution is
quadrupole GCMS. increased (FWHM is decreased), speci city is increased
and signal intensity is decreased. Resolution on Q1
Q Experimental and Q3 has minimal effect on electronic noise, but has
considerable effect on chromatographic interference,
Samples were prepared using the QuEChERS (Quick
which can be considered chemical noise. Depending on
Easy Cheap Ef fect ive Rugged a nd Safe) sa mple
the interference for a speci c analyte in a given sample
preparation method1). Analyses were conducted with
matrix, optimum sensitivity (signal/noise) relative to
a Shimadzu GCMS-TQ8030 GC/MS/MS operated in
resolution is empirical.
the MRM mode using conditions detailed Shimadzu
Calibration was conducted at three resolution settings
Application News GCMS-14022). MRM transitions and
on Q1 and Q3 using the mat rix-matched inter nal
optimized collision energies detailed in the Shimadzu
standard procedure. Five calibration standards were
GC/MS/MS Pesticide Database. 3) Individual 5-point
prepared in a blended pears extract over the range of
calibration curves were prepared for 36 pesticides from
1-200 ng/mL (ppb). Response factors were calculated
several chemical classes using the matrix-matched
and relative standard deviation (RSD) determined by the
internal standard procedure. A sample of organic blended
GCMSsolution software. The precision of the calibration
pears was used to minimize contribution of analytes from
was evaluated using the RSD of the response factors and
the sample matrix. Selectivity of the MRM technique was
the correlation coefficient (r) for each of the analytes.
evaluated as a function of mass spectral resolution on Q1
In general, the % RSD was < 25 % and correlation
and Q3 of the GCMS-TQ8030.
coefficient values for the multi-point calibration were
> 0.999 when using the resolution setting Q1 – Unit; Q3 –
QResults and Discussion Unit.
Chromatography
The chromatography of pesticides and the speci city of
the GCMS-TQ8030 operated in the MRM mode been
explained previously.4) A chromatogram showing the
separation of the 36 pesticides is shown in Fig. 4.2.1.
Fig. 4.2.1 Total Ion Chromatogram of Pesticide Standard – MRM Mode
91
4.2 Analysis of Pesticides in Baby Foods (2) - GC/MS/MS
Q1: Unit
Mycyclobutanil Cyprodanil
Q3: Unit
Q1: Unit
Q3: Low
Q1: Low
Q3: Low
Fig. 4.2.2 MRM Chromatograms for Trace Pesticides at Three Settings of MS Resolution
After acquisition of each of the multi-point calibrations, QConclusion

eight replicate injections each of the 1.0 and 5.0 ng/mL Detection of pesticides was demonstrated at low ng/mL
standards were analyzed to assess the precision and (ppb) levels in a QuEChERS extract of blended pears,
accuracy of measurement near the low end of the and linear, matrix-matched calibration was demonstrated
calibration range. The mean concentration and RSD from 1-200 ng/mL. Operation of the GCMS-TQ8030 in
for the replicate analyses are generally less than 20 % the MRM mode provided accurate, precise results for the
at 1 ppb and less than 10 % at 5 ppb.2) The precision of sample matrix. Precision and accuracy were demonstrated
the measurements was considered acceptable for this by replicate analyses of matrix spiked aliquots at 1 and
application. 5 ng/mL.
Chromatographic interferences show considerable T he r e s u lt s d e m o n s t r a t e t h a t ch r o m a t og r a p h ic
variability between compounds, but are minimized using interferences can be signi cantly reduced by increasing
Unit/Unit resolution setting (Q1 – unit; Q3 – unit). For the mass spectral resolution. Optimum sensitivity (signal/
some compounds, striking improvement in selectivity is noise) relative to resolution is empirical and variable with
obtained with increasing resolution; two examples are each analyte and matrix.
shown in Fig. 4.2.2.
[References]
1) AOAC Of cial Method 2007.01, Pesticide Residues in Foods by Acetonitrile Extraction and Partitioning with Magnesium Sulfate (2007)
2) Analysis of Pesticides in Baby Foods Using a GCMS-TQ8030 GC/MS/MS, PartⅡ Shimadzu Application News No. GCMS-1402
(December, 2013)
3) Shimadzu GCMSMS Pesticide Database (October, 2012)
4) Analysis of Organophosphorus Pesticides in Baby Foods Using a Triple-Quadrupole
GC/MS/MS System Shimadzu Application News No. GCMS-1304 (February, 2013)
92
Residual Pesticides
4.3 Analysis of Pesticides in Citrus Oil (1) - GC/MS/MS
Q Introduction Assessment of Optimum Mass Spectral Resolution

Contamination of consumer products with pesticides Variable resolution settings can be selected for the MRM
is a growing concern. Triple quadrupole GC/MS/MS transitions for a given analyte. As MS resolution is
operated in the Multiple Reaction Monitoring (MRM) increased (FWHM is decreased), speci city is increased
mode has emerged as a technique of choice for analysis and signal intensity is decreased. Resolution on Q1
of trace level contaminants in complex matrices such and Q3 has minimal effect on electronic noise, but has
as orange oil. A Shimadzu GCMS-TQ8030 was used considerable effect on chromatographic interference,
for trace analysis of 47 pesticides of various chemical which can be considered "chemical noise". Depending on
classes using the instrument con guration and operating the interference for a speci c analyte in a given sample
parameters described previously.1) Results were evaluated
matrix, optimum sensitivity (signal/noise) relative to
for calibration linearity, analytical precision, sensitivity,
and speci city. resolution is empirical. The chromatograms of all of
the analytes were examined visually to assess optimum
resolution. Based on visual comparison of the results, the
Q Experimental
resolution setting of Q1: unit; Q3: unit was chosen for the
Samples were diluted 10-fold in dichloromethane for
analyses.
analysis. The analyses were conducted using a Shimadzu
GCMS-TQ8030 triple quadrupole GC/MS/MS operated
in the multiple reaction monitoring (MRM) mode. 4XDOLWDWLYH6SHFL¿FLW\DQG&KURPDWRJUDSKLF
Calibration was conducted using the matrix-matched Interferences
internal standard procedure. Details of the analytical MRM chromatograms for the analytes were displayed
conditions are summarized in Shimadzu Application for calibration standards at the lowest calibration level.
News.2) MRM transitions details were taken from the For many of the analytes, minimal chromatographic
Shimadzu pesticide database.3) interference was observed at the lowest level, which was
A sample of organic orange oil was used as the sample de ned as the limit of quantitation (LOQ). For some other
matrix so it would be free from background pesticide
analytes, however, significant interference is observed
contamination.
even at higher concentrations, despite the specificity of
the MRM mode, owing to the complexity of the orange oil
QResults and Discussion matrix. Examples of interferences are presented Figures
Chromatography 4.3.2A and 4.3.2B.
The total ion chromatogram (TIC) acquired in the Q3
full-scan mode for the pesticide standard is shown in
Fig. 4.3.1A, and a chromatogram for organic orange oil is
shown in Fig. 4.3.1B, and shows a broad, intense signal
for limonene before 7.5 min. and intense chromatographic
peaks from 7.5 - 12 min.
Fig. 4.3.1A Total Ion Chromatogram of Pesticide Standard – Q3 Scan Mode
Limonene
Fig. 4.3.1B Total Ion Chromatogram of Organic Orange Oil – Q3 Scan Mode
93
4.3 Analysis of Pesticides in Citrus Oil (2) - GC/MS/MS
Fig. 4.3.2A MRM Chromatogram Carbofuran - 50 ppb in Orange Oil Fig. 4.3.2B MRM Chromatogram Metalaxyl - 50 ppb in Orange Oil
Calibration and Assessment of Precision QConclusion

Calibration was conducted using the matrix-matched Detection of pesticides was demonstrated at low ng/
internal standard procedure. Nine calibration standards mL (ppb) levels in orange oil using a Shimadzu GCMS-
were prepared in diluted organic orange oil over the TQ8030. Calibration was conducted using the matrix-
range of 0.5-500 pg for each analyte (corresponding to matched internal standard procedure. Nine calibration
5-5000 ppm in the orange oil samples. The precision standards were prepared and analyzed in diluted organic
of the calibration was evaluated using the RSD of the orange oil over the range of 0.5-500 pg. Precision and
response factors (< 20 %) and the correlation coef cient accuracy were demonstrated by replicate analyses of
(r) (> 0.999) for each of the analytes. After the multi-point matrix spiked aliquots at 5, 10 and 50 ng/mL. Results were
calibration, ten replicate injections of each of the lowest evaluated for calibration linearity, analytical precision,
level standards were analyzed to assess the precision of sensitivity, and speci city. A Shimadzu GCMS-TQ8030
measurement (< 10 %) and accuracy (80-120 % for most of system was shown to be a rapid, sensitive, and selective
the analytes) near the low end of the calibration range. In technique for analysis of various classes of pesticides in
a few cases, chromatographic interferences contributed to orange oil. Operation of the GC/MS/MS in the MRM
high-biased results (> 120 %). The precision and accuracy mode provided accurate, precise results for an extremely
for the multi-point calibration and the replicate analyses complex sample matrix.
were considered acceptable for this application.
[References]
1) Shimadzu Application News No. GCMS-1304 (February, 2013)
2) Shimadzu Application News No. GCMS-1404 (February, 2014)
3) Shimadzu GCMSMS Pesticide Database (October, 2012)
94
Residual Pesticides
4.4 Simultaneous Analysis of Residual Pesticides in Foods

via the QuEChERS Method (1) - GC/MS/MS
Q Explanation Q Experimental
Analytical standards (0.001 mg/L to 0.1 mg/L), as well as The European Union Reference Laboratory (EURL) has
samples (0.01 mg/L) created by pretreating paprika with reported their results on evaluating the validity of residual
the QuEChERS method and then adding pesticides to the pesticide analysis utilizing GC-MS/MS and LC-MS/MS1).
resulting solution, were measured using GC-MS/MS. In their report, the measurement of 66 pesiticides using
GC-MS/MS was recommended. Here we present selected
results of analysis of these pesticides using the triple
quadrupole GCMS-TQ8030.
Column : Rxi-5Sil MS (30 m length, 0.25 mm I.D., df = 0.25 μm)
GlassLiner : Sky Liner, Splitless Single Taper Gooseneck w/Wool (Restek Corporation, catalog # 567366)
[GC] [MS]
Injection Temp. : 250 °C
Interface Temp. : 250 °C
Column Temp. : 70 °C (2 min) - (25 °C/min) - 150 °C - (3 °C/min) - 200 °C -
Ion Source Temp. : 230 °C
(8 °C/min) - 280 °C/min (10 min)
Data Acquisition Mode : MRM (See the below.)
Injection Mode : Splitless
Flow Control Mode : Linear velocity (58.1 cm/sec.)
Injection Volume : 1 μL
MRM Monitoring m/z
Quantitative Transition Qualitative Transition Quantitative Transition Qualitative Transition
Com pound Name Precursor>Product CE (V) Precursor>Product CE (V) Compound Nam e Precursor>Product CE (V) Precursor>Product CE (V)
Diphenylam ine 169.10>77.00 26 169.10>115.10 30 Buprofezin 172.10>57.10 18 105.10>104.10 4
Ethoprophos 200.00>157.90 6 200.00>114.00 14 200.00>97.00 26 Bupirimate 273.10>193.20 8 273.10>108.00 18
Chlorpropham 213.10>171.10 6 213.10>127.10 18 beta-Endosulfan 240.90>205.90 14 238.90>203.90 14
Trifluralin 306.10>264.00 8 264.10>206.10 8 264.10>160.10 18 Oxadixyl 163.10>132.10 10 163.10>117.10 24
Dicloran 206.00>176.00 12 206.00>124.00 26 176.00>148.00 12 Ethion 231.00>174.90 14 231.00>128.90 26
Propyzam ide 172.90>144.90 16 172.90>109.00 26 T riazophos 161.10>134.10 8 161.10>106.10 14
Chlorothalonil 265.90>230.90 14 265.90>167.90 24 263.90>167.90 24 Endosulfan sulfate 386.90>252.90 10 386.90>216.90 26
Diazinon 304.10>179.10 12 179.20>137.20 18 Propiconazole-1 259.10>190.90 8 259.10>172.90 18 259.10>69.10 12
Pyrim ethanil 199.10>184.10 14 199.10>158.10 14 Propiconazole-2 259.10>190.90 8 259.10>172.90 18 259.10>69.10 12
T efluthrin 197.10>141.10 26 177.10>127.10 32 T ebuconazole 252.10>127.00 24 250.10>125.10 24
Pirim icarb 238.20>166.10 10 166.10>96.00 14 Iprodione 314.10>244.90 12 314.10>56.10 24
Chlorpyrifos-m ethyl 285.90>270.90 12 285.90>93.00 22 Brom opropylate 340.90>184.90 18 182.90>154.90 16
Vinclozolin 212.10>172.00 14 212.10>144.90 26 212.10>109.00 30 Bifenthrin 181.10>166.10 16 181.10>165.10 22 181.10>153.10 10
Parathion-m ethyl 263.10>109.00 18 263.10>81.00 26 Fenpropathrin 265.10>210.10 12 181.10>152.10 24 181.10>127.10 26
T olclofos-m ethyl 265.00>249.90 12 265.00>93.00 24 Fenazaquin 160.20>145.10 8 145.20>115.10 24 145.20>91.10 24
Metalaxyl 206.20>162.10 8 206.20>132.10 18 T ebufenpyrad 333.20>276.10 8 333.20>171.00 22
Fenitrothion 277.10>125.00 18 277.10>109.00 18 T etradifon 355.90>158.90 12 353.90>159.00 12 228.90>200.90 14
Pirim iphos-m ethyl 305.10>290.10 12 290.10>125.00 24 Phosalone 182.00>138.00 8 182.00>111.00 18 182.00>102.10 18
Dichlofluanid 332.00>167.10 6 224.00>123.00 12 Pyriproxyfen 136.10>96.00 12 136.10>78.00 24
Malathion 173.10>117.00 12 173.10>99.00 18 Cyhalothrin 181.10>152.10 24 163.10>127.00 14 163.10>91.00 22
Chlorpyrifos 196.90>168.90 14 196.90>107.00 26 Fenarim ol 251.00>139.00 18 139.10>111.00 16
Fenthion 278.10>125.00 22 278.10>109.00 18 Acrinathrin 289.10>93.10 12 181.10>152.10 24 208.10>181.10 8
Parathion 291.10>109.00 14 291.10>81.00 26 Perm ethrin-1 183.10>168.10 12 183.10>153.10 18 183.10>115.10 24
T etraconazole 336.10>218.00 18 336.10>204.00 26 Pyridaben 147.20>132.10 14 147.20>117.10 22
Pendim ethalin 252.20>162.10 12 252.20>161.10 12 Perm ethrin-2 183.10>168.10 12 183.10>153.10 18 183.10>115.10 24
Cyprodinil 225.20>224.10 6 224.20>208.10 18 Cyfluthrin-1 206.10>151.20 24 163.10>127.10 6 163.10>91.00 14
(E)-Chlorfenvinphos 323.10>266.90 14 267.00>159.00 18 Cyfluthrin-2 206.10>151.20 24 163.10>127.10 6 163.10>91.00 14
T olylfluanid 137.10>91.00 18 137.10>65.00 26 Cyfluthrin-3 206.10>151.20 24 163.10>127.10 6 163.10>91.00 14
Fipronil 367.00>227.90 26 367.00>212.90 26 Cyfluthrin-4 206.10>151.20 24 163.10>127.10 6 163.10>91.00 14
Captan 79.00>77.00 8 79.00>51.00 22 Cyperm ethrin-1 181.10>152.10 24 163.10>127.10 6 163.10>91.00 14
(Z)-Chlorfenvinphos 323.10>266.90 14 267.00>159.00 18 Cyperm ethrin-2 181.10>152.10 24 163.10>127.10 6 163.10>91.00 14
Phenthoate 274.10>125.00 18 274.10>121.10 12 Cyperm ethrin-3 181.10>152.10 24 163.10>127.10 6 163.10>91.00 14
Fol pet 147.10>103.10 10 147.10>76.00 26 Cyperm ethrin-4 181.10>152.10 24 163.10>127.10 6 163.10>91.00 14
Procymidone 283.10>96.10 12 283.10>67.10 24 Ethofenprox 163.20>135.00 10 163.20>107.10 18
Methidathion 145.10>85.00 8 145.10>58.00 18 Fenvalerate-1 125.10>99.00 22 125.10>89.00 22
alpha-Endosulfan 240.90>205.90 14 238.90>203.90 16 tau-Fluvarlinate-1 250.10>200.10 16 250.10>55.00 18
Mepanipyrim 222.20>220.10 8 222.20>193.10 26 Fenvalerate-2 125.10>99.00 22 125.10>89.00 22
Profenofos 337.10>266.80 16 207.90>63.00 26 tau-Fluvarlinate-2 250.10>200.10 16 250.10>55.00 18
Myclobutanil 179.10>152.00 8 179.10>125.00 16 Deltam ethrin-1 252.90>93.10 18 181.10>152.10 24
Flusilazole 233.10>165.10 18 233.10>152.10 18 Deltam ethrin-2 252.90>93.10 18 181.10>152.10 24
95
4.4 Simultaneous Analysis of Residual Pesticides in Foods
via the QuEChERS Method (2) - GC/MS/MS
QResults
Calibration curves for each pesticide obtained by analyzing six calibration standards (0.001 mg/L to 0.1 mg/L), the
mass chromatograms for the 0.01 mg/L samples, and the area repeatability (n=6) for each pesticide obtained from the
pesticide-spiked samples (0.01 mg/L) are shown below.
Chlorpyrifos Fenazaquin
Area (×1,000,000) (×10,000) Area (×1,000,000) (×100,000)
196.90>168.90 160.20>145.10
2.5 5.0 1.25
1.00 R = 0.9990831 196.90>107.00 R = 0.9990383 145.20>115.10
145.20>91.10
2.0 4.0 1.00
0.75
1.5 3.0 0.75
0.50
1.0 2.0 0.50
0.25 0.5 1.0 0.25
0.00 0.0
0.000 0.025 0.050 0.075 Conc. 16.75 17.00 17.25 17.50 0.000 0.025 0.050 0.075 Conc. 27.25 27.50 27.75 28.00
alpha-Endosulfan Fenvalerate-1
Area (×100,000) (×10,000) Area (×100,000) (×1,000)
240.90>205.90 2.5 125.10>89.00
R = 0.999708 1.00 238.90>203.90 R = 0.9995097 8.0 125.10>99.00
4.0
2.0
3.0 0.75 6.0
1.5
2.0 0.50 4.0
1.0
1.0 0.25 0.5 2.0
0.0 0.0
0.000 0.025 0.050 0.075 Conc. 20.25 20.50 20.75 21.00 0.000 0.025 0.050 0.075 Conc. 32.00 32.25 32.50 32.75
Fig. 4.4.1 Calibration Curves for Each Pesticide and the Mass Chromatograms for the 0.01 mg/L Samples
Table 4.4.1 Area Reproducibility for Each Pesticide (n=6)

Compound Name %RSD Compound Name %RSD Compound Name %RSD Compound Name %RSD
Diphenylamine 4.99 Chlorpyrifos 5.23 Buprofezin 4.92 Fenarimol 5.16
Ethoprophos 4.95 Fenthion 5.75 Bupirimate 5.47 Acrinathrin 2.03
Chlorpropham 6.26 Parathion 6.93 beta-Endosulfan 6.29 Permethrin-1 6.34
Trifluralin 5.33 Tetraconazole 6.96 Oxadixyl 5.74 Pyridaben 7.11
Dicloran 6.49 Pendimethalin 6.29 Ethion 6.18 Permethrin-2 6.24
Propyzamide 5.52 Cyprodinil 5.21 Triazophos 3.45 Cyfluthrin-1 4.44
Chlorothalonil 4.46 (E)-Chlorfenvinphos 5.35 Endosulfan sulfate 4.26 Cyfluthrin-2 3.77
Diazinon 5.45 Tolylfluanid 4.81 Propiconazole-1 6.02 Cyfluthrin-3 7.35
Pyrimethanil 3.18 Fipronil 6.76 Propiconazole-2 5.56 Cyfluthrin-4 8.19
Tefluthrin 5.13 Captan 5.74 Tebuconazole 7.59 Cypermethrin-1 8.58
Pirimicarb 5.00 (Z)-Chlorfenvinphos 5.52 Iprodione 1.72 Cypermethrin-2 3.71
Chlorpyrifos-methyl 5.27 Phenthoate 6.40 Bromopropylate 5.71 Cypermethrin-3 8.08
Vinclozolin 6.33 Folpet 6.56 Bifenthrin 5.29 Cypermethrin-4 2.48
Parathion-methyl 5.81 Procymidone 6.40 Fenpropathrin 4.00 Ethofenprox 5.03
Tolclofos-methyl 4.89 Methidathion 6.17 Fenazaquin 4.84 Fenvalerate-1 4.20
Metalaxyl 5.43 alpha-Endosulfan 6.27 Tebufenpyrad 5.62 tau-Fluvarlinate-1 2.16
Fenitrothion 5.10 Mepanipyrim 6.41 Tetradifon 6.09 Fenvalerate-2 5.65
Pirimiphos-methyl 5.35 Profenofos 5.92 Phosalone 5.90 tau-Fluvarlinate-2 2.14
Dichlofluanid 4.04 Myclobutanil 5.46 Pyriproxyfen 5.16 Deltamethrin-1 7.58
Malathion 6.31 Flusilazole 5.63 Cyhalothrin 5.38 Deltamethrin-2 7.32
[Reference]
1) EURL-FV Multiresidue Method using QuEChERS followed by GC-QqQ/MS/MS and LC-QqQ/MS/MS for Fruits and Vegetables
(European Reference Laboratory, 2010-M1)
96
Residual Pesticides
4.5 Scan/MRM Analysis of Residual Pesticides in Foods (1) - GC/MS/MS
The GCMS-TQ8030 is a triple quadrupole GC-MS/ For the evaluation, analytical standards (0.001 mg/L
MS system equipped with scan/MRM mode to allow to 0.1 mg/L) were used, as well as samples (0.01 mg/
simultaneous scan and MR M data measurements. L) created by pretreating paprika with the QuEChERS
Here we introduce the results of an investigation method, and then adding pesticides to the obtained
using the scan/MRM mode, where target pesticides solution. The pesticides specified as targets and their
were quantitatively determined using the MRM data, transitions were those recommended in the results of
and concentrations of the untargeted pesticides were the validity evaluations by the European Reference
estimated by applying the scan data to the Compound Laboratory1).
Composer Database Software Ver. 2.
Column : Rxi-5Sil MS (30 m length, 0.25 mm I.D., df = 0.25 μm)
Glass Liner : Sky Liner, Splitless Single Taper Gooseneck w/Wool (Restek Corporation, catalog # 567366)
[GC] [MS]
Column Temp. : 40 °C (2 min) - (8 °C/min) - 310 °C (5 min) Ion Source Temp. : 200 °C
Injection Mode : Splitless Data Acquisition Mode : Scan/MRM
Flow Control Mode : Linear velocity (40.0 cm/sec) Event Time : 0.1 sec (Scan), 0.3 sec (MRM)
Injection Volume : 1 μL Scan Mass Range : m/z 50 – 500
Scan Speed : 5,000 u/sec
MRM Monitoring m/z
Quantitative Transition Qualitative Transition Quantitative Transition Qualitative Transition
Com pound Name Precursor >Product CE (V) Precursor>Product CE (V) Compound Nam e Precursor>Product CE (V) Precursor>Product CE (V)
Diphenylam ine 169.10>77.00 26 169.10>115.10 30 Buprofezin 172.10>57.10 18 105.10>104.10 4
Ethoprophos 200.00>157.90 6 200.00>114.00 14 200.00>97.00 26 Bupirimate 273.10>193.20 8 273.10>108.00 18
Chlorpropham 213.10>171.10 6 213.10>127.10 18 beta-Endosulfan 240.90>205.90 14 238.90>203.90 14
Trifluralin 306.10>264.00 8 264.10>206.10 8 264.10>160.10 18 Oxadixyl 163.10>132.10 10 163.10>117.10 24
Dicloran 206.00>176.00 12 206.00>124.00 26 176.00>148.00 12 Ethion 231.00>174.90 14 231.00>128.90 26
Propyzam ide 172.90>144.90 16 172.90>109.00 26 T riazophos 161.10>134.10 8 161.10>106.10 14
Chlorothalonil 265.90>230.90 14 265.90>167.90 24 263.90>167.90 24 Endosulfan sulfate 386.90>252.90 10 386.90>216.90 26
Diazinon 304.10>179.10 12 179.20>137.20 18 Propiconazole-1 259.10>190.90 8 259.10>172.90 18 259.10>69.10 12
Pyrim ethanil 199.10>184.10 14 199.10>158.10 14 Propiconazole-2 259.10>190.90 8 259.10>172.90 18 259.10>69.10 12
T efluthrin 197.10>141.10 26 177.10>127.10 32 T ebuconazole 252.10>127.00 24 250.10>125.10 24
Pirim icarb 238.20>166.10 10 166.10>96.00 14 Iprodione 314.10>244.90 12 314.10>56.10 24
Chlorpyrifos-m ethyl 285.90>270.90 12 285.90>93.00 22 Brom opropylate 340.90>184.90 18 182.90>154.90 16
Vinclozolin 212.10>172.00 14 212.10>144.90 26 212.10>109.00 30 Bifenthrin 181.10>166.10 16 181.10>165.10 22 181.10>153.10 10
Parathion-m ethyl 263.10>109.00 18 263.10>81.00 26 Fenpropathrin 265.10>210.10 12 181.10>152.10 24 181.10>127.10 26
T olclofos-m ethyl 265.00>249.90 12 265.00>93.00 24 Fenazaquin 160.20>145.10 8 145.20>115.10 24 145.20>91.10 24
Metalaxyl 206.20>162.10 8 206.20>132.10 18 T ebufenpyrad 333.20>276.10 8 333.20>171.00 22
Fenitrothion 277.10>125.00 18 277.10>109.00 18 T etradifon 355.90>158.90 12 353.90>159.00 12 228.90>200.90 14
Pirim iphos-m ethyl 305.10>290.10 12 290.10>125.00 24 Phosalone 182.00>138.00 8 182.00>111.00 18 182.00>102.10 18
Dichlofluanid 332.00>167.10 6 224.00>123.00 12 Pyriproxyfen 136.10>96.00 12 136.10>78.00 24
Malathion 173.10>117.00 12 173.10>99.00 18 Cyhalothrin 181.10>152.10 24 163.10>127.00 14 163.10>91.00 22
Chlorpyrifos 196.90>168.90 14 196.90>107.00 26 Fenarim ol 251.00>139.00 18 139.10>111.00 16
Fenthion 278.10>125.00 22 278.10>109.00 18 Acrinathrin 289.10>93.10 12 181.10>152.10 24 208.10>181.10 8
Parathion 291.10>109.00 14 291.10>81.00 26 Perm ethrin-1 183.10>168.10 12 183.10>153.10 18 183.10>115.10 24
T etraconazole 336.10>218.00 18 336.10>204.00 26 Pyridaben 147.20>132.10 14 147.20>117.10 22
Pendim ethalin 252.20>162.10 12 252.20>161.10 12 Perm ethrin-2 183.10>168.10 12 183.10>153.10 18 183.10>115.10 24
Cyprodinil 225.20>224.10 6 224.20>208.10 18 Cyfluthrin-1 206.10>151.20 24 163.10>127.10 6 163.10>91.00 14
(E)-Chlorfenvinphos 323.10>266.90 14 267.00>159.00 18 Cyfluthrin-2 206.10>151.20 24 163.10>127.10 6 163.10>91.00 14
T olylfluanid 137.10>91.00 18 137.10>65.00 26 Cyfluthrin-3 206.10>151.20 24 163.10>127.10 6 163.10>91.00 14
Fipronil 367.00>227.90 26 367.00>212.90 26 Cyfluthrin-4 206.10>151.20 24 163.10>127.10 6 163.10>91.00 14
Captan 79.00>77.00 8 79.00>51.00 22 Cyperm ethrin-1 181.10>152.10 24 163.10>127.10 6 163.10>91.00 14
(Z)-Chlorfenvinphos 323.10>266.90 14 267.00>159.00 18 Cyperm ethrin-2 181.10>152.10 24 163.10>127.10 6 163.10>91.00 14
Phenthoate 274.10>125.00 18 274.10>121.10 12 Cyperm ethrin-3 181.10>152.10 24 163.10>127.10 6 163.10>91.00 14
Fol pet 147.10>103.10 10 147.10>76.00 26 Cyperm ethrin-4 181.10>152.10 24 163.10>127.10 6 163.10>91.00 14
Procymidone 283.10>96.10 12 283.10>67.10 24 Ethofenprox 163.20>135.00 10 163.20>107.10 18
Methidathion 145.10>85.00 8 145.10>58.00 18 Fenvalerate-1 125.10>99.00 22 125.10>89.00 22
alpha-Endosulfan 240.90>205.90 14 238.90>203.90 16 tau-Fluvarlinate-1 250.10>200.10 16 250.10>55.00 18
Mepanipyrim 222.20>220.10 8 222.20>193.10 26 Fenvalerate-2 125.10>99.00 22 125.10>89.00 22
Profenofos 337.10>266.80 16 207.90>63.00 26 tau-Fluvarlinate-2 250.10>200.10 16 250.10>55.00 18
Myclobutanil 179.10>152.00 8 179.10>125.00 16 Deltam ethrin-1 252.90>93.10 18 181.10>152.10 24
Flusilazole 233.10>165.10 18 233.10>152.10 18 Deltam ethrin-2 252.90>93.10 18 181.10>152.10 24
97
4.5 Scan/MRM Analysis of Residual Pesticides in Foods (2) - GC/MS/MS
QResults
An example of the results of the analysis of the analytical utilizing the Compound Composer Database Software (P/N: 225-
standards (0.001 mg/L to 0.1 mg/L) and the pesticide-spiked 13106-92). The simultaneous analysis database software contains
samples (0.01 mg/L) in scan/MRM mode are shown in Fig. 4.5.1. information (mass spectra, retention times, and calibration
As with Procymidone, shown in Fig. 4.5.1, strict quanti cation of curves) on more than 450 pesticides. As a result, it is possible to
the targeted pesticides could be performed by creating calibration identify pesticides from their estimated retention times and mass
curves from the MRM data. Furthermore, since the scan data was spectra without using analytical standards, and then calculate
sampled simultaneously, the pesticides could be con rmed from semi-quantitative values from the calibration curves. Also in this
the mass spectrum. investigation, it was possible to con rm the detection and semi-
For the untargeted pesticides, data analysis was performed quanti cation of untargeted pesticides such as Quinoxyfen.
Analysis Results for Target Compounds (×10,000)

1.0 96
Procymidone: Quantitative value 0.0098 mg/L 67
(×10,000) Area (×100,000) 0.5 283
83 255 285
3.5 50 109123137 162
283.10>96.10 5.0 177193 220 244 262 297
283.10>67.10 0.0
3.0 50 100 150 200 250 300
4.0
2.5
(×10,000)
2.0 3.0 1.0 96
1.5 2.0
67
1.0 0.5 283
1.0 53
285
0.5
77 110 124 145 186 212 240255
0.0 0.0
0.000 0.025 0.050 0.075 Conc. 50 100 150 200 250 300
25.25 25.50 25.75 26.00
Mass spectral confirmation is possible with the scan data.
Quantitative determination by creating calibration (Upper: Sampled mass spectrum; Lower: Mass spectrum in the library)
curves from the MRM data
(×1,000,000) Measurement Results for Pesticide-Spiked Samples
3.5
3.0
Scan chromatogram
2.5
2.0
1.5
1.0
0.5
MRM chromatogram
24.0 24.5 25.0 25.5 26.0 26.5 27.0 27.5 28.0 28.5
Screening Results for Other Pesticides Area ratio
Quinoxyfen: Quantitative value 0.0134 mg/L 3.0
(×10,000)
237.00 2.0
2.0 %
100 237
1.0
1.5
50
1.0 181 272 0.0
198 307
617588103 243 286 328 0.0 2.5 5.0 7.5 Conc. ratio
0 %
0.5 50 100 150 200 250 300 350 237
100
28.00 28.25 28.50 28.75 50 272

307
112133 161 274
57 75 208 244
Compound identification and semi-quantitative calculations 0
50 100 150 200 250 300 350
can be performed utilizing the information contained in the
simultaneous analysis database software. Information Registered in the Compound
Composer Database Software
Fig. 4.5.1 Scan/MRM Analysis Results

[Reference]
1) EURL-FV Multiresidue Method using QuEChERS followed by GC-QqQ/MS/MS and LC-QqQ/MS/MS for Fruits and Vegetables
(European Reference Laboratory, 2010-M1)
98
Residual Pesticides
4.6 Screening of Residual Pesticides in Food with Two

Different Columns (1) - GC/MS
In recent years, with increases in the number of Commercially-available oranges and soya beans were
pesticides and the diversification of substances under processed with the QuEChERS method using Restek
investigation, there have been calls for quick and high- Q-sep TM . A mixture of 138 pesticides was added to
accuracy screening for residual pesticides in foods the sample solutions, with the concentrations adjusted
using GC/MS. The Quick-DB database includes mass to 10 ng/m L. The pesticide-spiked samples were
spectra, retention times, and calibration curves for 478 subjected to Scan/SIM analysis using the analysis
pesticide components. It can be used to quickly calculate conditions stored in Quick-DB. Frequently detected
semi-quantitative values without requiring analytical components were analyzed with high sensitivity in SIM
standards. If a pesticide peak is detected, it is essential mode. For components with low detection frequency, a
to check for potential interference from co-eluting comprehensive analysis was performed in Scan mode.
contaminants to insure highly accurate screening. One of
the ways for checking is to analyze the samples with two
columns with different types of stationary phase. For this
purpose, the Twin Line MS system is very useful because
it enables the installation of two types of columns to one
MS. The Quick-DB contains information compatible with
two different columns, so that it can also be applied to the
Twin Line MS system. Here, residual pesticides in foods
were analyzed by applying Quick-DB and the Twin Line
MS system.
Instruments : GCMS-QP2010 Ultra with Twin Line MS System
Column 1 : Rxi-5Sil MS (30 mL., 0.25 mm I.D., df = 0.25 μm) (Restek Corporation, P/N: 13623)
Column 2 : Rtx-200MS (30 m L., 0.25 mm I.D., df = 0.25 μm) (Restek Corporation, P/N: 15623)
Glass Insert : Sky Liner, Splitless Single Taper Gooseneck w/Wool (Restek Corporation, P/N: 567366)
[GC] [MS]
Column Temp. : 60 °C (1 min) - (25 °C/min) - 160 °C - (4 °C/min) Ion Source Temp. : 200 °C
- 240 °C - (10 °C/min) - 290 °C (11 min) Solvent Elution Time : 1.5 min
Injection Mode : Splitless Measurement Mode : FAAST (Scan/SIM simultaneous
High-Pressure Injection : 250 kPa (1.5 min) measurement)
Carrier Gas Control : Linear velocity (40.0 cm/sec) Scan Mass Range : m/z 50 to 600
Injection Volume : 2 μL Scan Event Time : 0.15 sec
SIM Event Time : 0.3 sec
<Twin Line MS System>

The inlets of the two different columns are connect to two
different injection ports and the outlets are introduced
directly to the mass spectrometer interface at the same
time. One column is chosen for analysis while the other,
non-used column, has a reduced carrier gas owrate. This
enables application data from the different columns to
be acquired without venting the MS vacuum to change
columns. Moreover the retention times and retention
indices are the same as a single column system. A high-
capacity differential vacuum system provides the same
sensitivity as that obtained by a single column system.
Fig. 4.6.1 Twin Line MS System
99
4.6 Screening of Residual Pesticides in Food with Two
Different Columns (2) - GC/MS
QAnalysis Results
The pesticide-spiked samples (10 ng/mL) were analyzed the co-eluting contaminants were chromatographically
using the Twin Line MS and the data were processed separated from Carbaryl and Aldrin peaks (Fig. 4.6.2
utilizing the Quick-DB. Figs. 4.6.2, Figs. 4.6.3, and and Fig. 4.6.3 (right side)), and the semi-quantitative
Figs. 4.6.4 show the obtained mass chromatograms for 3 values were much more closer to 10 ng/ mL spike
selected compounds. The left shows those run on the Rxi- levels. Pyrimethanil were not impacted by co-eluting
5Sil MS and the right shows those on the Rtx-200 MS. contaminants with either of the columns. The results
Carbaryl and Aldrin peaks were impacted by close- or obtained from two different columns enhances the
co-eluting contaminants on the Rxi-5Sil MS (Fig. 4.6.2 reliability of screening results. These results demonstrated
and Fig. 4.6.3 (left side)). For these two compounds, the that the Twin Line MS allows the reliable determination of
calculated semi-quantitative values obtained from the pesticides in complex samples with the potential for matrix
calibration curves stored in the Quick-DB were higher interference, such as processed foods. This works for a
than the spike amount (10.0 ng/ mL) because of the semi-quantitative values using the Quick-DB.
interference. However, on the Rtx-200MS (on the right),
Carbaryl
(×10,000) (×10,000)
144.10 144.10
Quantitative value: Quantitative value:
2.5 115.10 13.8 ng/mL 2.5 115.10 10.6 ng/mL
116.10 116.10
2.0 2.0
1.5 1.5
1.0 1.0
0.5 0.5
13.75 14.00 14.25 14.50 14.00 14.25 14.50 14.75
Fig. 4.6.2 Mass Chromatogram of Carbaryl (10 ng/mL) Added to Liquid Soya Beans Extract (Left: Rxi-5Sil MS; Right: Rtx-200 MS)
Aldrin
(×1,000) (×1,000)
262.90 262.90
Quantitative value: Quantitative value:
264.90 264.90
17.3 ng/mL 5.0 9.3 ng/mL
7.5 293.00 293.00
4.0
5.0
3.0
2.5 2.0
1.0
15.3 15.4 15.5 15.6 15.7 15.8 15.9 16.0 16.1 10.75 11.00 11.25 11.50
Fig. 4.6.3 Mass Chromatogram of Aldrin (10 ng/mL) Added to Liquid Orange Extract (Left: Rxi-5Sil MS; Right: Rtx-200 MS)
Pyrimethanil
(×10,000) (×10,000)
6.0 198.10 198.10
199.10 Quantitative value: Quantitative value:
5.0 200.10 9.5 ng/mL 6.0 199.10 10.2 ng/mL
200.10
5.0
4.0
4.0
3.0
3.0
2.0
2.0
1.0 1.0
11.75 12.00 12.25 12.50 9.25 9.50 9.75 10.00
Fig. 4.6.4 Mass Chromatogram of Pyrimethanil (10 ng/mL) Added to Liquid Soya Beans Extract (Left: Rxi-5Sil MS; Right: Rtx-200MS)
100
Residual Pesticides
4.7 Easy Screening for Residual Pesticides in Processed

Q Explanation
The analysis of residual pesticides in processed foods surrogates suited to each pesticide. In analyzing residual
using GC-MS/MS, which provides excellent selectivity pesticides in processed foods, which contain a number
and sensitivity, has become a focus of attention. Before of contaminants, separating the pesticides from the
starting GC-MS/MS measurements, it is necessary contaminants can be impossible, even with GC-MS/
to optimize MRM transitions (precursor ions and MS. In this case, an effective approach to separating and
product ions) and collision energies (CE) for each detecting the pesticides is to perform the analysis with
pesticide measured, which is extremely labor intensive. two columns respectively, which differ in their separation
Furthermore, in order to calculate quantitative values, patterns. The information registered in Quick-DB is also
it is necessary to prepare standard samples and create compatible with analysis using two different columns for
calibration curves. The Quick-DB database contains residual pesticides in processed foods. In addition, if the
the opt i mal M R M condit ions ( M R M t ransit ions Twin Line MS system is used, the two columns can be
and CE), mass spectra, retention indices, calibration attached to the MS unit simultaneously, so data can be
curves and other information. This enables the semi- sampled from the different columns smoothly, without
quantitative analysis of pesticides without using standard compromising the MS vacuum. Here we report on the
samples. Pesticide surrogates are used as the internal results of applying Quick-DB and the Twin Line MS
standard substances for calibration curves. Favorable system to the analysis of residual pesticides in curry.
quantitative accuracy is achieved by selecting the
Q Experimental
Using the Restek Q-sep TM , commercially-available retort- analysis under the analysis conditions registered in Quick-
pouch curry was pretreated via the QuEChERS method. DB. The two columns indicated in Analytical Conditions were
The sample solution obtained was spiked with 230 standard installed to a single GC-MS with the Twin Line MS system. The
pesticide samples at a concentration of 10 ng/mL. The retention times for the pesticide components were estimated
pesticide-spiked samples were then subjected to Scan/MRM based on the analysis results for the n-alkane standard sample.
Instrument : GCMS-TQ8030 (Twin Line MS System)
Column 1 : Rxi-5Sil MS (30 m L., 0.25 mm I.D., df = 0.25 μm) (Restek Corporation, P/N: 13623)
Column 2 : Rtx-200MS (30 m L., 0.25 mm I.D., df = 0.25 μm) (Restek Corporation, P/N: 15623)
Glass Insert : Sky Liner, Splitless Single Taper Gooseneck w/Wool (Restek Corporation, P/N: 567366)
[GC] [MS]
Column Temp. : 60 °C (1 min) - (25 °C/min) - 160 °C - (4 °C/min) Ion Source Temp. : 200 °C
- 240 °C - (10 °C/min) - 290 °C (11 min) Solvent Elution Time : 1.5 min
Injection Mode : Splitless Measurement Mode : FAAST
High Pressure Injection : 250 kPa (1.5 min) (Scan/MRM simultaneous
Carrier Gas Control : Linear velocity (40.0 cm/sec) measurement)
Injection Volume : 2 μL Scan Event Time : 0.15 sec
Scan Mass Range : m/z 50 to 330
QAnalysis Results
The liquid food extract spiked with pesticides was semi-quantitative values with respect to the additive
analyzed, and data processing was performed with concentration. Then the pesticides were classified into
Quick-DB. The analysis results are shown in Fig. 4.7.1. those with a ratio under 50 %, 50 % to 200 %, and over
When semi-quantitative analysis was performed using 200 %, to nd the distribution. The results are shown in
the calibration curves registered in Quick-DB, favorable Fig. 4.7.2. A signi cant 83 % of components had a semi-
semi-quantitative values were obtained, close to the quantitative value 50 % to 200 % that of the concentration
additive concentration of 10 ng/mL for many of the of the standard pesticide samples added. From this, it is
components. To evaluate the quantitative accuracy for evident that semi-quantitative analysis can be performed
this analysis method, ratios were calculated for the with high accuracy.
101
4.7 Easy Screening for Residual Pesticides in Processed
6% 11 %
(14 compounds) (24 compounds)
83 %
(192 compounds)
As a result of calculation of the semi-quantitative values using the calibration

curves preregistered in Quick-DB, favorable quantitative values were obtained.
(*The concentration of the internal standard is indicated as 1 ng/mL.)
< 50 % 50 -200 % 200 % <
Fig. 4.7.1 Analysis Results for the Pesticide-Spiked Samples Fig. 4.7.2 Percentage Distribution of Semi-Quantitative Values
(10 ng/mL concentration) with Respect to the Additive Concentration
In the analysis of residual pesticides in foods, when dimethoate. With the Rxi-5Sil MS, there was an impact
pesticide peaks are detected, it is necessary to check from contaminants, but with the Rtx-200MS, there was
whether contaminants have been misidentif ied as not. Semi-quantitative value obtained from the calibration
pesticides, and whether contaminant overlap has in ated curves registered in Quick-DB was favorable, 9.6 ng/mL,
the size of the quantitative values. One confirmation for the use of the Rtx-200MS column. In this way, even
method is to analyze the samples with columns with for pesticides of which separation from contaminants
different separation patter ns, and then check that is dif cult, separation is possible if using columns with
essentially the same quantitative values are obtained different separation patterns, enabling highly reliable
for the pesticides detected in the respective columns. semi-quantitative analysis.
As an example, Fig. 4.7.3 shows the analysis results for
(×1,000) (×1,000)
143.00>111.00 1.50 143.00>111.00
1.25 125.00>79.00 125.00>79.00
1.25
1.00
1.00
0.75 0.75 Quantitative Value
9.6 ng/mL
0.50 0.50
0.25 0.25
11.00 11.25 11.50 11.75 12.00 12.25 12.50 12.75
Fig. 4.7.3 Chromatograms for Liquid Curry Extract, Spiked with Dimethoate (10 ng/mL Concentration)
(Left: Rxi-5Sil MS; Right: Rtx-200MS)
High-accuracy semi-quantitative analysis was achieved quickly and easily, by attaching two columns to the GCMS-
TQ8030 utilizing the Twin Line MS system, and then screening for residual pesticides in processed foods using Quick-DB.
102
Residual Pesticides
4.8 High Throughput LC-MS/MS Analysis of Carbendazim in

Orange Juice (1) - LC/MS/MS
A new high throughput LC-MSMS method was developed Orange juice samples were diluted 40 to 50x with water
to facilitate increased testing for Carbendazim at low ppb and centrifuged at 3,000 rpm for one minute before
levels. Carbendazim is a broad spectrum fungicide used analysis. Isocratic and gradient reversed phase high speed
in cereal and fruit crops. Although its use is banned in the methods were developed for the analysis of Carbendazim
US, there are a number of countries that still legally use on a 2.0 × 50 mm 1.6 micron column coupled to a tandem
Carbendazim to help control molds such as black spot. quadrupole mass spectrometer. A high throughput
Recently, orange juice imported from Brazil has tested injector that features a seven second injection time was
positive for Carbendazim. Since a tolerance level has not used to help speed the sample throughput.
been established by the FDA, carbendazim is considered Three commercial brands of orange juice tested positive
an unlawful pesticide residue. A number of commercial for Carbendazim at low ppb levels. An organic brand
orange juices were tested from the local area and found of orange juice did not contain any detectable levels of
to contain Carbendazim in low ppb levels. A novel high Carbendazim and was used to prepare the matrix matched
throughput method LC-MSMS method was developed calibration curves. Good linearity was obtained from low
that allows the analysis of Carbendazim to be completed ppb levels up to a 1 ppm concentration. A blank injected
in less than one minute to facilitate the testing of multiple after the 1 ppm standard showed no carryover. The use of
samples. UHPLC with a high speed injector allowed an analysis to
be completed within a one minute timeframe.
cps
×10000 area
×10000 area
retention time Spiked amount(ppb)
Fig. 4.8.1 Chromatogram of Orange Juice Spiked with 1 ppb Carbendazim and Calibration Curve
103
4.8 High Throughput LC-MS/MS Analysis of Carbendazim in
Orange Juice (2) - LC/MS/MS
Table 4.8.1 shows the levels of carbendazim measured in different orange juice brands. Fig. 4.8.2 is the chromatogram of
an orange juice matrix blank injected after 1 ppm standard. Fig. 4.8.3 and Fig. 4.8.4 are representative chromatograms
of two brands of orange juice. The MM brand contained 8.1 ppb carbendazim, while none was detected in the certi ed
organic orange juice.
Table 4.8.1 Levels of Carbendazim cps
retention time
Fig. 4.8.2 Chromatogram of Orange Juice Matrix Blank
cps cps
retention time retentiom time
Fig. 4.8.3 Chromatogram of Brand MM Orange Juice Fig. 4.8.4 Chromatogram of Certi ed Organic Orange Juice
QConclusion
Carbendazim was detected in four commercial brands of orange juice at low ppb levels. Levels found were below
FDA limits for a positive test. The calibration curve was linear up to 500 ppb, and the method was sensitive enough to
detect carbendazim below the most stringent regulatory threshold. No carryover was detected and the method run time
was under one minute, thanks to the extremely high speed autosampler which completed each injection cycle in just 7
seconds. Sample preparation only required dilution and ltration for rapid, ef cient analysis.
104
Cd
Ca
Pb
5. Inorganic Metals
5.1 Measurement of Lead in Sugar (1) - AA
Q Explanation QAnalytical Method and Conditions

The Japanese pharmacopeia specifies that analysis of Quantitation was conducted by the standard addition
lead content in re ned white sugar be conducted by the met hod. T he i nje ct ion volu mes of t he pre pa red
electrothermal atomization (furnace) method. Since high measurement solution, dilution-blank solution and Pb
sensitivity sample analysis is possible even at micro standard solution (20 ppb) were adjusted respectively
levels, this method is effective for trace level analysis even using the autosampler. Tables 5.1.1 – 3 indicate the
for toxic elements other than lead. instrument used for the analyses and the main analytical
Here we introduce an example of such an analysis based conditions.
on the method speci ed in the Japanese pharmacopeia.
Table 5.1.1 Instrument and Optics Parameters
Q Pretreatment Atomic absorption spectrophotometer unit: AA-7000
T he s a m ple u s e d fo r t he a n a ly si s c o n si s t e d of Instrument
Atomizer unit: GFA-7000
commercially available granulated sugar. Since atomic
Analysis wavelength 283.3 nm
absorption spectrometry requires that the sample be in
Slit width 0.7 nm
solution in order to conduct measurement, pretreatment
Current 10 mA
of the ref ined sugar is necessary. Here we used a
high pressure acid digestion vessel as indicated in the Lamp mode BGC-D2
pretreatment procedure prescribed in the pharmacopeia.

The high pressure acid digestion vessel consists of an
internal PTFE vessel and an external metal or ceramic Table 5.1.2 Furnace Program
high pressure vessel. In the actual preparation, 0.050 g Temperature Time Gas Flowrate
of sample was rst accurately weighed into the internal Ý& V
Mode Sensitivity
/PLQ
PTFE vessel. To this, 0.5 mL of nitric acid was added for 1 60 3 RAMP REGULAR 0.10
measurement of the toxic metals. The vessel was then 2 120 20 RAMP REGULAR 0.10
placed in the external high pressure vessel, and this was 3 250 10 RAMP REGULAR 0.10
heated for 5 hours in a 150 °C thermostatic chamber. After
4 600 10 RAMP REGULAR 1.00
cooling, puri ed water was accurately added to bring the
5 600 10 STEP REGULAR 1.00
volume to 5 mL, and this was used as the measurement
6 600 3 STEP HIGH 0.00
solution. In addition, a blank solution was prepared by
adding puri ed water to 0.5 mL of the above-mentioned 7* 2200 3 STEP HIGH 0.00
nitric acid to bring the volume to 5 mL. 8 2500 2 STEP REGULAR 1.00
7* Atomization stage
Graphite tube: Pyrolysis tube
Table 5.1.3 Autosampler Standard Addition Parameter Settings

Addition Sample Diluent Pb: 10 ppb Total
Concentration
0 ppb 14 ѥL 6 ѥL 0 ѥL 20 ѥL
105
5.1 Measurement of Lead in Sugar (2) - AA
QResults
The measurement results were under the quantitation
limit. Fig. 5.1.1 shows an overlay of some typical peak
profiles, and Fig. 5.1.2 shows the calibration curve
using the blank solution. The quantitation limit in this
measurement is 0.2 ppb for the concentration in aqueous
solution, which converts to 0.03 ppm in the solid sample.
This easily satis es the criterion value of 0.5 ppm.
Addition
concentration
Fig. 5.1.2 Calibration Curve of Blank Solution
Blank
Time (sec)
Fig. 5.1.1 Peak Pro les (partial)
In addition, Pb was added to the sample solution to bring

the Pb concentration of the solution to 1.3 ppb (0.13 ppm
in solid), and this was also measured as a sample. The
results are shown in Fig. 5.1.3 and Table 5.1.4. The value
was 0.13 ppm in the solid, indicating good correlation.
The 1.43 dilution factor in Table 5.1.4 was calculated from Fig. 5.1.3 Calibration Curve Using Sample Addition
the total injection volume of 20 μL with respect to the
sample injection volume of 14 μL (20/14).
Table 5.1.4 Sample Addition Results and Concentration Computation
Sample Conc. Setting Conc. Sample Total Dilution Actual Actual
Abs. Coefficient Conc.
ID (ppb) (ppb) Amt. Volume Factor Conc. Unit
Blank
Added Conc: 0 ppb
Added Conc: 1 ppb
Added Conc: 2 ppb
Added Conc: 3 ppb
Added Sample
106
Cd
Pb
Inorganic Metals
Ca
5.2 Measurement of Minerals in Dietary Supplements (1) - AA
Q Explanation Q Measurement Conditions

Recently, the development and sales of a variety of dietary The measurement wavelength, type of ame, and matrix
supplements have increased dramatically against the modifier used are shown in Table 5.2.1. The N2O-C2H 2
background of rising public interest regarding health. ame was used for measurement of Ca and Mo, and the
Here we introduce the method of analysis of minerals in air-C2H2 ame was used for all the other elements. La was
dietary supplements as specified in the Pharmacopoeia added as a matrix modifier for measurement of Ca and
of the United States (USP 32), where the supplement Mg, and ammonium chloride was added for measurement
market now stands at about three trillion yen (33 billion of Mo and Se.
dollars). As one example, in the case of tablets of oil -
and water- soluble vitamins with minerals, the sample
preparation and measurement methods are speci ed for
the quantitation of the minerals Ca, Cr, Cu, Fe, K, Mg,
Mn, Mo, Se, and Zn, in which ame atomic absorption Table 5.2.1 Measurement Conditions
spectroscopy is used for conducting the quantitation. Element Wavelength Flame Matrix Modifier
Ca 422.7 nm N2O-C2H2 0.1 % La
QSample Preparation Cr 357.9 nm Air-C2H2
The sample preparation differs for (1) Ca, Cr, Cu, Fe, Cu 324.7 nm Air-C2H2
K, Mg, Mn, Zn and (2) Mo, Se in the above supplement. Fe 248.3 nm Air-C2H2
For the elements in group (1), at least 20 tablets are K 766.5 nm Air-C2H2
crushed and a quantity corresponding to 5 tablets are Mg 285.2 nm Air-C2H2 0.1 % La
transferred to a porcelain crucible. After ashing at 550 °C Mn 279.5 nm Air-C2H2
in a muff le furnace, hydrochloric acid is added and Mo 313.0 nm N2O-C2H2 2 % Ammonium Chloride
the contents are heated to dissolve the residue. Adjust
Se 196.0 nm Air-C2H2 2 % Ammonium Chloride
the final solution to 0.125 N hydrochloric acid. For the
Zn 213.8 nm Air-C2H2
elements in group (2), at least 20 tablets are crushed, and
a quantity corresponding to 1000 μg of the measurement
element is weighed. This is decomposed using nitric acid
and perchloric acid, and is nally brought to a xed 2 %
solution of ammonium chloride.
Table 5.2.2 Element Concentrations for Calibration Curves
QStandard Concentrations of Elements STD (ѥg/mL)
Element
Accordi ng to the USP, calibration cu r ves are to 1 2 3 4 5 6 8
be generated using standard solutions having the Mn 0.5 0.75 1 1.5 2
concentrations shown in Table 5.2.2, and quantitation K 0.5 1 1.5 2 2.5
is conducted using a calibration curve approximated Zn 0.5 1 1.5 2 2.5
by a straight line using a standard solution prepared for
Cu 0.5 1 2 3 4
the concentration indicated in bold type in the Table.
Cr 1 2 3 4
Examples of the target element calibration curves are
Fe 2 4 5 6 8
shown in Fig. 5.2.1 – 10, but 0 μg/mL is not included in
accordance with USP. In the case of Zn, since the high Mg 0.2 0.3 0.4 0.5 0.6
concentration of the standard solution causes curvature of Ca 1 1.5 2 2.5 3

the calibration curve at normal sensitivity, the angle of the Mo 5 10 25
burner was changed and measurement was conducted at Se 5 10 25
lower sensitivity to improve the linearity.
107
5.2 Measurement of Minerals in Dietary Supplements (2) - AA
Fig. 5.2.1 Calibration Curve of Ca Fig. 5.2.6 Calibration Curve of Mg
Fig. 5.2.2 Calibration Curve of Cr Fig. 5.2.7 Calibration Curve of Mn
Fig. 5.2.3 Calibration Curve of Cu Fig. 5.2.8 Calibration Curve of Mo
Fig. 5.2.4 Calibration Curve of Fe Fig. 5.2.9 Calibration Curve of Se
Fig. 5.2.5 Calibration Curve of K Fig. 5.2.10 Calibration Curve of Zn
108
Cd
Pb
Inorganic Metals
Ca
5.3 Measurement of Cadmium and Lead in Food Additives (1) - AA

A food additive is defined in Japan's Food Sanitation The standard solutions for atomic absorption analysis
Act as "an item to be used for the purpose of storage or were prepared by diluting a 1000 mg/L standard solution
processing of food, which is added to, mixed with, or to obtain 1 μg/L of cadmium and 10 μg/L of lead,
diffused into food in any manner." Food additives are used respectively. The calibration curves were generated using
for a variety of purposes, as, for example, preservatives, an autosampler to adjust the injection volumes of standard
sweeteners, coloring agents, and stabilizers. Test methods solution in a stepwise manner. In addition, 5 μL of a
and component standards have been established for many
palladium nitrate solution (50 mg/L palladium content)
of these, and have been published as a food additives
compendium titled "Japan's Speci cations and Standards was added as a matrix modi er to all of the samples. The
for Food Additives." One of the purity test items is heavy main conditions that were used for the spectrometer and
metal testing (in terms of lead content), for which the atomization are shown in Tables 5.3.1 and 5.3.2.
eighth edition of the compendium adopts a colorimetric
method using Nessler cylinders. However, in the ninth Table 5.3.1 Optics Parameters
edition, a different test method is under review, in
which the element lead is handled individually. Here, Cd Pb
we introduce an example of analysis of cadmium (Cd) Analytical wavelength 228.8 nm 283.3 nm
and lead (Pb) in Ơ -cyclodextrin (cyclic oligosaccharide),
Slit width 0.7 nm
a substance used in functional foods, pharmaceuticals,
Ignition mode BGC-D2
cosmet ics, et c. T he a n alysis wa s conduct e d by
electrothermal atomic absorption spectrometry (ETAAS)
using the AA-7000 atomic absorption spectrophotometer. Table 5.3.2 Atomizing Parameters
Cd Pb
QSample Preparation Ashing temperature 700 ʝ 800 ʝ
Sample digestion was conducted using the ETHOS One Atomizing temperature 2200 ʝ
microwave sample preparation system (Milestone Srl). Standard solution 0.2, 0.5, 1.0 2.5, 5, 10
concentration (ppb)
Compared to pretreatment using dry ashing or an open
system such as wet digestion, microwave digestion Tube type Platform
permits quick digestion of the sample, making it unlikely Sample injection volume 20 ѥL
that contamination or volatilization of the measurement 5 ѥL of 50 ppm
0DWUL[PRGLÀHU None
palladium nitrate
element will occur. The digestion process ow is shown
in Fig. 5.3.1. For validity assessment of the pretreatment
and measurement, the same process was conducted on a
sample spiked with standard solution prior to digestion. With the microwave digestion method, since much of the
Preparation was conducted so that the spiked solid acid that is added remains, it is not uncommon for the
concentrations were 0.05 μg/g of Cd and 0.5 μg/g of Pb. acid concentration in the sample solution to be more than
10 %. High acid concentration is a factor that can lead to
a decrease in repeatability and sensitivity. The platform
Transfer 0.5 g sample to digestion container. tube used for this measurement (see Fig. 5.3.2) is resistant
to the effects of acidity and coexisting substances in the
Add small amount of distilled water and 8 mL nitric matrix because the sample is injected into a plate having
acid, then mix. a recess (platform) that is mounted in the tube and then
uniformly heated by radiant heat from the outer wall.
Seal container, set in ETHOS One, digest for approx.
Fig. 5.3.3 shows the changes in absorbance of a lead
1 hour.
standard solution caused by varying the nitric acid
After cooling, transfer to a vessel, add distilled water concentration. A fairly constant absorbance was found to
to a volume of 50 mL. be obtained up to a nitric acid concentration of 20 %.
Measure the sample by electrothermal atomic

absorption spectrometry.
Fig. 5.3.1 Flowchart of Sample Decomposition
109
5.3 Measurement of Cadmium and Lead in Food Additives (2) - AA
Table 5.3.3 Measurement Results of Cd and Pb in Ơ -Cyclodextrin

Element Cd Pb
Measured value < 0.003 ѥg/g < 0.07 ѥg/g
Spike and recovery rate 105 % 99 %
Abs
Fig. 5.3.2 Sectional View of Platform Tube
0.18
0.16
0.14
Absorbance
0.12
0.1
0.08
0.06
0.04
0.02
0
0 5 10 15 20
Nitric Acid Concentration (%) Fig. 5.3.4 Calibration Curve of Cd
Fig. 5.3.3 Sensitivity Variation of Pb 10 μg/L Standard Solution

Due to Changes in Nitric Acid Concentration When Abs
Using a Platform Tube
QResults and Conclusion

The sample measurement results are shown in Table
5.3.3. Neither of the elements was detected in the
sample. Calculation of the lower limit of quantitation as
a concentration in a solid at an absorbance of 0.01 Abs
yielded 0.003 μg/g for cadmium and 0.07 μg/g for lead.
Good values were obtained in spike and recovery testing,
and high-sensitivity analysis of heavy metals was possible
using electrothermal atomic absorption spectrometry
with a platform tube, without adverse effects from the
acid concentration. The calibration curves are shown in Fig. 5.3.5 Calibration Curve of Pb
Figs. 5.3.4 and 5.3.5, respectively, and the peak profiles
are shown in Fig. 5.3.6. The AA-7000 Series features a Abs Abs
lineup that includes not only dedicated instruments for Cd Pb
the flame method and electrothermal method, but also a STD 1 μg/L
dual-use instrument that offers automatic switching of the
STD 10 μg/L
atomization method, thereby supporting a wide range of
application requirements. Regarding the Ơ -cyclodextrin
that was measured in this application, the eighth edition Acid added
of the Specifications and Standards for Food Additives Acid added
speci es a separate reference value for lead (1 μg/g or less).

For pretreatment, after ashing 10 g of sample, nitric acid is
added to bring the solution to a volume of 10 mL, and ame Acid not
added
Acid not
added
atomic absorption is speci ed as the measurement method.
When the AA-7000 flame method is used to analyze the 0 3sec 0 3sec
sample prepared in this manner, the expected detection
limit of lead in a solid sample is about 0.2 μg/g. Fig. 5.3.6 Peak Pro les
110
Cd
Pb
Inorganic Metals
Ca
5.4 Measurement of Heavy Metals (Cd, Pb) in Pet Food (1) - AA
Q Explanation Processing for f lame measurement was conducted

In 2007, an incident occurred in which pet food was found according to the Test Method for Pet Animal Feed
to contain melamine that had been mixed in with the pet (established by the Food and Agricultural Materials
food in the country where it was produced. However, this Inspection Center, the Ministry of Agriculture, Forestry
was discovered only after many pet cats and dogs died as a and Fisheries of Japan, No. 1764, September 1, 2009).
result of melamine poisoning. As a result of this incident, Fig. 5.4.2 shows the f low chart for sample digestion,
the Japanese Ministry of the Environment and the Ministry and Fig. 5.4.3 the flow chart for solvent extraction for
of Agriculture, Forestry and Fisheries established a study Flame AA, respectively. The solvent extraction process
group to address this situation, and in 2008, the Act on is intended for removal of coexisting substances and the
Ensuring of Safety of Pet Animals Feed was enacted. Based concentration of analyte elements.
on this law, an ordinance pertaining to standards of pet
animal feed components was promulgated (Amendment, Transfer 10 g of sample to 100 mL tall beaker.
September 1, 2011, Ministry of Agriculture, Forestry and
Fisheries, Ministry of Environment, Ordinance No. 3), and Ashing by electric furnace (up to 500 °C maximum)
has been in effect since March 1, 2012. Also cited in this Let cool, then add 10 mL HCl, and water to about 30 mL.
ordinance are mycotoxins, organochlorine compounds, and
Heat for several minutes.
three heavy metal elements, cadmium, lead and arsenic.
The concentrations of heavy metals in pet foods for sale are Let cool, adjust volume to 100 mL.
required to meet the criteria of Table 5.4.1, assuming a 10 %
&ROOHFWÀOWUDWHVL[ÀOWHUSDSHUDQGXVHDVVDPSOH
moisture content.
Table 5.4.1 Regulated Values for Heavy Metals in Pet Food Fig. 5.4.2 Flow Chart of Sample Digestion for Flame AA
Cd 1 ppm or less
Pb 3 ppm or less
As 15 ppm or less Transfer 30 mL sample solution to separatory funnel containing
14 mL phosphoric acid.
Here, we introduce an example of analysis of Cd and
Pb in pet food using the AA-7000 Atomic Absorption Add 5 mL potassium iodide solution (68 w/v%) and SXULÀHG water
to bring volume to about 50 mL.
Spectrophotometer.
Gently shake to mix, and let stand 5 minutes.
QSample Preparation
Accurately add 10 mL MIBK, and after shaking vigorously, let stand.
The sample consisted of dry pet food that was nely ground in
a food processor. Sample digestion for electrothermal atomic Collect MIBK layer in test tube and measure by Flame AA.
absorption was conducted using the ETHOS One microwave
sample preparation system (Milestone Srl). The digestion Fig. 5.4.3 Flow Chart of Sample Extraction for Flame AA
process ow is shown in Fig. 5.4.1. For validity assessment,
the same preparation process was conducted on a sample
spiked with standard solution prior to digestion. Preparation QAnalytical Method and Conditions
was conducted so that the spiked solid concentrations were The standard solution for electrothermal measurement
0.5 ppm of Cd and 1 ppm of Pb. by atomic absorption analysis was prepared by diluting
a 1000 mg/L standard solution to obtain 2 ppb (ƫg/L)
of Cd and 20 ppb of Pb (ƫg/L). A calibration curve was
Transfer 0.5 g sample to digestion container. generated using an autosampler to prepare stepwise
Add small amount of distilled water and 8 mL nitric acid, then mix. increasing injection volumes. In addition, 5 μL of a
Seal container, set in ETHOS One, digest for approx. 1 hour.
palladium nitrate solution (100 ppm (mg/L) palladium
content) was added as a matrix modifier to all of the
After cooling, transfer to a vessel, add distilled water to a volume to 50 mL. samples. The standard solution used for the flame AA
Conduct measurement by electrothermal atomic absorption. method was prepared using solvent extraction similarly
as for the sample solution. The main conditions that were
Fig. 5.4.1 Flow Chart of Sample Digestion for ETAAS used for the spectrometer and atomization are shown in
(Electrothermal Atomic Absorption Spectrometry) Tables 5.4.2 - 4.
111
5.4 Measurement of Heavy Metals (Cd, Pb) in Pet Food (2) - AA
Table 5.4.2 Optics Parameters Table 5.4.6 Limit of Quantitation of Cd and Pb

Cd Pb Element Cd Pb
Analytical wavelength 228.8 nm 283.3 nm Electrothermal method 0.003 ppm 0.07 ppm
Slit width 0.7 nm Flame method 0.01 ppm 0.3 ppm
Ignition mode BGC-D2
Abs Abs
Table 5.4.3 Atomizing Parameters for ETAAS Pb
Cd
Cd Pb
Ashing temperature 600 °C 900 °C
Atomizing temperature 2200 °C 2400 °C
Standard solution
0.5, 1.0, 2.0 5, 10, 20
oncentration (ppb)
Tube type Platform
0DWUL[PRGLÀHU 5 ѥL of 100 ppm palladium nitrate
Fig. 5.4.4 Calibration Curves by ETAAS

Table 5.4.4 Atomizing Parameters for Flame AA
Cd Pb
Abs Abs
Flame type Air – Acetylene
Cd Pb
$FHW\OHQHÁRZUDWH 0.8 L/min
StandarGVROXWLRQ
0.05, 0.10, 0.20 0.25, 0.50, 1.00 Spiked
FRQFHQWUDWLRQ(ppm) Spiked
The standard solution concentration refers to the

concentration in the solvent.
UnSpiked UnSpiked
QResults and Conclusion
Table 5.4.5 shows the sample measurement results. The
values were converted to indicate the concentrations in
0 3 sec 0 3 sec
the solid sample. Table 5.4.6 shows the lower limits of
quantitation. The calculated concentrations in the solid Fig. 5.4.5 Peak Pro les of Sample and Spiked Sample by ETAAS
sample are based on the absorbance values of 0.01 Abs and
0.004 Abs obtained using the Electrothermal and Flame The measurement results obtained using microwave
methods, respectively. Fig. 5.4.4 and Fig. 5.4.5 show the digestion – electrothermal atomic absorption were in
calibration curves and measurement solution peak pro les good agreement with the measurement results obtained
by the Electrothermal method. by the method prescribed in the Test Method for Pet
Animal Feed, and excellent spike and recovery results
were also obtained. Further, compared to the dry ashing
Table 5.4.5 Measurement Results for Cd and Pb in Dog Food
– solvent extraction pretreatment operations, preparation
Element Cd Pb of the measurement solution by microwave digestion can
Reference value 1 ppm or less 3 ppm or less
be accomplished more quickly, and combined with the
electrothermal absorption method using the AA-7000,
Electrothermal method 0.19 ppm (94 % ) 0.26 ppm (106 % )
heavy metals could be analyzed with high sensitivity. Not
Flame method 0.20 ppm < 0.3 ppm only does the AA-7000 support analysis by the ame and
Values in parentheses indicate spike/recovery rate. electrothermal methods, the lineup includes a model that
permits automated switching between both atomization
methods to accommodate a wide range of requirements.
112
Cd
Pb
Inorganic Metals
Ca
5.5 Measurement of Cadmium in Brown Rice (1) - AA

The standard for restricting the amount of cadmium Measurement was conducted using the calibration
in rice was strengthened in Japan from a maximum of curve method. A 1000 mg/L standard solution for
10 mg/kg to a maximum of 0.4 mg/kg as speci ed in the atomic spectrometry was diluted to prepare a 2 μg/L
Food Sanitation Law according to the Ministry of Health, standard solution for the measurement. The calibration
Labour and Welfare noti cation No. 183 (noti cation of curve was generated by changing the injection volume
April 8, 2010). This standard was applied on February 28, of this standard solution in stepwise fashion using an
2011. Although this restriction previously applied only to autosampler. In addition, 5 μL of palladium nitrate
brown rice, it now includes polished rice. Here we present solution (100 mg/L Pd) was added to each of the samples,
an example of analysis of cadmium in a standard sample including the standard solution, as a matrix modi er.
(NIES No. 10-a) of brown rice by the high-sensitivity
electrothermal atomic absorption method using the AA- The main measurement conditions are shown in Tables
7000. For an example of cadmium analysis in rice by the 5.5.1 and 5.5.2.
ame method and by the ICP emission spectrometry, refer
to Reference 1 and Reference 2, respectively. Table 5.5.1 Optics Parameters
Analysis wavelength 228.8 nm
QSample Preparation Slit width 0.7 nm
After weighing out 1 g of sample into a beaker, nitric
Low 8 mA
acid and hydrogen peroxide were added and thermal Current values
High 100 mA
decomposition was conducted. The decomposition flow
is shown in Fig. 5.5.1. This pretreatment process was Lamp mode BGC-SR
repeated 6 times on the same sample to evaluate the
repeatability of the preparation process.
Table 5.5.2 Atomizing Parameters
Temperature Time Heating Gas Flowrate
No.
(ºC) (sec) Mode (L/min)
Weigh 1 g sample into a tall beaker
1 60 3 RAMP 0.1
2 150 15 RAMP 0.1
←Add about 10 mL distilled water
3 250 10 RAMP 0.1
←Add 10 mL nitric acid and mix well Temperature Program 4 600 10 RAMP 0.2
5 600 15 STEP 0.2
6 600 3 STEP 0.0
Cover with watch glass and heat (At about 180° for 30 - 60 min)
7 2200 3 STEP 0.0
8 2400 2 STEP 1.0
←Add 5 mL nitric acid and 5 mL hydrogen peroxide Atomization stage is No. 7
Sample injection volume ѥ/
Palladium nitrate solution
Cover with watch glass and heat (At about 230° for several hours) 0DWUL[PRGLÀHU SDOODGLXPFRQFHQWUDWLRQSSPѥ/
Tube type High density graphite tube
←At near dry state, add a small amount (about 0.5 mL) nitric Signal processing Peak height
acid, and repeat until solution is nearly colorless
Cool
Adjust volume to 50 mL (measurement solution)
Fig. 5.5.1 Sample Decomposition Flow Chart
113
5.5 Measurement of Cadmium in Brown Rice (2) - AA
Q Measurement Results
Abs
Table 5.5.3 shows the sample measurement results. At a
Blank 0.2 ppb 0.4 ppb 0.6 ppb 0.8 ppb
low concentration of 1/10 the standard value, excellent
results were obtained for both concent ration and
repeatability.
Fig. 5.5.2 and Fig. 5.5.3 show the calibration curve
and segments of the standard solution peak profiles,
respectively.
Fig. 5.5.4 shows the peak profiles of the respective
samples.
Table 5.5.3 Measurement Results of Cd in Brown Rice 0 2 0 2 0 2 0 2 0 2

(sec)
NIES No. 10-a
(Guaranteed value 0.023 ± 0.003 mg/kg)
Process 1 0.024 mg/kg Fig. 5.5.3 Peak Pro les of Standards
Process 2 0.024 mg/kg
Process 4 0.023 mg/kg Abs Abs Abs
Process 6 0.023 mg/kg Preparation 1 Preparation 2 Preparation 3
Average 0.024 mg/kg
Standard deviation 0.0004 mg/kg
Relative standard
1.7 %
deviation (%RSD)
Each value is an average of n=2.
r=0.9997
Abs=0.1961 × Conc+0.00156 0 2 0 2 0 2 (sec)
Abs Abs Abs
Preparation 4 Preparation 5 Preparation 6
0 2 0 2 0 2 (sec)
Fig. 5.5.2 Calibration Curve Fig. 5.5.4 Peak Pro les of Samples
[References]
1) Shimadzu Application News No. A427 “Determination of Cadmium in Brown Rice by Flame Atomic Absorption” (2010)
2) Shimadzu Application News No. J87 “Multi-Element Simultaneous Determination of Nutrients as well as Hazardous Elements in
Brown Rice by ICPE-9000” (2007)
114
6. Toxin Inorganic Poison
$QDO\VLVRI'LDUUKHWLF6KHOO¿VK3RLVRQ'63- LC/MS
Q Explanation
Diarrhetic Shellfish Poison (DSP) is a substance that groups, their analysis is dif cult. In Japan, DSP is of cially
accumulates in the digestive gland of the shellfish when analyzed by a bioassay method called the mouse unit method.
they ingest poisonous planktons such as Dinophysis However, HPLC analysis combined with f luorescence
fortii or Dinophysis acuminata. When humans ingest derivatization is also applicable.
shell sh affected by DSP, they suffer acute gastroenteritis Introduced here is an example of analysis of OA, DTX-1 and
accompanied by such symptoms as vomiting, diarrhea and PTX-6 (Fig. 6.1.1) using LC/MS. The ESI mass spectra of the
stomachache. Although the amount ingested normally does positive and negative ions of OA were shown in Fig. 6.1.2.
not lead to death in humans, it is known that DSP does not In the negative ion ESI method, the deprotonated molecule
decompose at normal household cooking temperatures. (M-H) – is clearly detected. In the positive ion spectrum, the
Typical DSP includes Okadaic acid (OA), Dinophysistoxin molecule with the attached sodium ion (M+Na)+, as well as
(DTX), Pectenotoxin (PTX), and Yessotoxin. Since they fragment ions where 1 - 4 water molecules have detached
have complex structures and no suitable chromophore from the protonated molecule, are detected.
Fig. 6.1.1 Structures of Okadaic Aid (OA), Dinophysistoxin-1 (DTX-1) and Pectenotoxin-6 (PTX-6)
Inten.(×100,000)
8.0
7.0
6.0
5.0
4.0
3.0
2.0
1.0
0.0
Inten.(×100,000)
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Fig. 6.1.2 Positive and Negative ESI Mass Spectra of Okadaic Aid (OA)
115
$QDO\VLVRI'LDUUKHWLF6KHOO¿VK3RLVRQ'63- LC/MS
The results of selected ion monitoring (SIM) analysis using ion detection). As for PTX-6, the sensitivity decreases to
m/z of the deprotonated molecule (M-H) – in the negative about one-fifth in the negative ion detection compared to
ion detection and m/z of the sodium ion-added molecule the positive ion detection, and PTX has isomers that do not
(M+Na)+ in the positive ion detection yielded good linearity contain carboxylic acid. Although these factors make the
in the concentration range of 13-1625 ng/mL, with a spectrum comparatively complex, positive ion detection is
quantitation limit of 13 ng/mL (excluding PTX-6 in negative more suitable when PTX needs to be analyzed.
(×1,000,000)
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0
(×100,000)
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 19.0 20.0 21.0 22.0 23.0
Fig. 6.1.3 SIM Chromatograms of DSP using Positive and Negative ESI
Column : 6KLPSDFN932'6PP/ðPP,'
Mobile Phase A : 5 mmol/L Ammonium Acetate + 0.1 % Formic Acid-Water
Mobile Phase B : Acetonitrile
Time Program : %PLQńPLQ
Probe Voltage : N9(6,1HJDWLYH0RGH N9(6,3RVLWLYH0RGH
Nebulizer Gas Flow : 1.5 L/min ł
Drying Gas Pressure : 0.2 MPa ł
CDL Temp. : 200 °C ł
Block Heater Temp. : 200 °C ł
CDL & Q-array Voltage : Using default values ł
SIM : m/z 803.25, 887.15, 817.25 for Negative Mode
m/z 906.25, 911.15, 634.25, 850.30, 827.20, 864.30, 841.20 for Positive Mode
116
Toxin Inorganic Poison
+LJK6SHHG$QDO\VLVRI$ÀDWR[LQVLQ)RRG- LC

A atoxins are mycotoxins that are extremely carcinogenic Column : Shim-pack XR-ODSⅡ
and acutely toxic, and because they are subject to food PP/ðPP,'ƫP
contamination monitoring, their measurement is routinely Mobile Phase : :DWHU0HWKDQRO$FHWRQLWULOH YYY
conducted by HPLC and other analytical methods. Here Flowrate : 1.0 mL/min
we introduce an example of ultra-high-speed analysis of Column Temp. : 50 °C
a atoxins in food using a combination of the Prominence Injection Volume : ƫ/
RF-20Axs high-sensitivity uorescence detector and the Detection : RF-20Axs Ex. at 365 nm, Em. at 450 nm
Nexera Ultra High Performance LC system, in which the RF Cell : Conventional cell
aflatoxins B1, B2 , G1 and G 2 are analyzed using direct Cell Temp. : 25 °C
high-sensitivity uorescence detection without conducting
TFA derivatization.
mV
Peaks
Q$QDO\VLVRI$ÀDWR[LQV6WDQGDUG6ROXWLRQ 0.175 1. Aflatoxin G2 (5 ng/L)
2. Aflatoxin G1 (20 ng/L)
Fig. 6.2.1 shows a chromatogram of a standard mixture 0.150 3. Aflatoxin B2 (5 ng/L)
of the 4 a atoxins. For the analytical column, the Shim- 4. Aflatoxin B1 (20 ng/L)
3
pack XR-ODSⅡ (100 mm L. × 3.0 mm I.D., 2.2 μm) was 0.125
used. The concentrations of B1 and G1 were each 20 ng/L 0.100
(20 ppt), and those of B2 and G2 were each 5 ng/L (5 ppt). 1
The mixture was prepared in a solution of water / 0.075
4
acetonitrile = 9/1 (v/v). Use of the RF-20Axs allowed high 0.050
sensitivity detection of B1 and G1 without undergoing 2
derivatization with TFA. 0.025
0.000
Although an injection volume of 8 μL was used in the 0.0 1.0 2.0 3.0 min
analysis shown in Fig. 6.2.1, we investigated the peak
shapes and separation performance using larger sample Fig. 6.2.1 Chromatogram of a Standard Mixture of A atoxins
(8 μL injected)
injection volumes for analyses at even lower trace levels.
Fig. 6.2.2 shows the chromatograms of the same standard
mixture of 4 a atoxins used in the analysis of Fig. 6.2.1, mV
1.00
but with injection volumes of 8 μL, 30 μL, and 50 μL, Peaks 8 μL
respectively. Even with a 50 μL injection volume, good Same as Fig. 6.2.1 30 μL
50 μL
separation was obtained without any degradation of peak 0.80
3
shape. The detection limits (with SN ratio = 3.3) using the
50 μL injection were 1 ng/L (1 ppt) for a atoxin B1, and 0.60
2 ng/L (2 ppt) for a atoxin G1. 1
0.40
4
0.20 2
0.0 1.0 2.0 3.0 min
Fig. 6.2.2 Chromatograms of a Standard Mixture of A atoxins-

Comparison of Injection Volume
117
+LJK6SHHG$QDO\VLVRI$ÀDWR[LQVLQ)RRG- LC
Q Effect of Cell Temperature Control

It is generally known that uorescence intensity is easily Sample 50 g
affected by the surrounding temperature, speci cally that (Aflatoxin Standard Solution)
it is diminished or “quenched” at higher temperatures.
200 mL Water / Acetonitrile = 1/9(v/v)
Fig. 6.2.3 shows the relationship between the RF-20Axs
Keep in dark at room
cell temperature and the peak height ratio (based on a temperature for 5 min
peak height of 1 at 20 °C). From these results, it is clear
that aflatoxins B1 and B2 are particularly affected by
Mixing for 30 min
room temperature fluctuation. Because the RF-20Axs
is equipped with a cell temperature control feature as Filtration
standard, high accuracy analysis is possible without the
adverse effect of room temperature uctuation, even in (8 mL)
the high-sensitivity analysis of a atoxins.
Clean-up by multi-functional column
“MycoSep 226 AflaZon+”
1.100 Fraction
1.050
Peak Height Ratio
1.000 (1 mL)
0.950
0.900 B1 Evaporation by N2 gas
0.850 B2 0.5 mL Water / Acetonitrile = 9/1(v/v)
0.800
G1
0.750 Sample solution
G2
0.700
20 ˚C 25 ˚C 30 ˚C 35 ˚C 40 ˚C
Cell Temperature (˚C) Fig. 6.2.4 Sample Preparation
Fig. 6.2.3 Relationship between Cell Temperature and Peak Height Ratio
QAnalysis of Food Sample

mV
We conducted sample preparation of commercially Peaks
available wheat our according to the procedure shown 4.00 1. Aflatoxin G2
2. Aflatoxin G1
in Fig. 6.2.4. In addition, the same preparation was also 3. Aflatoxin B2
conducted for the same our, but spiked with a standard 4. Aflatoxin B1
mixture of 4 a atoxins (B1 and G1 at 0.8 μg/kg, and B2 and 3.00
G2 at 0.2 μg/kg). These samples were analyzed using the 3

same analytical conditions as shown on the previous page.
2.00 1
Fig. 6.2.5 shows the respective chromatograms.
4
2
1.00
Spiked
Unspiked
0
0.0 1.0 2.0 3.0 min
Fig. 6.2.5 Chromatograms of Wheat Flour Samples (8 μL injected)

(Upper: Spiked, Lower: Unspiked)
118
6.3 $QDO\VLVRI$ÀDWR[LQ%1, B2, G1 and G2 at High Sensitivity (1) - LC
Q Explanation Q'HULYDWL]DWLRQRI$ÀDWR[LQVZLWK7ULÀXRURDFHWLF$FLG
A atoxins are toxins produced by the fungus Aspergillus. Typically, af latoxins B1 and G1 are converted to the
In addition to being acutely toxic, they are also known hydroxyl-derivative af latoxins B 2a and G 2a using
to be carcinogenic, so their inspection in foods is tri uoroacetic acid (TFA) to increase their uorescence
necessary1). Up to now, a atoxin B1 is the only a atoxin intensity in HPLC analysis. Fig. 6.3.1 shows the structures
that has been regulated in Japan. However, beginning of the 4 aflatoxins and those of the B2a and G 2a TFA
October 2011, the restriction extends to the total of derivatives.When analyzing af latoxins in food, TFA
a atoxin B1, B2, G1 and G2.2). derivatization is done for both the standard solution and
Here, we introduce two cases of analysis of these 4 the sample solution.
aflatoxins (B1, B2 , G1, G 2), using the Prominence RF-
20A XS high-sensitivity f luorescence detector, first
by f luorescence detection using trif luoroacetic acid
derivatization, and then direct detection without TFA
derivatization.
O O O
O O O
O HO O O
O O O
O TFA O
O O O O
CH3 CH3 CH3
Aflatoxin B1 Aflatoxin B2a Aflatoxin B2
O O O O O O
O O HO O O O O
O O O
TFA O O
O O O O
CH3 CH3 CH3
Aflatoxin G1 Aflatoxin G2a Aflatoxin G2
Fig. 6.3.1 Structures of A atoxins B1, B2, G1, G2, and Tri uoroacetic Acid-Derivatized Forms (B2a, G2a)
119
Q Analysis of Standard Solution Af ter QAnalytical Conditions

'HULYDWL]DWLRQZLWK7ULÀXRURDFHWLF$FLG Column : Shim-pack FC-ODS (150 mm L. ðPP,'ƫP
Fig. 6.3.3 shows chromatograms of standard solutions Mobile Phase : Water / Methanol / Acetonitrile = 6/3/1 YYY
of the 4 aflatoxins (B1, B2 , G1, G 2) obtained following Flowrate : 0.8 mL/min
TFA derivatization (Fig. 6.3.2) with 20 μL injections. Column Temp. : 40 °C
Regarding the st andard solution B 2a peak in the Detection : RF-20AXS, Ex at 365 nm, Em at 450 nm
RF Cell : Conventional cell
chromatogram on the right in Fig. 6.3.3, a peak area
Cell Temp. : 25 °C
repeatability (n=6) of 1.2 %RSD was obtained, and the Injection Volume : ƫ/
detection limit (S/N ratio=3.3, 20 μL injection) was
calculated to be 0.4 ng/L (8 fg). Ultra-trace levels of Aflatoxin Standard Solution
a atoxins can accurately be detected with high sensitivity Evaporation by N2 gas
using the RF-20A XS. 0.2 mL TFA
Standing in dark at room temperature for 15 min
0.8 mL Water / Acetonitrile = 9/1 (v/v)
HPLC (20 μL)
Fig. 6.3.2 Derivatization with TFA
mV µV
30.0 Peaks Peaks
1 400
1. Aflatoxin B2a (B1) : 2.0 µg/L 1. Aflatoxin B2a (B1) : 20 ng/L
2. Aflatoxin B2 : 0.5 µg/L 2. Aflatoxin B2 : 5 ng/L
25.0 1
3. Aflatoxin G2a (G1) : 2.0 µg/L 3. Aflatoxin G2a (G1): 20 ng/L
4. Aflatoxin G2 : 0.5 µg/L 300 4. Aflatoxin G2 : 5 ng/L
20.0
3 3
15.0 200
10.0 2
100 4 2
4
5.00
0
0.00
0.0 2.0 4.0 6.0 8.0 10.0 12.0 min 0.0 2.0 4.0 6.0 8.0 10.0 12.0 min
Fig. 6.3.3 Chromatograms of A atoxin Standard Solutions After Derivatization with Tri uoroacetic Acid (20 μL injected)
(Left) B1 and G1: 2.0 μg/L, B2 and G2: 0.5 μg/L, (Right) B1 and G1: 20 ng/L, B2 and G2: 5 ng/L
QAnalysis of Standard Solution by Direct Detection
Fig. 6.3.4 shows chromatograms of standard solutions of the 4 a atoxins (B1, B2, G1, G2) obtained with 20 μL injections,
without TFA derivatization. The analytical conditions were the same as those shown on this page. Regarding the B1 peak
in the chromatogram on the right in Fig. 6.3.4, a peak area repeatability (n=6) of 2.7 %RSD was obtained, and the detection
limit (S/N ratio=3.3, 20 μL injection) was calculated to be 3 ng/L (60 fg). This demonstrated that testing for a atoxin B1
and G1 can be done with suf cient sensitivity using direct detection with the RF-20A XS, without TFA derivatization.
mV μV
8.00 Peaks 2 Peaks 2
1. Aflatoxin B1 : 2.0 μg/L 1. Aflatoxin B1: 20 ng/L
2. Aflatoxin B2 : 0.5 μg/L 2. Aflatoxin B2: 5 ng/L
3. Aflatoxin G1 : 2.0 μg/L 60.0 3. Aflatoxin G1: 20 ng/L
4
6.00 4. Aflatoxin G2 : 0.5 μg/L 4 4. Aflatoxin G2: 5 ng/L
1
1 40.0
4.00
3
3 20.0
2.00
0.00
0.00
0.0 5.0 10.0 15.0 min 0.0 5.0 10.0 15.0 min
Fig. 6.3.4 Chromatograms of A atoxin Standard Solutions by Direct Detection (20 μL injected)
(Left) B1 and G1: 2.0 μg/L, B2 and G2: 0.5 μg/L, (Right) B1 and G1: 20 ng/L, B2 and G2: 5 ng/L
120
QAnalysis of Food mV
For a food application example, a atoxin standard solution Peaks
was added to commercially available corn our so that the 1. Aflatoxin B2a (B1)
12.5 2. Aflatoxin B2
respective a atoxin concentrations in the sample became 0.8 3. Aflatoxin G2a (G1)
μg/kg for B1 and G1, and 0.2 μg/kg for B2 and G2. The pseudo 4. Aflatoxin G2
contaminated sample was then analyzed. Fig. 6.3.5 shows 10.0
the sample preparation procedure. The contaminants were
1
removed using a multifunctional column. 7.5
Analysis was conducted using both TFA derivatization and
direct detection without derivatization. Fig. 6.3.6 shows the 3
5.0
chromatograms obtained from analysis of commercially
available corn flour that was spiked and unspiked (blank) 4
2
with the af latoxin standard solution, in this case using 2.5

Spiked
TFA derivatization. Fig. 6.3.7 shows the chromatograms
obtained using direct detection analysis without conducting Unspiked (Blank)
0.0
derivatization.
0.0 5.0 10.0 15.0
min
Sample 50 g
Fig. 6.3.6 Chromatograms of Corn Flour Using Derivatization with
(Aflatoxin Standard Solution) TFA (20 μL injected)
200 mL Water / Acetonitrile = 1/9 (v/v) (Upper) Spiked with A atoxin Standard, (Lower) Unspiked
Placed in dark room and
maintained at ambient temperature for 5 minutes
mV
Mixing for 30 min Peaks

1. Aflatoxin B1
3.5 2. Aflatoxin B2
Filtration
3. Aflatoxin G1
3.0 4. Aflatoxin G2
(8 mL)
Clean-up by multi-functional column 2.5

“MycoSep 226 AflaZon+” 2
2.0
Fraction 4
(1 mL) (1 mL) 1.5 1

3
Evaporation by N2 gas Evaporation by N2 gas 1.0
0.1 mL TFA
0.5 Spiked
Placed in dark room and maintained
at ambient temperature for 15 minutes Unspiked (Blank)
0.0
0.4 mL Water / Acetonitrile 0.5 mL Water / Acetonitrile
= 9/1 (v/v) = 9/1 (v/v) 0.0 5.0 10.0 15.0
min
Sample solution for Fig. 8 Sample solution for Fig. 9
Fig. 6.3.7 Chromatograms of Corn Flour by Direct Detection

Fig. 6.3.5 Sample Preparation (20 μL injected)
(Upper) Spiked with A atoxin Standard, (Lower) Unspiked
[References]
Mobile Phase : $:DWHU0HWKDQRO$FHWRQLWULOH YYY 1) Handling of Foods Containing Mycotoxins (Af latoxins)
B: Acetonitrile (Notification No. 128, issued on March 16, 1971 by the
Gradient Elution Method Environmental Health Bureau, Ministry of Health, Labour
Time Program : %PLQń90 % and Welfare; revised on March 26, 2002 by the Food
PLQńPLQ Inspection Division [No. 0326001], Japan)
2) Handling of Foods Containing A atoxins (Noti cation 0331,
(The other analytical conditions are the same as those on No. 5, issued on March 31, 2011 by the Food Safety Division,
the previous page) Ministry of Health, Labour and Welfare, Japan)
121
6.4 High Speed Analysis of Nivalenol and Deoxynivalenol (1) - LC

Nivalenol and deoxy n ivalenol a re t r ichothecene Column : 3KHQRPHQH[6\QHUJLƫP+\GUR53c
mycotoxins which are produced by Fusarium fungi, PP/ðPP,'ƫP
and are known to be associated with digestive system Mobile Phase : A: PPRO/ 6RGLXP 3KRVSKDWH
disorders and toxicity to the immune system if they %XIIHUS+
enter the food chain through contaminated cereal crops. B: Acetonitrile
Here we introduce an example of high-speed analysis of C: Methanol
nivalenol and deoxynivalenol* using the ultra-fast LC A / B / C = 18/1/1 YYY
system Prominence UFLC with the high-speed, high Flowrate : 0.9 mL/min
resolution, high-performance Phenomenex Synergi Column Temp. : 40 °C
Hydro-RP column. Injection Vol. : ƫ/
* The standard mixture of nivalenol and deoxynivalenol, and Detection : SPD-20AV at 220 nm
the our extract solution were provided by the Japan Grain UV Cell : Semi-micro Cell
Inspection Association.

Fig. 6.4.1 shows the st r uct u res of nivalenol and
deoxy n ivalenol. Because t hese a re h ig h ly pola r
compounds, we used the Phenomenex Synergi Hydro-
RP (2.5 μm particle diameter) high-speed, high resolution
ODS column, in which the bonded phase is endcapped
with a polar group to provide enhanced retention, while
also providing increased longevity with highly aqueous
mobile phases. Fig. 6.4.2 shows the analysis results of
the standard mixture of nivalenol and deoxynivalenol
(prepared by dissolving each in acetonitrile at 1.0 mg/L,
and then diluting with mobile phase), using a 20 μL
injection. The peak in the vicinity of 2 minutes on the
chromatogram derives from dissolved oxygen in the
sample solution1), but does not interfere with the complete
separation of nivalenol and deoxynivalenol when using
the Phenomenex Synergi Hydro-RP column.
Fig. 6.4.2 Chromatogram of a Standard Mixture of Nivalenol
and Deoxynivalenol (1.0 mg/L each, 20 μL injected)
Fig. 6.4.1 Structures of Nivalenol and Deoxynivalenol
122
6.4 High Speed Analysis of Nivalenol and Deoxynivalenol (2) - LC
QAnalysis of Flour QAnalytical Conditions

Fig. 6.4.3 shows the results of analysis of a our extract Column : 3KHQRPHQH[6\QHUJLƫP+\GUR53c
solution contaminated with trichothecene mycotoxins. PP/ðPP,'ƫP
Here, the gradient elution method was used for the Mobile Phase : $DEF YYY
purpose of conducting column washing. The our sample : %DEF YYY
preparation procedure is shown in Fig. 6.4.4. In Japan, a: PPRO/6RGLXP3KRVSKDWH
the provisional standard criteria value for deoxynivalenol %XIIHUS+
content in our is set to 1.1 ppm2), but the concentration of b: Acetonitrile
deoxynivalenol in this sample was 0.60 ppm. c: Methanol
Time Program : % PLQ ń 100 % (4.01-
PLQńPLQ
Column Temp. : Ý&
Detection : SPD-20AV at 220 nm
Sample 50 g
Fig. 6.4.3 Chromatogram of Flour Extract
[References]
1) “Test Method for Deoxynivalenol” (Food Safety Noti cation No. 071702, Ministry of Health, Labour and Welfare, July 17, 2003, Japan)
2) “Provisional Standard Criteria Value for Deoxynivalenol” (Food Safety Noti cation No. 0521001, Ministry of Health, Labour and
Welfare, May 21, 2002, Japan)
123
6.5 Analysis of Arsenic in Foods (1) - EDX

X-ray uorescence spectrometers enable quickly, easily, Instrument : EDX-700
and non-destructively analyzing solids, powders, liquids, X-Ray Tube : Rh target
and other samples. In particular, they are useful for Filter : Ni or without
detecting and quantitatively analyzing arsenic (As), Voltage - Current : N9²ƫ$DXWR
cadmium (Cd), and potassium (K), and other elements in Atmosphere : Air
toxic substances in foods. The following is an example Measurement Diameter : 10 mm
Measurement Time : 40 sec
of analyzing arsenic levels of sodium arsenite mixed in
Dead Time : 25 %
oolong tea, juice, and curry. EDX-700/800 models include
five types of primary filters as standard accessories,
which are required for trace analysis of substances such QAnalysis Results
as arsenic. An example of trace analysis is also described Qualitative analysis results for oolong tea, juice, and
below. curry are shown in Fig. 6.5.1, Fig. 6.5.2, and Fig. 6.5.3.
Quantitative analysis values are indicated in Table 6.5.1.
QSamples Equations were balanced assuming H 2O (water) as the
Samples were prepared by mixing sodium arsenite primary ingredient and ignoring all non-arsenic elements.
(NaAsO2) with commercially marketed oolong tea, juice,
and curry (in retort packaging) to an arsenic concentration Table 6.5.1 Quantitative Value of As in Foods
of about 0.4 %.
Element Oolong Tea Juice Curry
About 6 mL of untreated samples were placed in liquid
As 0.39 % 0.35 % 0.33 %
containers with 6 μm thick PET film adhered to the H2O 99.61 % 99.65 % 99.67 %
bottom.
Analyte Result Std. Dev. Calculation Line Intensity Analyte Result Std. Dev. Calculation Line Intensity
AS 0.390 % 0.002 Quant.-FP AsKa 76.119 AS 0.352 % 0.002 Quant.-FP AsKa 69.052
H2O 99.610 % - Balance - - H2O 99.648 % - Balance - -
Fig. 6.5.1 Qualitative and Quantitative Analysis of Oolong Tea Fig. 6.5.2 Qualitative and Quantitative Analysis of Juice
124
6.5 Analysis of Arsenic in Foods (2) - EDX
Analyte Result Std. Dev. Calculation Line Intensity

AS 0.329 % 0.002 Quant.-FP AsKa 64.851
Fig. 6.5.4 Qualitative Analysis With and Without Ni Filter
H2O 99.671 % - Balance - -
Table 6.5.2 Detection Limits* of As in Aqueous Solution

Fig. 6.5.3 Qualitative and Quantitative Analysis of Curry
With Ni Filter Without Ni Filter
0.9 ppm 2.0 ppm

Q EDXRF Analysis of Trace Arsenic Using
Primary Filter
Trace analysis requires using a primary filter. Using a *Detection limits calculated as follows:
primary lter enables reducing the scattering of primary
X-rays from the X-ray tube to obtain measurements with C : Standard value
good S/N ratios. Consequently, heavy metals such as
¥
C I back I net : Net intensity
L.L.D.= 3
arsenic can be detected down to a few ppm. The following I net T I back : Background intensity
T : Measurement time
is an example of analyzing trace arsenic in an aqueous
solution using a nickel primary lter. The nickel lter is
also required for trace analysis of 29Cu, 30Zn ... 42Mo, 73Ta,
74W ... and 92U as well. QAnalysis Results
Qualitative analysis results from the 10 ppm aqueous
arsenic solution with and without using a nickel lter are
QSamples shown overlaid in Fig. 6.5.4.
A 10 ppm aqueous solution was used, containing a Based on these results, detection limits were calculated
standard 1000 ppm solution of arsenic for atomic for with and without using a nickel filter, as shown in
absorption spectrometry, diluted by 100 times. Table 6.5.2.
125
7. Foreign Matters Offensive Odors
7.1 High Sensitivity Analysis of 2,4,6-Trichloroanisole in
Wine (1) - GC/MS/MS
Q Introduction
2,4,6-trichloroanisole (TCA) emitted from wine corks samples, or thermal desorption. The HS-20 headspace
can taint wine and cause an objectionable odor. Due sampler includes a trap function that is able to concentrate
to the low threshold value for sensing the odor, highly headspace gases. Here we introduce an example of high-
sensitive measurements are required for monitoring. sensitivity measurement of TCA in wine using an HS-
Conventionally, TCA was measured using methods such trap GC/MS system. The structure of TCA is illustrated in
as purge and trap, which is very effective in concentrating Fig. 7.1.1 and the mass spectrum is shown in Fig. 7.1.2.
%
100
196.9
209.9
50
166.9 198.9
109.0
62.1 73.0 97.0 147.1 160.1 177.0
0
50.0 75.0 100.0 125.0 150.0 175.0 200.0
Fig. 7.1.1 Structure of TCA Fig. 7.1.2 Mass Spectrum of TCA
HS-20 GCMS-QP2010 Ultra
Mode : Trap GC Unit
Equilibrating Time : 30 min Column : Rxi-5ms 0.32 mm I.D. × 60 mL., df = 1.0 ѥm
Oven Temp. : 60 °C Column Temp. : 50 °C (1 min) - 10 °C/min - 300 °C (5 min)
Sample Line Temp. : 260 °C Carrier Gas Control : Constant pressure
Transfer Line Temp. : 260 °C Carrier Gas Pressure : 180 kPa
Trap Equilibrating Temp. : 80.0 °C Injection Mode : Splitless
Trap Cooling Temp. : 80.0 °C Sampling Time : 3 min
Trap Desorbing Temp. : 280.0 °C Additional Flow
Vial Pressurizing Time : 2.0 min APC1 : 100 kPa
Pressure Equilibrating Time : 0.1 min APC3 : 50 kPa
Load Time : 0.1 min MS Unit
Load Equilibrating Time : 0.1 min Interface Temp. : 280.0 °C
Dry Purge : 5 min Ion Source Temp. : 230.0 °C
Injection Time : 20 min Solvent Elution Time : 14 min
Needle Flush : 20 min Measurement Start Time : 15 min
Injection Cycle : 3 cycles Measurement End Time : 20 min
Cycle Time : 50 min Measurement Mode : SIM
Sample Loading Volume : 5 mL Selected Ions (m/z) : 211.9, 209.9, 196.9, 194.9
Event Time : 0.2 sec
126
Foreign Matters Offensive Odors
7.1 High Sensitivity Analysis of 2,4,6-Trichloroanisole in

Wine (2) - GC/MS/MS
QSensitivity QLinearity and Repeatability
A wine sample spiked with the equivalent of 1 ng/L Linearity was con rmed by adding speci c concentrations
TCA was measured by SIM using the HS-trap method of trichloroanisole to wine (from 1 to 100 ng/L, as shown
(Fig. 7.1.3). The results show how the system was able to in Fig. 7.1.6). The results showed good linearity. 3 ng/L of
analyze low concentrations of TCA with high sensitivity. trichloroanisole was added to wine to test the repeatability
A wine sample spiked with the equivalent of 100 ng/L (n=5) of peak area (Table 7.1.1). Results showed good
TCA was measured by SIM using the headspace-GC/MS repeatability, with a CV value not exceeding 5 %.
method and HS-trap method, as shown in Fig. 7.1.4 and
Fig. 7.1.5. A comparison of both shows that the HS-trap
method provided about 10 times higher sensitivity. Area(×10,000)
R 2= 0.99991
(×1,000)
2.0 211.90 5.0
209.90
1.5
2.5
1.0
0.5
0.0
16.75 17.00 17.25 0 50 Conc.
Fig. 7.1.3 SIM Chromatogram of TCA in Wine Measured Using HS-Trap
(Wine spiked with 1 ng/L TCA) Fig. 7.1.6 Linearity of TCA (Wine spiked with 1-100 ng/L TCA)
(×1,000)
211.90 Table 7.1.1 Area Repeatability of TCA ( n=5, wine spiked with 3 ng/L TCA)
209.90
2.0 AREA 1 2 3 4 5 Average %RSD
TCA
3,103 3,051 2,925 3,020 2,742 2,968 4.79 %
1.5 m/z 211.9
1.0
QConclusion
0.5 We presented here an example of high-sensitivity
16.75 17.00 17.25
measurement of trichloroanisole in wine using an HS-
trap GC/MS system. The results showed that the system
Fig. 7.1.4 SIM Chromatogram of TCA in Wine Measured Using
was easily able to measure even a few nanograms per
Conventional Headspace GC/MS
(Wine spiked with 100 ng/L TCA) liter. This also confirmed that an HS-trap-GC/MS
system using the HS-20 headspace sampler is effective in
(×10,000) monitoring trichloroanisole in wine.
3.0 211.90
209.90
2.0
1.0
16.75 17.00 17.25

Fig. 7.1.5 SIM Chromatogram of TCA in Wine Measured Using HS-Trap
(Wine spiked with 100 ng/L TCA)
127
7.2 Rapid Determination of Melamine in Food (1) - LC

Melamine is used as the main raw material of melamine Column : Shim-pack XR-ODS
resin, a thermosetting plastic, but recently it is receiving PP/ðPP,'ƫP
considerable attention due to numerous incidents of food Mobile Phase : A:PPRO/6RGLXP3KRVSKDWH%XIIHUS+
contamination with this substance. Analysis of melamine containing 10 mmol/L Sodium 1-octanesulfonate
in food can be performed using HPLC, LC/MS(/MS), B: Acetonitrile
and GC/MS, etc., depending on the objective, but among $% YY
these, HPLC allows for easy, multi-specimen screening Flowrate : 1.2 mL/min
analysis. Here we introduce a rapid analytical technique Column Temp. : 40 °C
for determining melamine in food using the ultra-fast Injection Vol. : ƫ/
LC, Prominence UFLC together with a simple sample Detection : SPD-M20A at 235 nm
preparation procedure. Flow Cell : Conventional cell
QAnalysis of Standard Solution mA

Melamine (Fig. 7.2.1) is a high-polarity substance with a
triazine ring, but since it is not suf ciently retained using
a typical reversed-phase mode, we used the reversed-
phase ion pairing mode for this analysis. Detection was
conducted with a photodiode array detector, and the
Shim-pack XR-ODS high-performance, high-resolution 100
column (particle size 2.2 μm) was used for the analytical 235
column. Fig. 7.2.2 shows the UV spectrum of melamine.
Quantitation was conducted at the maximum absorption
wavelength of 235 nm. Fig. 7.2.3 shows the chromatogram
of a melamine standard solution (1000 μg/L). 0
200 250 300 350 nm
Fig. 7.2.2 UV Spectrum of Melamine

H2N N NH2
mAU
N N
5 Peak
1. Melamine 1
NH2
Fig. 7.2.1 Structure of Melamine
0 2 4 6 min
Fig. 7.2.3 Chromatogram of Melamine Standard Solution (1000 μg/L)
128
7.2 Rapid Determination of Melamine in Food (2) - LC
QLinearity and Sensitivity QAnalysis of Melamine in Food

The linearity of melamine detection using standard Melamine equivalent to 2 mg/kg was added to each
solution concentrations of 10 μg/L to 100 μg/L is portion of commercially available milk and wheat our,
shown in Fig. 7.2.4. Excellent linearity is obtained with and sample preparation was conducted according to the
a correlation coefficient (R 2) greater than 0.9999. The procedure of Fig. 7.2.5 ( nal prepared liquid containing
quantitation limit (S/N=10) and detection limit (S/N=3.3) 20 μg/L concentration of melamine in each). The
with injection of 10 μL were 16 μg/L and 5 μg/L, analytical results are shown in Fig. 7.2.6 and Fig. 7.2.7.*
respectively. The recovery of added melamine in this analysis was
97 % for the milk, and 73 % for the wheat our.
* It is recommended to rinse the column (for example, mobile

3000 phase: A / B = 1/1) after completion of each analysis.
2500
mAU
Peak Area
2000 Q Peak
1. Melamine (Spiked)
1500
1000
0.2 1
500
Milk
0 Spiked at 2 mg/kg
0 25 50 75 100
Concentration (ѥg/L) Milk Blank
0
Fig. 7.2.4 Linearity (10 μg/L to 100 μg/L)

0 2 4 6 min
QSample Preparation
A simple method based on methanol deproteinization Fig. 7.2.6 Chromatograms of Milk
was examined as a food preparation method for rapid
screening analysis. Fig. 7.2.5 shows the sample preparation
procedure for analysis of milk and wheat our. mAU
QPeak
0.6 1. Melamine (Spiked)
Milk 0.5 g Wheat Flour 0.5 g
Add 2 mL Water
Fill up to 10 mL with Methanol
0.4
Shake Wheat Flour
1 Spiked at 2 mg/kg
&HQWULIXJHDWUSPIRUPLQDWÝ&
0.2
Supernatant Residue
Wheat Flour Blank
Dilute Five Times with Water
0
Filtrate with 0.2 ѥm Filter
0 2 4 6 min
HPLC
Fig. 7.2.5 Sample Preparation Fig. 7.2.7 Chromatograms of Wheat Flour
129
7.3 Analysis of Contaminant Adhering to Frozen Pizza (1)
- FTIR/EDX/EPMA
Q Explanation Table 7.3.1 Instruments and Analytical Conditions
The steady stream of consumer complaints related to Instruments : IRPrestige-21, AIM-8800
foods re ects continuing high concern for food safety. To Resolution : 4 cm-1
address these concerns and speci c contamination-related Accumulation : 45
complaints, it is important to quickly and accurately Apodization : Happ-Genzel
report the analysis results and clearly elucidate the Detector : MCT
contamination pathway. Here, using a Fourier transform
infrared spectrometer (FTIR) and an energy dispersive (2)
X-ray fluorescence spectrometer (EDX), in addition to (5)
an electron probe micro analyzer (EPMA), we present
(1)
the results of analysis of a contaminant adhering to the
surface of frozen pizza.
(3) (4)
Q Photograph of Contaminant Adhering to

Surface Frozen Pizza
T he phot og r aph i n Fig. 7.3.1 shows t he sit e of
contamination on the frozen pizza. It was discovered
Fig. 7.3.2 Microscope Photograph of Measurement Sites
when the package of this commercially available frozen
pizza was opened. The foreign substance was subjected
to complex analysis using a Fourier transform infrared Abs
spectrometer (FTIR), an energy dispersive X-ray
uorescence spectrometer (EDX), and an electron probe 1
micro analyzer (EPMA).
(1)
(2)
0.5
(3)
(4)
(5)
4000 3000 2000 1500 1000 1/cm
Fig. 7.3.3 Infrared Spectra of Contaminants
It is evident from Fig. 7.3.3 that the infrared spectra appear

differently depending on the location. Among the spectra
of Fig. 7.3.3, searches were conducted using the spectra (1),
Fig. 7.3.1 Photograph of Contaminant Adhering to Frozen Pizza (4) and (5). The results are shown in Fig. 7.3.4 - Fig. 7.3.6.
Abs
QAnalysis by FTIR 1
Some of the foreign substance was scraped off the frozen
pizza, and after rolling it onto the diamond cell, the
infrared spectrum was measured by transmission infrared
microscopy. Measurements were conducted at multiple 0.5 Contaminant 1
sites on the contaminant. Fig. 7.3.2 shows a photograph
indicating the measurement locations, and Fig. 7.3.3
shows the overlaid infrared spectra that were obtained at
Fluorine compound
the respective sites. Measurements were conducted using 0
4000 3000 2000 1500 1000
30 × 30 μm focal regions. The analytical conditions used 1/cm
are shown in Table 7.3.1.
Fig. 7.3.4 Search Result 1
130
7.3 Analysis of Contaminant Adhering to Frozen Pizza (2)

- FTIR/EDX/EPMA
Abs
Table 7.3.2 Quantitative Results for Contaminated Site by FP Method (%)
Fe Cr Ni Si Ca Al Ba Mn
1
59.42 12.07 8.97 8.91 4.69 3.55 1.72 0.79
From the results shown in Fig. 7.3.7 in which Fe, Cr and Ni

0.5
Contaminant 4
were detected in the contaminant as principal components,
and considering the quantitative results, the contaminant is
presumed to consist of stainless steel (SS). Further, since Al,
0
Starch Si, Ca and Ba were also detected, it is possible that a ceramic
4000 3000 2000 1500 1000 1/cm or other pigment-containing material may also be included.
Since Na, P, S, Cl, K and Ca were also detected at the un-
Fig. 7.3.5 Search Result 2 contaminated site, these are assumed to be of food origin. As
for the F that is derived from the uorine compound and was
Abs detected by FTIR, it was not clearly detected here, probably
1 due to the small relative mass within the analysis radius and
the overlap with the FeLǂ line.
0.75
0.5
QAnalysis by EPMA
Contaminant 5
After scraping off a portion of the contaminant and coating its
0.25 surface with gold, we conducted mapping of each element within
a micro-region measuring 400 × 400 μm. Fig. 7.3.8 shows the
Linseed oil
0 mapping results for the principal elements that were detected.
4000 3000 2000 1500 1000 1/cm The SS constituents Fe and Cr that were detected by EDX were
detected over a wide range. In addition, the F that was detected by
Fig. 7.3.6 Search Result 3 FTIR was detected in a localized perimeter. Thus, the results are
As can be seen from the respective search results, there were consistent with the obtained FTIR and EDX results.
search hits on a uorine compound, starch, and linseed oil. Of
these, the starch and linseed oil are expected components of the
pizza dough. The uorine compound is an industrial product,
and since it is also used in cooking utensils, it is apparently a
contaminant derived from an external source.
QAnalysis by EDX
Analysis of the intact contaminant adhering to the frozen pizza
was conducted using a 3 mm analysis diameter. Fig. 7.3.7 shows
the qualitative results comparing the normal and contaminated
parts of the frozen pizza. In addition, a comparative pro le of the
normal and contaminated sites of Fig. 7.3.7 was calculated, and Fig. 7.3.8 Results of Elements Mapping for Contaminant Part
quantitative analysis of the detected elements was conducted by
the FP method. The obtained results are shown in Table 7.3.2. QConclusion
Complex qualitative analysis using FTIR, EDX and EPMA
was conducted to identify a contaminant adhering to the
surface of frozen pizza, and the results indicated detection of
SS constituents and a uorine compound. EPMA mapping
results indicated that the SS constituents (Fe, Cr) were
scattered over a region of approximately 10 μm. On-site
- Normal Site
veri cation in the actual manufacturing process is essential
- Contaminated Site to identify the source of contamination, but from the
photograph of Fig. 7.3.1, the contaminant appears to be some
type of burnt substance. The above analysis suggested that
the contaminant consists of burnt cooking oil mixed with
a uorine compound originating from a cooking utensil or
Fig. 7.3.7 6C-92U Qualitative Results for Contaminated and Normal Sites manufacturing machine, in addition to SS powder.
131
7.4 Introduction of a Search System for Contaminants in
Tap Water (1) - FTIR/EDX
Q Explanation or absence of metallic luster, and measurement technique
Various contaminants, both organic and inorganic, can be present in pertaining to the contaminant materials are also recorded in
tap water. It is therefore necessary to identify not only contaminants the database. Furthermore, the elemental information obtained
that are organic, but also those that are inorganic in nature. Without from EDX analysis results is also stored in this database, thereby
information on both of these types of contaminants, adequate permitting details to be viewed using the EDX pro le database.
determination is not possible. Here, we present the determination of In addition to the qualitative and quantitative information, the
organic and inorganic contaminants in tap water using both infrared EDX pro le database also includes photographs of the samples.
spectroscopy (FTIR) for analysis of organic substances, and X-ray The procedure for identifying contaminants using the tap water
uorescence analysis (EDX) for inorganic elements. contaminant search system is shown in Fig. 7.4.1. This sequence
we present the analysis of these two types of contaminants in tap is used for analysis regardless of the category of the system
water and introduces how a tap water contaminant search system where the contaminant was discovered. Here, we used the ow
can be utilized for identi cation of the substances. chart scheme to analyze contaminants in tap water.
QContaminants Found in Tap Water QAnalysis of Organic Material

Contaminants due to foreign matter in tap water can originate First, observation of contaminant A that was detected in tap
from a variety of sources, including the tap water system materials water revealed a lusterless, black-colored material, suggesting
themselves, minerals, and microorganisms. In particular, major that it was an organic substance. In the case of organic
sources of contamination include rubber and metallic components contaminants, following the procedure for "Organic Samples"
from seals that have degraded due to aging of the water supply shown in Fig. 7.4.1, FTIR analysis was conducted first.
equipment. These types of contaminants should be quickly identi ed Contaminants found in tap water are typically visible, so it is
to allay the concerns of users. A combination of FTIR and EDX simple to conduct measurement using a single re ection ATR
analysis can effectively provide useful information for elucidating accessory. The analytical conditions are shown in Table 7.4.1,
the identity of a foreign substance. FTIR analysis can be used to and a photograph of the instrument used is shown in Fig. 7.4.2.
identify organic compounds and some inorganic compounds, while The search results obtained using the infrared spectrum
EDX analysis can be used to obtain qualitative and quantitative database are shown in Fig. 7.4.3. From this, it is clear that
information for such substances as iron rust and scale (calcium and components of the spectrum of the contaminant matches well to
magnesium deposits), which are dif cult to identify from infrared a "Discharge pipe packing" (principal component is SBS).
spectra. In addition, this approach is effective in narrowing down and Table 7.4.1 Instrument and
identifying the possible contaminant candidates when seals contain Analytical Conditions
the same principal components (organic substances) but different
additives. Another strong advantage is that the sample need only be Instrument : ,5$IÀQLW\
about 1 mm in size for analysis by either FTIR or EDX. MIRacle10 Ge,
Diamond
Organic Samples Inorganic Samples Resolution : 4.0 cm-1
EDX Analysis Accumulation: 40
When organic
Apodization : Happ-Genzel
information is required:
Fig. 7.4.2 Photograph of IRAf nity-1
FTIR Analysis Detector : DLATGS with MIRacle 10 ATR
Infrared Spectrum Database Accessory Attached
When inorganic
information is required:
Abs
EDX Analysis
EDX Profile Database
(1) (2) (3) (4)
Contaminant Identification Search Result
Fig. 7.4.1 Procedure of Contaminant Analysis by Tap Water

Contaminant Search System Contaminant A
QThe Features of Tap Water Contaminant

Search System 4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800
1/cm
The tap water contaminant search system comprises two

databases, an infrared spectrum database and an EDX pro le Details of infrared spectrum database search result:
database. The infrared spectrum database includes the infrared "Discharge pipe 32 packing_Gray_Inside"
spectra of contaminants collected from tap water, in addition Materials: Styrene butadiene styrene (SBS), Calcium carbonate
to the infrared spectra of commercially available tap water (CaCO3), Magnesium silicate (TALC, 3Mg4SiO2H2O)
system maintenance parts. Since searches are conducted Major elements: Cl, Ca, Si, Mg, S, Zn, Ti
using a database created from actual contaminants found in Color: Gray Shape: Resin/Ring-shaped Hardness: Soft
tap water, the search suitability rate is quite good. In addition Metallic luster: No Technique: ATR (Diamond)
to qualitative results, the color, shape, hardness, presence Fig. 7.4.3 Infrared Spectra and Search Results for Contaminant A
132
7.4 Introduction of a Search System for Contaminants in

Tap Water (2) - FTIR/EDX
Next, as indicated in procedure (2) of Fig. 7.4.1, EDX analysis was conducted to
obtain element information, and the "Discharge pipe packing" determined in the
FTIR analysis result was matched against the EDX pro le database. The pro le used
for matching is shown in Fig. 7.4.4. Primarily, the elements Ca, Si, and Mg were
clearly detected. These results clearly support the presence of calcium carbonate and
magnesium silicate (talc), as shown in details of infrared spectrum database search Fig. 7.4.5 Photograph of EDX-800HS Energy Dispersive X-Ray
result-Fig. 7.4.3. Here, when the main component is an organic substance (in this Fluorescence Spectrometer
case SBS) as identi ed in a search of the infrared spectra, the quantitative results
obtained from the EDX pro le database are calculated using the FP method.
Fig. 7.4.6 Qualitative and Quantitative Results for Contaminant B by EDX

Abs
Fig. 7.4.4 Qualitative and Quantitative Results for "Drain Fitting Seal" by EDX Search Result
QAnalysis of Inorganic Materials
Next, observation of contaminant B in tap water revealed a powder sample Contaminant B
with metallic luster, suggesting that contaminant B was an inorganic material.
Inorganic contaminants are rst analyzed by EDX, as indicted in the procedure
of Fig. 7.4.1 for Inorganic Samples. The analytical conditions are shown in 4000 3600 3200 2800 2400 2000 1800 1600 1400 1200 1000 800 1/cm
Table 7.4.2, and a photograph of the instrument used is shown in Fig. 7.4.5. The
EDX analysis results are shown in Fig. 7.4.6. The principal component of this Details of infrared spectrum database search result: "Iron rust_3"
contaminant was detected as Fe, and is assumed to consist of iron rust based on Materials: Iron (Ⅲ) oxide hydroxide (Fe(OH)3) Major elements: Fe, S, Si, P
the quantitative results. In addition, contaminant B was measured by the FTIR Color: Yellow Shape: Sand-shaped Hardness: Soft Metallic luster: No
single re ection ATR method, and the infrared spectrum database was used to Technique: ATR (Ge)
conduct a search, as outlined in procedure (3) of Fig. 7.4.1. The infrared spectrum Fig. 7.4.7 Infrared Spectra and Search Results for Contaminant B
search results are shown in Fig. 7.4.7. These results indicated a similarity of the
contaminant to iron rust. Also, this similarity is supported by the "Iron Rust_3" QConclusion
hit in the FTIR search result using the EDX pro le database. This analysis shows Here, we introduced a tap water contaminant search system using
that tap water contaminants can be identi ed more accurately using the EDX FTIR analysis and EDX analysis. Even when a commercially available
pro le database together with the infrared spectrum database. product is provided with information about materials that are included,
Table 7.4.2 Instrument and Analytical Conditions information related to additives is often not known, making it dif cult
Instrument : EDX-800HS to identify such materials as rubber and plastics. However, the infrared
X-Ray Tube : Rh target spectrum database prepared here includes qualitative information
: IRU&O related not only to principal components but also to additives, in addition
Filter
to elemental information detected by EDX. Also included is a sample
Tube Voltage : >N9@&6F>N9@7L8 database containing information collected from actual contaminants,
Tube Current : Auto as well as cross-section and surface measurements of seals, which
Atmosphere : Vacuum have a relatively high detection rate in tap water. Thus, this system
Measurement Diameter : 1 [mmφ] can be utilized as a contaminant analysis tool not only for tap water-
Measurement Time : >VHF@7L8>VHF@&6F>VHF@&O related utilities, but across a wide range of elds, such as environmental
Dead Time : Max 25 [%] (including contract analysis), petrochemical, and food elds.
133
,GHQWL¿FDWLRQRI&RQWDPLQDQWV$QLPDO+DLU- MCE
Q Explanation
If a contaminant is discovered during the process of
manufacturing foods, medicines, and cosmetics, etc., Specimen (animal hair, etc.)
it is essential to identify the contaminant not only to
clarify the cause or source of the contamination but for DNA extraction
establishing a standard operating procedure to prevent
the recurrence of such contamination as required in
hygiene control. Presumptive inspection of contaminants Purified DNA
is typically conducted by microscopic observation, but
since this requires specialized knowledge and experience, PCR
it is dif cult for inexperienced individuals to make a clear
assessment and to determine the source of contamination,
human or animal. Especially, with respect to animal hair, PCR product
a very common contaminant, it is extremely dif cult to
determine the type of contamination by visual inspection. Electrophoresis
Reliable methodology is therefore required to solve this MultiNA
problem. The Aichi Industrial Technology Institute Food
Research Center has developed a DNA testing method for Identification of animal type
identifying the type of animal associated with specific
hair contaminants(*). The animal species that can now
be detected reliably with this method include 6 types of Fig. 7.5.1 Experimental Procedure for Identi cation of Contaminants
farm animals (cow, pig, chicken, horse, sheep, and goat), 3 (Animal Hair) Using DNA Testing
types of pets (dog, cat, and rabbit), and 3 types of rodents
(sewer rat, black rat, and house mouse), for a total of 12
animal types. Here we introduce examples of analysis of QReagents/Kits
animal types by DNA testing using a combination of the * QIAGEN Fast Cycling PCR Kit
abovementioned method together with Shimadzu’s MCE- (Qiagen) 203743
202 MultiNA microchip electrophoresis system. * Primer Sets for identi cation of animals
(each 12 types) (Bex)
Q Experimental Procedure
* QIAamp DNA Micro Kit
The DNA extraction method and PCR conditions used
(Qiagen) 56304
were in accordance with the “Animal Hair DNA Testing
Protocol”(*) developed by the Aichi Industrial Technology * DNA-500 kit
Institute Food Research Center. The samples used for (Shimadzu Corp.) P/N: 292-27910-91
PCR consisted of DNA samples of cow, pig, chicken, * GelStar® Nucleic Acid Stain
horse, sheep, goat, dog, cat, rabbit, sewer rat, black rat, (Takara Bio) F0535
and house mouse that were kindly provided by the Aichi
* 25 bp DNA Ladder
Industrial Technology Institute Food Research Center.
(Invitrogen) 10597-011
In addition to these, DNA was also extracted from the
hair of 4 breeds of dog (Shiba, chihuahua, corgi, and
Afghan hound), a cat (Norwegian forest cat), and a rabbit
(Lionhead rabbit). Following PCR, the amplified DNA
was subjected to size analysis using the MultiNA. The
animal types were identi ed from the analysis results (See
procedure owchart, Fig. 7.5.1).
* Animal Hair DNA Testing Protocol

(Aichi Industrial Technology Institute Food Research Center)
http://www.pref.aichi.jp/cms les/contents/0000016/16149/
protocol0821.pdf
Patent Application Number: Special Application 2007-240023
Invention Name: Animal Identi cation Primer Set and Primer Kit
The Primer Set is manufactured and marketed by Bex Co., Ltd.
with the permission of Aichi Prefecture.
http://www.bexnet.co.jp
134
,GHQWL¿FDWLRQRI&RQWDPLQDQWV$QLPDO+DLU- MCE
QResults
Fig. 7.5.2 shows the analysis results for the PCR products results of 1 to 12 (Fig. 7.5.2). In addition, with the
of the 12 animal types (cow, pig, chicken, horse, sheep, goat, electrophoresis results of a to f (Fig. 7.5.2), DNAs of lengths
dog, cat, rabbit, sewer rat, black rat, and house mouse) and which can be used to identify the animal types were also
dog breeds (Shiba, chihuahua, corgi, and Afghan hound), detected from the respective hair of dog (Shiba, chihuahua,
cat (Norwegian forest cat), and rabbit (Lionhead rabbit). corgi, and Afghan hound), rabbit (Lionhead rabbit), and cat
Characteristic lengths of PCR products (cow (137 bp), pig (Norwegian forest cat). By combining the use of the Primer
(230 bp), chicken (159 bp), horse (183 bp), sheep (224 bp), goat Sets and the MultiNA microchip electrophoresis system as
(160 bp), rabbit (167 bp), dog (122 bp), cat (220 bp), sewer rat described in this analysis example, contaminants can be
(237 bp), black rat (102 bp), and house mouse (116 bp)) were identified from minute quantities of biological substances
ampli ed for the 12 animal types, respectively. The DNA of such as hair, etc., often discovered in manufacturing
the 12 types of animals was detected from the electrophoresis processes and products.
L 1 2 3 4 5 6 7 8 9 10 11 12 L a b c d e f
L : 25 bp DNA Ladder L : 25 bp DNA Ladder

1 : cow a : dog (chihuahua)
2 : pig b : dog (corgi)
3 : chicken c : dog (Afghan hound)
4 : horse d : dog (Shiba)
5 : sheep e : rabbit (Lionhead)
6 : goat f : cat (Norwegian forest cat)
7 : rabbit
8 : dog
9 : cat
10 : sewer rat
11 : black rat
12 : house mouse
Fig. 7.5.2 Analytical Results of PCR Products
135
7.6 Analysis of Odor in Frozen Shrimp (1) - GC/MS
Q Explanation
The smell of food is an important characteristic related to Packing material
its taste, aroma, and freshness. The headspace technique,
in which the gas released during heating of the sample is
analyzed, has typically been used in the aroma analysis Collection tube
CarbotrapB+Carboxen1000
of foods. The headspace technique usually relies on
heating of the sample to transfer the target substances
to the vapor phase. However, in the case of perishable Nitrogen gas
N2 100mL/min
foods, there is always a danger that heating will cause
Collection time: 5min
sample degeneration. Although the headspace technique
Collection volume: 500mL
can be conducted using a lower heating temperature, this
approach would normally result in reduced sensitivity. An
alternate approach when analyzing samples which could Fig. 7.6.1 Schematic Diagram of Sampling Method
be affected by thermal degradation involves concentrating
the volatilized gas at or near room temperature using
the thermal desorption technique. Here we introduce
an example of analysis of trace quantities of substances Q2. Sample Introduction
volatilized from frozen shrimp at room temperature. Analysis of the volatile odor substances of frozen shrimp
was conducted using the thermal desorption technique.
QAnalysis Outline The thermal desorption technique uses a collection tube
1. Sampling which is connected to the instrument; the collection
Volatile substances were collected. tube, containing volatile substances collected from the
2. Sample introduction sample, is thermally desorbed and introduced into the
Volatile substances were introduced into GC/MS. GC/MS. Since the sample width (bandwidth) of the
3. Data analysis volatile substances widens at the time of desorption from
3.1 Qualitative analysis the collection tube, typically, a secondary trap tube is
Volatile substances were identi ed from their mass installed and the volatile substances are cooled and re-
spectra.
concentrated to narrow the bandwidth, thus achieving
3.2 Quantitative analysis of the generated gas
For each substance identified, a calibration curve higher chromatographic resolution. The analytical
was generated using standard samples of known conditions are shown on next page. Sample introduction
concentration. The volatilized compounds in when using thermal desorption is as illustrated in the
the shrimp samples were calculated using the schematic diagram of Fig. 7.6.2.
calibration curves.
Q1. Sampling Method

The frozen sample (5 g) was enclosed in a container
and set aside for 30 min to defrost at room temperature. Collection tube Secondary trap tube Column
Nitrogen gas was then passed through the container

at 100 mL/min. A total of 500 mL of the expelled gas Heat Cooling during trap
was passed through the collection tube containing Heating during desorption
adsorbent packing for trapping the target substances,
thereby concentrating the volatile odor substances. Since
this analysis targets trimethylamine, a collection tube
packed with CarbotrapB+Carboxen1000 (SHIMADZU Fig. 7.6.2 Schematic Diagram of Thermal Desorption
PN 223-57474-91) was used. TENAX-TA, which is a
frequently used packing for thermal desorption analysis,
was not used in this case due to its weak retention of
trimethylamine. Weak retention is cause for concern due
to the possible breakthrough and loss of the analyte. The
type of collection tube must be selected according to the
chemical properties of the analytes.
136
7.6 Analysis of Odor in Frozen Shrimp (2) - GC/MS
Trimethylamine
TIC
750000
11
4
500000
1
250000
10
2
9
876
3
12
13
0
5.0 10.0 15.0 20.0 25.0 30.0
Fig. 7.6.3 Total Ion Current Chromatogram of Frozen Shrimp

Q3. Data Analysis Table 7.6.1 Results of Similarity Search
3.1 Qualitative Analysis Peak compound name
The total ion current chromatogram obtained from analysis of 1 Trimethylamine
the frozen shrimp is shown in Fig. 7.6.3. The principle peak was 2 &DUERQGLVXOÀGH&62)
identi ed by conducting a similarity search with the NIST08 3 'LPHWK\OVXOÀGH
Mass Spectral Library. The identification results are shown 4 Acetone
5 Tetrahydrofuran (THF)
in Table 7.6.1. Trimethylamine, carbon disul de and dimethyl 6 Isopropyl alcohol
sul de, etc. were con rmed. 7 Chloromethoxymethane
8 Ethanol
QAnalytical Conditions 9 Acetonitrile
-TD- 10 Chloroform
11 Toluene
Instrument : TD-20 12 1-Butanol
Desorp Temp. : Ý& 13 Pyridine
Desorp Flowrate : 60 mL/min
Desorb Time : 10 min 3.2 Quantitative Analysis of Generated Gas
1st Trap Tube : CarbotrapB+Carboxen1000 Quantitative analysis was conducted to determine the amount
6+,0$'=831 of generated gas associated with the detected peak number 1,
2nd Trap : TENAX-TA trimethylamine (retention time at approximately 3 minutes). A
6+,0$'=831
trimethylamine standard substance was diluted with methanol,
Trap Low Temp. : Ý&
Trap High Temp. : Ý& and quantities corresponding to 0.1, 0.3 and 1 μg were added
Valve Temp. : Ý& to the collection tube. Analyses were then conducted using
Line Temp. : Ý& the same procedure as with the actual sample. Fig. 7.6.4 shows
IF Temp. : Ý& the calibration curve generated using the area values of the
mass chromatogram of m/z 58. For the quantitative calculation
Instrument : GCMS-QP2010 Plus results, quantities added to the collection trap were assumed to
-GC- correspond to the collected headspace volume of 500 mL.
Column : RESTEK Stabilwax
PðPP,'GI ƫP Area
Column Temp. : Ý&PLQ²Ý&PLQ²Ý&PLQ (×1,000,000) 1.25
Carrier Gas : +H&RQVWDQW/LQHDU9HORFLW\0RGH
Linear Velocity : 36 cm/s 1.00
Injection Method : Split
0.75
Split Ratio : 1:15
-MS- 0.50
Interface Temp. : Ý& 0.25
Ion Box Temp. : Ý&
Ionization Method : EI 0.00
Scan Range : m/z 35 - 550 0 0.5 1
Scan Interval : 0.5 s Amount Generated (μg)
Fig. 7.6.4 Calibration Curve of Trimethylamine (m/z 58)
137
8. Food Properties
8.1 Melting of Chocolate - TA

The melting process of various edible fats and oils is Instrument : DSC-60
measured by DSC. Six types of crystals exist in cocoa Sample : Chocolate
oil, a component of chocolate. Of these, V type crystal Sample Amount : 22.87 mg
is said to possess good thermal stability. Since V type Atmospheric Gas : Nitrogen
crystal melts at about 34 °C, DSC measurement can be Gas Flowrate : 30 mL/min
used to know the condition of V type crystal contained [Temperature Program]
in a particular sample of chocolate. Fig. 8.1.1 shows a Heating Rate : 3 °C/min
DSC curve of the chocolate sample heated at 3 °C/min.
Fig. 8.1.2 shows a DSC curve of the same chocolate
sample, reheated after cooling the melted sample to -50 °C
to harden it. It is evident that the peak at 30.4 °C has
completely disappeared.
'6&
mW
0.00 –36.25 J/g
3.39 Ý&
13.99 Ý&
– 2.00
– 4.00
30.44 Ý&
– 6.00
0.00 50.00
7HPS>Ý&@
Fig. 8.1.1 Chocolate Measurement (1st time)
'6&
mW
2.00
–23.45 J/g
0.00
–11.71 Ý&
7.92 Ý&
– 2.00
16.30 Ý&
19.60 Ý&
– 4.00
– 6.00
0.00 50.00
7HPS>Ý&@
Fig. 8.1.2 Chocolate Measurement (2nd time)
138
Food Properties
8.2 Gelatinization of Starch - TA

Starches gelatinize when heated with water. The Instrument : DSC-60
gelatinization reaction can be analyzed by DSC because Sample : Flour
it is accompanied by endothermic reaction. Here we Sample Amount : 4.21 mg
conducted measurement of our starch (17.4 %). Atmospheric Gas : Nitrogen
Gas Flowrate : 30 mL/min
[Temperature Program]
Heating Rate : 5 °C/min
DSC
mW
1.00
0.00
Ý&
–1.00
20.00 40.00 60.00 80.00 100.00

7HPS>Ý&@
Fig. 8.2.1 Gelatinization Temperature of Flour (17.4 %)

Here we conducted measurement of corn starch (19.9 %). Instrument : DSC-60
It is known that when sucrose and salt are added to starch, Sample : Corn
the gelatinization temperature changes. Sample Amount : 4.97 mg
Atmospheric Gas : Nitrogen
Heating Rate 5 °C/min
DSC
mW
1.00
0.00
68.90 ÝC
–1.00
40.00 60.00 80.00
7HPS>Ý&@
Fig. 8.2.2 Gelatinization Temperature of Corn (19.9 %)
139
8.3 DSC Measurement of Liquor - TA

Whiskey was sealed in a hermetic cell and measured Instrument : DSC-60
by DSC. The exothermic peak at -74.2 °C is due to Sample Name : Whiskey
crystallization. The peak at -65.9 °C shows the melting Sample Weight : 13.7 mg
of ethanol, -49.8 °C the eutectic point of ethanol and Atmospheric Gas : Nitrogen
water, and -25.6 °C the melting of water. The height of the Gas Flowrate : 30 mL/min
eutectic peak varies depending on the storage period of [Temperature Program]
the malt. Heating Rate : 10 °C/min
'6&
mW
2.00
²Ý&
0.00
– 2.00
²Ý&
– 4.00
²Ý&
²Ý&
– 6.00
–100.00 – 50.00 0.00
7HPS>Ý&@
Fig. 8.3.1 DSC Curve for Whiskey

Brandy was sealed in a hermetic cell and measured by Instrument : DSC-60
DSC. Sample Name : Brandy
Sample Weight : 9.11 mg
Atmospheric Gas : Nitrogen
Heating Rate : 10 °C/min
'6&
mW
0.00 ²Ý&
–88.44 Ý&
²Ý&
–5.00
–10.00
²Ý&
–100.00 0.00
7HPS>Ý&@
Fig. 8.3.2 DSC Curve for Brandy

140
Food Properties
8.4 DSC Measurement of Fish Meat - TA

DSC analyzed the freshness of carp meat. With the most fresh Instrument : DSC-60
carp meat, an exothermic peak at 42.8 °C and endothermic Sample Name : Carp Meat
peaks at 56.3 °C and 75.8 °C were observed (Fig. 8.4.1). With Sample Weight (Fig. 8.4.1) : 25.4 mg
carp meat after 6 hours, a minute exothermic peak at 37.0 °C (Fig. 8.4.2) : 24.5 mg
and endothermic peaks at 54.3 °C and 75.7 °C were observed (Fig. 8.4.3) : 26.5 mg
(Fig. 8.4.2). With carp meat after 24 hours, endothermic Atmospheric Gas : Nitrogen
peaks at 42.3 °C, 55.1 °C and 73.1 °C were observed but no Gas Flowrate : 30 mL/min
exothermic peak was observed (Fig. 8.4.3). The endothermic [Temperature Program]
peaks at 42 °C and around 73 °C are due to the denaturation Heating Rate : 5 °C/min
of myosin and actin, respectively. The exothermic peak at
around 40 °C corresponds to the shrinkage of myosin and
actin caused by ATP remaining in the sh meat. The amount
of ATP remaining varies with the storage period.
'6& '6&
mW mW
1.00 1.00
Ý&
0.00 0.00
42.28 Ý&
Ý& Ý& Ý& Ý&
–1.00 –1.00
50.00 100.00 50.00 100.00

7HPS>Ý&@ 7HPS>Ý&@
Fig. 8.4.1 DSC curve for Fresh Fish Meat Fig. 8.4.3 DSC Curve for Fish Meat after 24 Hours
'6&
mW
1.00
36.97 Ý&
0.00
Ý& Ý&
–1.00
50.00 100.00
7HPS>Ý&@
Fig. 8.4.2 DSC Curve for Fish Meat after 6 Hours
141
8.5 Protein Denaturation and Texture Analysis for Chicken (1) - TA
Q Explanation QProtein Denaturation at Different Heating Times

The deliciousness of food is greatly in uenced by taste After confirming that the temperature at the center of
due to chemical interactions with the tongue for the basic frying chicken breasts was 70 °C, the chicken breasts
tastes of sweetness, saltiness, acidity, bitterness, and were kept warm for 0 min, 1 hour, 2 hours, and 4
umami (also referred to as savoriness), in addition to the hours, respectively, at which times the chicken breasts
physical sensations of hardness and softness and juiciness were immediately frozen by shock freezing. Before
sensed by the teeth. In particular, texture, which includes conducting DSC measurement of the frozen samples,
the sensations of juiciness and softness, is an important they was defrosted and placed in an aluminum sealed
factor related to the deliciousness of fried foods, such as cell, and heated at a rate of 10 °C/min up to 100 °C. An
fried chicken. Here we conducted an overall evaluation endothermic peak due to denaturation of the protein actin
of texture in fried chicken heated over different time is seen within one hour of heat retention. This means
periods. DSC was to measure protein denaturation, and a that un-denatured protein still remains. On the other
precision universal testing machine was used to measure hand, when heat retention continues beyond 2 hours,
the hardness and elasticity. no endothermic peaks are seen, and the softness is lost.
Thus, this correlates to the experience that the longer heat
Q Protein Denaturation of Chicken retention continues, the dryer the texture becomes.
Chicken breast seasoned with spices was placed in an
aluminum sealed cell, and heated at rate of 10 °C/min.
Endothermic peaks were seen at 57 °C, 64 °C and 78 °C.
It is presumed that the endothermic peaks correspond
to denaturation of myocin at 57 °C, connective tissue at
64 °C, and actin at 78 °C.
'6& DSC
mW mW
1.00
No heat retention
2.00 Ý&
Unheated Heat retained 1 hour Ý&
0.50
Ý& Ý& Ý& Heat retained 2 hours
1.00
Heat retained 4 hours
0.00
20.00 40.00 60.00 80.00 100.00 40.00 60.00 80.00 100.00
7HPS>Ý&@ 7HPS>Ý&@
Fig. 8.5.1 DSC Curve of Chicken Fig. 8.5.2 DSC Curves of Fried Chicken Subjected to Different
Heat Retention Times
142
Food Properties
8.5 Protein Denaturation and Texture Analysis for Chicken (2) - TM
QHardness and Elasticity Due to Different 45

Heat Retention Time 40
Next, the chicken breast ber was evaluated for hardness 35
by orthogonal shearing, and for elasticity. Table 8.5.1 30
4 hours
shows the testing conditions, and Fig. 8.5.4 shows the
Force (N)
25
2 hours
force and time graph. Fig. 8.5.6 shows the definition of 20
parameters used to indicate texture. Hardness is indicated 15
1 hour
by the maximum force first applied, and elasticity is 10
0 min
indicated as a ratio of the recovery times after applying 5
the load twice. Table 8.5.2 shows the hardness and 0
elasticity calculated based on these test results. It is -5
0 0.4 0.8 1.2 1.6 2 2.4 2.8 3.2 3.5
clear that while the hard-ness rose marginally up to a Time (sec)
heat retention time of 1 hour, the specimen suddenly
becomes harder after heat retention exceeds 2 hours. This Fig. 8.5.4 Relation between Force and Time
also correlates to the DSC measurement result in which
protein denaturation is seen within one hour.
Fig. 8.5.5 Starting Status of Shear Test
H : Hardness A2
A1
: Cohesiveness
B : Brittleness T2
: Elasticity
T1
C : Adhesive force T2
H × T1 : Gumminess
A3 : Adherence
A2 T2
Force T1 : Dent H × A1 × T1 : Chewiness
Fig. 8.5.3 Photograph of AGS-X System
Table 8.5.1 Testing Conditions
Testing Instrument AGS-X

Load Cell 1 kN
Stress
Jig Tooth-shaped push rod Time

Test Speed 750 mm/min
Clearance 5 mm (shear indentation amount)
Number of Load Cycles 2 cycles
Fig. 8.5.6 Texture-Related Parameters
Software TRAPEZIUMX (Texture)
Sample Dimensions About 20 × 20 × 10 mm
Table 8.5.2 Test Results
Heat Retention Time Hardness (N) Elasticity

0 min 8.87 1.33
1 hour 11.50 2.05
2 hours 16.83 2.98
4 hours 32.77 5.72
143
8.6 Texture Analysis of "Soumen" Japanese Vermicelli (1) - TM
Q Explanation QTensile Test

The evaluation of the mechanical properties of foods, The specimens were grasped in grips (sponge was
such as strength and hardness, is becoming widely used attached to the grip faces to prevent destruction of the
for the numerical comparison and control of food texture. specimens), and the grips were mounted in the tester
Here we introduce the tensile test and cutting test of through universal joints. Tensile test was performed under
Japanese soumen vermicelli to evaluate the texture. the following conditions:
Soumen vermicelli is originated in Nara Prefecture in 1) Force measurement Load cell (1 N)
Japan. It was made by hand by kneading wheat, salt, and 2) Extension measurement Internal extensometer in tester
water; applying food oil and starch; and then stretching, 3) Test speed 50 mm/min
drying, and maturing. Nowadays, it is generally machine-
made. According to JAS (Japan Agricultural Standards) Fig. 8.6.2 and Fig. 8.6.3 show an overview of the grips and
standards, noodles less than 1.3 mm diameter are a specimen mounted in the tester.
"soumen," those from 1.3 mm to 1.7 mm diameter are
"hiyamugi," and larger noodles are classi ed as "udon."
QTesting Equipment and Specimens

Two types of commercially available soumen (Sample
A, Sample B) were used as specimens for these tests.
However, the diameters varied between 0.8 mm and Grip face
(with sponge)
1.3 mm due to the degree of drying (the state in which
it was sold). Therefore, the noodle diameters were
measured with Vernier calipers to select specimens of
approximately the same diameter. The specimens were
added to boiling water and boiled for three minutes and Specimen
then washed in cold water for ten seconds. Ten specimens
each of Sample A and Sample B were tested within
five minutes. Testing was performed using a Shimadzu Fig. 8.6.2 Grips for Tensile Test
EZ Test tabletop tester (Fig. 8.6.1).
Upper grip
Specimen
Lower grip
Fig. 8.6.1 EZ Test Tabletop Tester Fig. 8.6.3 Overview of Tensile Test
144
Food Properties
8.6 Texture Analysis of "Soumen" Japanese Vermicelli (2) - TM
Fig. 8.6.4 shows the tensile test results as force-displacement (extension) curves. (Curves are superimposed for all ten
specimens.) The results indicate a maximum force of 164 mN and break displacement of 56 mm for Sample A and
120 mN maximum test force and break displacement of 37.8 mm for Sample B. Sample A exhibits greater extension and
strength than Sample B. (All values are averages of ten samples.)
Sample A Sample B
Force (mN)
Force (mN)
Displacement (mm) Displacement (mm)
Fig. 8.6.4 Tensile Test Results
QCutting Test
Cutting test can be performed as an evaluation method
that approximates biting through foods. For cutting test,
the specimen is placed on the compression plate and the
cutting test jig (tooth-shape press: R 0.2 mm knife-edge
Cutting test jig
tip) is pressed down on the sample from above. The test (tooth-shape press)
conditions are as follows:
1) Force measurement Load cell (1 N) Specimen
2) Indentation measurement Internal extensomete in tester
3) Test speed 5 mm/min
Compression plate
Fig. 8.6.5 shows a specimen mounted in the tester. Fig. 8.6.5 Overview of Cutting Test
Sample A Sample B
Force (mN)
Force (mN)
Displacement (mm) Displacement (mm)
Fig. 8.6.6 Cutting Test Results

Fig. 8.6.6 shows the cutting test results as force-displacement (indentation) curves. (Curves are superimposed for all ten
specimens.) The results indicate a maximum force of 207 mN for Sample A and 129 mN maximum test force for Sample B. (All
values are averages of ten samples.) The tensile and cutting test results above provide a numerical evaluation that Sample A has a
rmer texture than Sample B. The results con rm that the materials tester is effective for quanti cation for functional testing.
145
8.7 Measurement of Texture Characteristics of Rice - TM
Q Explanation Table 8.7.3 Test Conditions

The EZ Test Shimadzu Texture Analyzer is used for Test Speed 50 mm/min
various purposes such as numerical conversion of texture Pressing Amount 15 mm
characteristics of foods such as chewiness, firmness, Temperature 28 °C
and palatability; quality evaluation based on the change Humidity 60 %
of hardness; and strength evaluation of food packaging.
The Analyzer is usable for such a wide range of purposes
because a wide variety of jigs can be selectively used
according to the type of test being performed. We
conducted texture characterization of rice using the
Texture Analyzer.
QSamples and Testing Machines

Three types of rice; blended rice, Koshihikari (one of
Fig. 8.7.2 Test Piece Placed
the most popular brands of rice), and rice with barley
were used as samples in the measurement. Fig. 8.7.1 QTest Results
shows the appearance of the EZ Test Shimadzu Texture Table 8.7.4 shows "Summary of Test Results (Average Values)"
Analyzer used in the measurement. The analyzer has an and Fig. 8.7.3 shows a "Force-Time" measurement example.
exceptionally operable, compact frame and is perfect for
texture characterization of foods. Table 8.7.1 shows the Table 8.7.4 Summary of Test Results (Average Values)
process to make test pieces and Table 8.7.2 shows the Cohesion Cohesiveness
con guration of the system used for the measurement. Sample Hardness [N]
Strength [N] [Nmm]
Blended Rice 7.86 0.54 0.967
Koshihikari 16.3 1.09 2.78
Rice with Barley 7.67 0.33 0.603
Hardness : The maximum force loaded when compressed
Cohesion Strength : The maximum force required to pull off the jig
after compression
Cohesiveness : A value calculated by multiplying the force
required to pull off the jig after compression by
the distance
20
15
Fig. 8.7.1 Appearance of Texture Analyzer 10
Blended Rice
N Koshihikari
Rice with Barley
Table 8.7.1 Test Piece Making Process 5
(1) Rice was cooked and left as is for one hour. 0

(2) Rice was made into 10 g units. 00 10 20 30 40 50 60
-5
Each unit of rice was then formed with a mold into a 20 mm SEC
(3)
high cylindrical form with a diameter of 25 mm.
Fig. 8.7.3 Force-Time Graph
Table 8.7.2 System Con guration QSummary
Main Unit EZ Test Koshihikari showed over twice as high values as compared
Load Cell Capacity 100 N to the other two types of rice for all items; hardness,
Jig 50 mm dia. compression plate cohesiveness, and cohesion strength. Cohesiveness is
Software TRAPEZIUMX Texture believed to closely indicate the sensation people fell when
they actually eat rice. The test result for cohesiveness clearly
indicates Koshihikari's characteristics. The use of the EZ Test
QTest Conditions Texture Analyzer is recommended for evaluating food
Table 8.7.3 shows test conditions. Fig. 8.7.2 shows how a texture because it provides easier texture characterization as
test piece is placed. compared to sensory evaluations and is highly operable.
146
Food Properties
8.8 Measurement of Texture of Pork - TM
Q Explanation 1.2
Nowadays people like tender meat more than chewy meat. 1.05
Therefore, various measures are taken to make pork tender, for
0.9
example by soaking it in water or in protein degrading enzyme
solution to quickly achieve an adequate level of tenderness.
Test force (N)

0.75
Two cuts of store-bought Boston pork butt were soaked for 20 0.6
hours, one in water and the other in a protein degrading enzyme
solution. A penetrating strength test was then performed on 0.45
them and their textures were converted into numerical values for 0.3
comparison. Fig. 8.8.1 is the penetrating test force-penetrating
0.15
depth curve of store-bought Boston pork butt. Fig. 8.8.2 shows
how the penetrating strength test was performed. 0
0 2 4 6 8 10 12 14 16 18 20
Stroke (mm)
5 Fig. 8.8.3 Curve of Pork Soaked in Water for 20 Hours
4.5
1.2
4
1.05
3.5
Test force (N)
3 0.9
2.5 0.75
Test force (N)
2 0.6
1.5 0.45
1
0.3
0.5
0.15
0
0 2 4 6 8 10 12 14 16 18 20
0
Stroke (mm) 0 2 4 6 8 10 12 14 16 18 20
Stroke (mm)
Fig. 8.8.1 Penetrating Test Force-Penetrating Depth Curve on
Store-Bought Boston Pork Butt Fig. 8.8.4 Curve of Pork Soaked in Enzyme Solution for 20 Hours
In these tests, the strength was measured at the point where a
5 mm diameter penetration elasticity jig penetrated a 20 mm
thick cut of Boston Pork Butt to a depth of 10 mm at a speed of
100 mm/min. When the results shown in Fig. 8.8.3 and Fig. 8.8.4
are compared, the jig went deeper into the pork soaked in
enzyme solution for 20 hours as compared to the pork soaked in
water for 20 hours when the same test force was used, indicating
the former is more tender. When Fig. 8.8.1 and Fig. 8.8.3 are
compared, approximately four times the strength is necessary to
penetrate store-bought pork to the same depth as pork soaked in
water for 20 hours, clearly indicating the difference in hardness.
As shown in these gures, the texture of pork was converted
into numerical values from the pork penetrating test strength-
penetrating depth curve. Table 8.8.1 below shows the maximum
penetrating test strength of these three tests.
Table 8.8.1
Fig. 8.8.2 Penetrating Strength Test Being Performed on Pork Average Value of
Sample
Maximum Test Force N
The EZ Test Shimadzu Table-Top Universal Tester was Store-Bought Boston Pork Butt 3.90
used in the test. Fig. 8.8.3 is the penetrating test force- Boston Pork Butt Soaked in Water
0.98
penetrating depth curve of pork soaked in water for 20 for 20 hours
hours. Fig. 8.8.4 shows the results of soaking meat in a Boston Pork Butt Soaked in Enzyme
0.49
protein degrading enzyme solution for 20 hours. Solution for 20 hours
147
8.9 Texture Evaluation of Care Food (1) - TM
Q Explanation QTest results

The development of care food, which is processed to be The results obtained from testing 3 types of food products
soft and easy to eat, is steadily progressing to the point are shown in the force-time graphs of Fig. 8.9.2 - Fig. 8.9.4.
that the shape, color and taste are nearly indistinguishable
from ordinary food, except that the texture is softened to
enable crushing using just the gums and tongue. These 1.0
commercially available products are a great boon to the 0.8
elderly and individuals with internal mouth injuries who
may have inadequate strength for chewing and drinking, 0.6
and are helping these disadvantaged groups to better
Force (N)
0.4
enjoy the pleasures of tasting and eating. Meanwhile,
according to the Health Promotion Law in Japan, 0.2
the Japan Consumer Affairs Agency is authorized to 0.0
conduct reviews of the suitability of "foods for people
with dysphagia" as regulated special purpose foods, and -0.2
based on these reviews, permit the display of such foods. -0.4
Here, we conducted measurements of commercially 0 1 2 3 4 5
available care food based on the test methods specified Time (sec)
for reviewing the suitability of foods for people with
dysphagia. We then classified the food according to Fig. 8.9.2 Food A (vegetable soup)
the specific standard based on the obtained hardness,
adhesiveness, and cohesiveness data.
1.0
QTesting Equipment and Specimens 0.8
The inst r ument used for this evaluation was the
Shimadzu EZ Test Texture Analyzer, with a 5 N test 0.6
force measurement load cell. The samples used consisted
Force (N)
0.4
of 3 commercially available care food products (sample
names: A - C). 0.2
0.0
QTest Conditions
An overview of the measurement setup is shown in -0.2
Fig. 8.9.1. The sample diameter was 40 mm, and the -0.4
sample was loaded to a height of 15 mm in the 20 mm 0 1 2 3 4 5
high sample container. Using a plastic plunger 20 mm Time (sec)
in diameter and 8 mm in height, compression testing
was conducted twice at a speed of 10 mm/sec with a Fig. 8.9.3 Food B (rice gruel)
clearance of 5 mm. The test was conducted using a sample
temperature of 20 °C. 1.0
0.8
0.6
Force (N)
0.4
0.2
0.0
-0.2
-0.4
0 1 2 3 4 5
Time (sec)
Fig. 8.9.1 Overview of Texture Test Fig. 8.9.4 Food C (sukiyaki)
148
Food Properties
8.9 Texture Evaluation of Care Food (2) - TM
The results obtained from the series of measurements are shown in Table 8.9.1.
Table 8.9.1
Hardness Adhesiveness Cohesiveness
Permissible Standard
Sample Measured Value Measured Value
Judgment Judgment Measured Value Judgment (Overall Judgment)
(N/m2) (J/m3)
Food A 0.84 × 103 Ⅰ, Ⅱ, Ⅲ 0.19 × 103 Ⅰ, Ⅱ, Ⅲ 0.82 Ⅰ, Ⅱ Ⅲ
Food B 1.97 × 103 Ⅰ, Ⅱ, Ⅲ 0.41 × 103 Ⅰ, Ⅱ, Ⅲ 0.76 Ⅰ, Ⅱ Ⅲ
Food C 1.88 × 103 Ⅰ, Ⅱ, Ⅲ 0.08 × 103 Ⅰ, Ⅱ, Ⅲ 0.52 Ⅰ, Ⅱ Ⅲ
The measured values for hardness, adhesiveness and would be presumed to be Ⅱ. However, the permissible
cohesiveness that are within the range of the permissible standard becomesⅢ in the nal evaluation, because food
standard are indicated with a □ that surrounds the B and food C contain non-homogeneous ingredients.
standards in the judgment eld. Food A does not satisfy Thus, by de ning the numerical value and comparing it
the hardness standards Ⅰ and Ⅱ, so the permissible with the judgment standards, the judgment can be made as
standard was designated as Ⅲ. In the case of foods B and to which permissible standard it corresponds.
C, item Ⅰ is not satisfied, so the permissible standard
[Reference]
(Permissible Standards of Foods for People with Dysphagia Note 1))
Note 1) Regarding permission to display special usage foods (Excerpted from Ministry of Health, Labour and Welfare
Food Safety Noti cation No. 0212001)
Standard*1 Permissible StandardⅠ*2 Permissible StandardⅡ *3 Permissible Standard Ⅲ *4

Hardness
(Resistance during compression at 2.5 × 103 - 1 × 104 1 × 103 - 1.5 × 104 3 × 102 - 2 × 104
constant rate) (N/m2)
Adhesiveness (J/m3) 4 × 102 or less 1 × 103 or less 1.5 × 103 or less
Cohesiveness 0.2 - 0.6 0.2 - 0.9 -
*1 Within the permissible range of the standard under conditions of either of ambient temperature or the normal temperature at which eating
takes place.
*2 Homogeneous items (for example, jellied foods)
*3 Homogeneous items (for example, jellied or smooth foods). However, this excludes foods that satisfy permissible standardⅠ.
*4 Includes non-homogeneous foods (for example, rice gruel which is easily clumped or collected, soft paste-like or jellied foods).
However, this excludes foods that satisfy permissible standardⅠor permissible standardⅡ.
149
8.10 Particle Size Distribution Measurement of Chocolate (1) - PT
Q Explanation
There are several possible parameters for expressing particle size analyzer to measure the size distribution of
the taste of a piece of chocolate. For example, it can particles in chocolate. Chocolate is a mixture of cocoa
be described in terms of taste characteristics such mass, which consists of ground cocoa beans mixed with
as sweetness or bitterness, but it is also possible to milk, sugar, cocoa butter, and other ingredients. This can
characterize chocolate in terms of its texture in the be considered as a mixture of various particles in fat.
mouth, for instance, how readily it melts in the mouth. Consequently, the particle size distribution presumably
Included in such parameters is "tongue texture," for varies depending on the dispersion conditions. In this
which particle size distribution provides a potential index example, isopropanol at about 45 °C was used as the
for expressing this parameter in numeric terms. Here dispersant.
we introduce the use of a SALD-2300 laser diffraction
Fig. 8.10.1 SALD-2300 Laser Diffraction Particle Size Analyzer
Funnel Dispersant : ,VRSURSDQRO&
Dispersing Agent : None
Dispersing Method : Stirred using a magnetic stirrer
Refractive Index : 1.70 - 0.05i
Laser Light Batch Cell
Stroke of
Vertical Motion
Stirring Plate
Fig. 8.10.2 Batch Cell
150
Food Properties
8.10 Particle Size Distribution Measurement of Chocolate (2) - PT
QTest Samples and Results

Three kinds of chocolate were prepared as samples. results indicate that C contains larger particles than B
Sample A was milk chocolate, B was milk chocolate and B contains larger particles than A. When the samples
from a different manufacturer, and C was chocolate were actually eaten, the mouth texture of Sample A was
removed from a chocolate chip cookie. Each sample was extremely smooth, B was slightly less smooth than A, and
cut into thin pieces with a box cutter knife and placed in C felt clearly grainy on the tongue. Put simply, the results
a 50 mL beaker. Then 45 °C isopropanol was added to indicate that an adequately smooth mouth texture can
dissolve (disperse) the samples into a suspension, which be achieved if all the particles are smaller than 50 μm in
was stirred for about 2 minutes with a magnetic stirrer. diameter. In contrast, chocolate containing particles with
This suspension was used as the stock solution. Part of diameters larger than 100 μm clearly imparts a grainy
this stock solution was sampled and added to a batch sensation. Of course, smoothness on the tongue is not the
cell filled with ambient-temperature isopropanol until only factor that determines how good a chocolate product
the appropriate concentration was achieved. Fig. 8.10.3 tastes, but this example shows how measuring the particle
shows the particle size distribution results obtained from size distribution provides a measurement scale that can be
measurement of the samples as described above. The used for evaluation.
q3 (%)
10 10
9 9
Chocolate B Chocolate C
Normalized Particle Amount (Diff)
8 8
7 7
6 Chocolate A 6
5 5
4 4
3 3
2 2
1 1
0 0
0.01 0.06 0.1 0.5 1 5 10 50 100 500 1000 5000
Particle Diameter (μm)
Refractive
File Name Sample ID Sample # Absorbance
Index
1 A A bc ipa st2m 0.10 1.60-0.02i
2 B B bc ipa st2m 0.08 1.60-0.02i
3 C C bc ipa st2m 0.12 1.60-0.02i
Median D Modal D Mean V 10 %D 50 %D 90 %D 0 %D 0 %D 0 %D 0 %D 0 %D 0 %D

Std Dev
ѥP ѥP ѥP ѥP ѥP ѥP ѥP ѥP ѥP ѥP ѥP ѥP
1 6.969 14.994 6.771 0.377 2.049 6.969 20.764 0.000 0.000 0.000 0.000 0.000 0.000
2 15.895 30.617 13.265 0.449 2.906 15.895 45.539 0.000 0.000 0.000 0.000 0.000 0.000
3 42.862 127.664 36.861 0.534 6.351 42.862 163.880 0.000 0.000 0.000 0.000 0.000 0.000
Fig. 8.10.3 Particle Size Distributions of Several Kinds of Chocolate
151
9. 9DULHW\,GHQWLÀFDWLRQ Genetic
Analysis Irradiated Food
,GHQWL¿FDWLRQRI5LFH9DULHWLHVí0&(
Q Explanation QAnalytical Conditions for PCR-RFLP Products

Rice is one of Japan's agricultural products for which Instrument : MCE-202 "MultiNA"
package labeling of the variety name and production Analysis Mode : DNA-1000 on-chip mode
region are obligator y accordi ng to the Japanese
Agricultural Standard. However, since mislabeling can be
problematic, various technologies have been developed
to scienti cally verify label content. One method that is Polished rice
widely used for assessing the rice variety is to amplify
a specific gene region from extracted rice DNA using
DNA extraction
PCR, and then to analyze the PCR product using an
electrophoresis analyzer. Here we introduce a method of
conducting multiplex PCR to identify rice varieties using
an identi cation kit capable of distinguishing among 112 Extracted DNA
commercially available varieties from DNA extracted
from polished rice. The electrophoresis patterns of the Multiplex PCR by Variety
obtained PCR products were determined using the MCE- Identification Kit
202 "MultiNA" Microchip Electrophoresis System.
PCR products
Using the rice DNA extraction kit (Takara Bio), DNA
Analysis of PCR products-MultiNA
was extracted from 20 polished grains each of 5 types
of rice (Koshihikari, Akitakomachi, Kinuhikari, Kirara
397, and Hitomebore). Using the extracted DNA as a
template, 4 sets of multiplex PCR (sets A, B, C, and D) Acquisition of appearance patterns
were conducted using the reagent provided with the rice of PCR products
variety identi cation kit (Kokken). Each PCR product was
analyzed using the MultiNA.
Collation of patterns
QReagents/Kits
Rice DNA Extraction Kit (scaled to 20 grains polished rice)
(Takara Bio, 9103) Identification of rice varieties
Kome Bugyo Series ② Variety Identi cation Kit (Ver. 2)
(Kokken, KK-KB-02-02)
DNA-1000 Kit (Shimadzu, P/N 292-27911-91) Fig. 9.1.1 Rice Variety Identi cation Procedure
SYBR® Gold nucleic acid gel stain
(invitrogen, S11494)
ƴ× 174 DNA / HaeⅢ Markers (Promega, G1761)
152
9DULHW\,GHQWLÀFDWLRQ Genetic Analysis Irradiated Food
,GHQWL¿FDWLRQRI5LFH9DULHWLHVí0&(
QResults
After conducting 4 sets (sets A, B, C, and D) of multiplex products were determined from electrophoresis analysis
PCR using the rice variety identification kit (Kokken) results of the multiplex PCR of the 4 sets, and the different
with the templates of DNA extracted from the 5 varieties rice varieties were identi ed by comparing the obtained
of rice, the obtained PCR products were analyzed with patterns with the expected pattern of each variety of
the MultiNA. The results are shown in Fig. 9.1.2. Five rice. The tool that automates the pattern comparing is
pairs of primers from set A, 4 pairs from set B, 5 pairs provided by the kit manufacturer. The all patterns of the
from set C, and 5 pairs from set D were used to conduct PCR products obtained from the analysis results using
multiplex PCR. The PCR products obtained with the the MultiNA correspond are consistent with the expected
primer pairs from each set are shown in the respective pattern of each variety. MultiNA is the powerful tool for
control lanes. The appearance patterns for 19 PCR electrophoretic analysis of multiplexed PCR products.
Akitakomachi
Akitakomachi
Hitomebore
Hitomebore
Koshihikari
Koshihikari
Kirara 397
Kirara 397
Kinuhikari
Kinuhikari
Control
Control
Ladder
Ladder
Set A Set B
Akitakomachi
Akitakomachi
Hitomebore
Hitomebore
Koshihikari
Koshihikari
Kirara 397
Kirara 397
Kinuhikari
Kinuhikari
Control
Control
Ladder
Ladder
Set C Set D
Fig. 9.1.2 Analytical Results of Multiplex PCR of Extracted DNA from Rice Using MultiNA
153
9.2 ,GHQWL¿FDWLRQRI7KXQQXVí0&(
Q Explanation Q Experimental Procedure

Consumer concern over food safety has steadily risen in The DNA extraction and PCR conditions conformed to
recent years. Responding to this concern, the Japanese those presented in the Manual for Distinguishing Among
Agricultural Standard (Law Concerning Standardization Tuna Fish Stocks* produced by the Food and Agricultural
and Proper Labeling of Agricultural and Forestry Materials Inspection Center and the National Research
Products) was revised. The Quality Labeling Standard Institute of Fisheries Science, Fisheries Research Agency.
System was established, requiring the clear and accurate DNA was extracted from pieces of Atlantic blue n tuna,
display of food product name, country of origin, etc. It is southern blue n tuna, Ơ and ơ bigeye tuna, yellow n tuna
the responsibility of the manufacturer or the distributor and albacore tuna. PCR was conducted using the DNA
to accurately convey this information as a product extracted from the various types of tuna as templates.
selection guideline to consumers. For example, seafood Primers specific to the tuna mitochondrial DNA were
belonging to the tuna species, which is consumed in used for PCR ampli cation. The obtained PCR products
large quantities by the Japanese, is dif cult to distinguish were processed using restriction enzymes (Alu I, Mse I,
among the various types when presented in the fresh or Tsp509 I). Electrophoresis of the obtained enzyme digest
processed seafood state. Therefore, misidentification, fragments was conducted using the MultiNA, and the sh
inaccurate labeling and disguise during the distribution varieties were distinguished based on the differences in
process are considered to be social problems, therefore the fragment patterns.
requiring a technique that can quickly, simply and
accurately distinguish among product varieties. Here we （*）Manual for Identi cation of Thunnus Species
introduce the procedure for distinguishing the differences Food and Agricultural Materials Inspection Center, National
Research Institute of Fisheries Science, Fisheries Research
among fish stock of Atlantic bluefin tuna (Thunnus
Agency
thynnus), southern bluefin tuna (T. maccoyii), Ơ and ơ http://www.famic.go.jp/technical_information/hinpyou/
bigeye tuna (T. obesus), yellowfin tuna (T. albacares), pdf/maguro_manual.pdf (Japanese)
and albacore tuna (T. alalunga) using the PCR-RFLP
(Polymerase Chain Reaction -Restriction Fragment
Length Polymorphism) method as described in the
manual produced by the Food and Agricultural Materials Specimen
Inspection Center and the National Research Institute of
Fisheries Science, Fisheries Research Agency. The MCE- DNA extraction
202 "MultiNA" microchip electrophoresis analyzer was
used for detection of the separation patterns of the PCR-
RFLP products used for distinguishing the differences DNA purification
between these types of sh stocks.
PCR
PCR products
Restriction enzyme processing
PCR-RFLP products
MultiNA electrophoresis
Identification of tuna varieties
Fig. 9.2.1 Experimental Procedure of Identi cation of Thunnus

Using PCR-RFLP Method
154
9.2 ,GHQWL¿FDWLRQRI7KXQQXVí0&(
QReagents/Kits QResults
· DNA-500 Kit (Shimadzu) Fig. 9.2.2 shows the results of analysis of the PCR-RFLP
· SYBR® Gold nucleic acid gel stain (Invitrogen) products from Atlantic bluefin tuna (Thunnus thynnus),
· 25 bp DNA Ladder (Invitrogen) southern blue n tuna (T. maccoyii), Ơ and ơ bigeye tuna (T.
· DNeasy Blood & Tissue Kit (Qiagen) obesus), yellow n tuna (T. albacares), and albacore tuna (T.
· Alu I (New England Biolabs Japan) R0137S alalunga) by MultiNA. The Atlantic blue n tuna, ơ bigeye
· Mse I (New England Biolabs Japan) R0525S tuna, and albacore tuna show distinctive fragment patterns
· Tsp 509 I (New England Biolabs Japan) R0576S as a result of Alu I restriction enzyme processing, allowing
them to be distinguished (Alu I processing marked with+
in Fig. 9.2.2). However, the southern blue n tuna, Ơ bigeye
tuna, and yellowfin tuna show the same fragmentation
QAnalytical Conditions for PCR-RFLP Products pattern. Therefore, we next performed restriction enzyme
Instrument : MCE-202 "MultiNA" processing using Mse I. The southern blue n tuna showed
Analysis Mode : DNA-500 on-chip mode a distinct fragmentation pattern as a result of restriction
enzyme processing using Mse I, allowing its identi cation
(Mse I processing marked with+ in Fig. 9.2.2). As for the
remaining Ơ bigeye tuna and yellowfin tuna, 2 types of
distinct fragmentation patterns were observed as a result of
Tsp 509 I restriction enzyme processing, allowing these to
be easily distinguished (Tsp 509 I processing marked with+
in Fig. 9.2.2). The excellent sensitivity, separation and
repeatability of the analysis data obtained with the MultiNA
demonstrate that it is a powerful and fully automated tool
for determining intra-species genetic variation.
Southern bluefin tuna
★Southern bluefin tuna

★Atlantic bluefin tuna
α Bigeye tuna
α Bigeye tuna
★α Bigeye tuna
★Albacore tuna
Yellowfin tuna
Yellowfin tuna
★Yellowfin tuna
★β Bigeye tuna
Ladder
Ladder
Ladder
Alu I Processing Mse I Processing Tsp509 I Processing
Fig. 9.2.2 Analytical Results for PCR-RFLP Products from Thunnus

155
9.3 Spectroscopic Measurement and Multivariate Analysis
IRU&ODVVL¿FDWLRQRI%HHU7\SHVí89
Q Explanation for each sample is indicated on their product labels as some
Beer is an alcoholic beverage that is quite popular, and "value" or "range" within the above-mentioned ranges.
consumed in large quantities. Furthermore, the market T he deg rees of absor pt ion i n t he ult raviolet a nd
is now bustling with beverages such as non-alcoholic nearinfrared regions in these data approximately re ect the
beer and low-malt beer with beer-like flavors. Beer, low- given content values for protein and alcohol, respectively.
malt beer, and non-alcoholic beer can all be considered
types of beer. A great many varieties of these have
been produced and marketed with modifications to the QAnalytical Conditions
ingredients to adjust such characteristics as alcohol and Instrument : Shimadzu UV-3600 Ultraviolet-Visible
calorie content. From the standpoint of spectrometric Near-Infrared Spectrophotometer
analysis—assuming that the absorption spectra should Measurement : 230 nm – 1900 nm
reflect specific characteristics depending on the types and Wavelength Range
quantities of ingredients in different beers—we were very Scan Speed : Medium
interested to see the kinds of differences that would occur Sampling Pitch : 1.0 nm
upon actual examination. Here, we introduce the results Photometric Value : Absorbance
of our investigation into the differences in absorption Slit Width : 3 nm
spectra obtained from measurement of a variety of beers Detector Switching : 870 nm, 1650 nm
using the UV-3600 Ultraviolet-Visible Near-Infrared Wavelengths
Spectrophotometer. Also presented here is our attempt to
classify different types of beer using multivariate analysis.
Abs.
4.0
QSamples and Measurement Results
The absorption spectra of 14 types of commercially 3.0
available beers (4 types of beer, 6 types of low-malt beer,
4 types of non-alcoholic beer) were measured using the
UV-3600. Degassing was conducted using ultrasonic 2.0
irradiation for 3 minutes, and measurement was conducted
using a 2 mm optical path length quartz cell and a blank 1.0
sample consisting of air. The measurement results are
shown in Fig. 9.3.1. In addition, expanded spectra of the
0.0
ultraviolet region (230 - 400 nm) and the near-infrared 230.0 500.0 1000.0 1500.0 1900.0
region (1400 - 1500 nm and 1650 - 1750 nm) are shown nm
in Figs. 9.3.2 through 4, respectively. The large peak in
the vicinity of 1450 nm in Fig. 9.3.3 is due mainly to the Fig. 9.3.1 Absorption Spectra of Beers, Low-Malt Beers, and
absorption of water, while the peak in the vicinity of 1695 Non-Alcoholic Beers (Thick Line: Beers; Thin Line:
nm in Fig. 9.3.4 is attributed mainly to ethanol absorption. Low-Malt Beer; Dotted Line: Non-Alcoholic Beers)
For reference, the absorption spectra of water and ethanol
(99.5 %) are shown in Fig. 9.3.5. It is clear that the peak Abs.
indicated by the arrow near 1450 nm corresponds to the 4.0
peaks in Fig. 9.3.3, and that the peak indicated by the arrow
near 1695 nm corresponds to the peaks in Fig. 9.3.4. Due 3.0
to the apparent signal saturation in the ultraviolet region
of this data, the absorption spectra were measured in the
ultraviolet region (230 - 400 nm) once again after diluting 2.0
all of the samples 5-fold with distilled water. Those results
are shown in Fig. 9.3.6. The peaks that can be seen in 1.0
the 230 - 300 nm region are believed to be due mainly to
absorption of the protein contained in beers.
0.0
230.0 250.0 300.0 350.0 400.0
The alcohol content in the samples is, for beer: 5 %; low- nm
malt beer: 3 - 5.5 %; non-alcoholic beer: 0 %. The protein
content per 100 mL is, for beer: 0.2 - 0.4 g; and for low- Fig. 9.3.2 Expanded Spectra of Fig. 9.3.1 (230 - 400 nm) (Thick
malt and non-alcoholic beer: 0 - 0.3 g. The alcohol content Line: Beers; Thin Line: Low-Malt Beers; Dotted
Line: Non-Alcoholic Beers)
156

Abs.
3.0
Q&ODVVL¿FDWLRQRI%HHU7\SHVE\0XOWLYDULDWH$QDO\VLV
Non-alcoholic beer We attempted to classify the beers using multivariate
Low-malt beer
2.8 analysis. Using the 5-fold dilution absorbance data (230 -
400 nm) and the undiluted sample absorbance data (401
2.6
- 1870 nm), we conducted principal component analysis
2.4 (PCA)1). The obtained score plot2) is shown in Fig. 9.3.7.
Beer
"A" corresponds to beer, "B" to low-malt beer, and "C"
2.2 to non-alcoholic beer. The samples of A, B, and C are
2.0 clearly grouped accordingly. The closer the points are to
1400.0 1420.0 1440.0 1460.0 1480.0 1500.0
nm each other on the score plot, the greater the corresponding
Fig. 9.3.3 Expanded Spectra of Fig. 9.3.1 (1400 - 1500 nm) (Thick Line: Beers; samples should resemble one another. Accordingly,
Thin Line: Low-Malt Beers; Dotted Line: Non-Alcoholic Beers) A1 and A3, and C1 and C2 should be similar to each
other, and in fact, as can be seen from their respective
Abs.
0.7 ultraviolet spectra shown in Fig. 9.3.8, they are similar.
Looking at the loading plot3) shown in Fig. 9.3.9 reveals
the characteristics of the various groups. As shown in
Fig. 9.3.9, loading vector components3) corresponding to
0.6 Beer
the data components of the ultraviolet region are plotted
Low-malt beer to the right (or upper right) of the center. This indicates
that the further to the right a sample is plotted in Fig. 9.3.7,
the greater its ultraviolet absorption will be. The beer
0.5
Non-alcoholic beer samples A1 - A4, which are actually distributed in that
1650.0 1660.0 1680.0 1700.0 1720.0 1740.01750.0
nm direction, display high ultraviolet absorbance as shown
Fig. 9.3.4 Expanded Spectra of Fig. 9.3.1 (1650 - 1750 nm) (Thick Line: Beers; in Fig. 9.3.6. Also, in the loading plot of Fig. 9.3.9 there
Thin Line: Low-Malt Beers; Dotted Line: Non-Alcoholic Beers) are many loading vector components plotted in the upper
Abs.
left quadrant from 1400 - 1480 nm, which corresponds
3.0 to the absorption of water. This means that the further
to the upper left a sample is plotted on the score plot,
the closer that sample is to pure water, and the lower
2.0
the alcohol content. The non-alcoholic beer samples C1
- C4, which are actually distributed in that direction,
1.0
display high absorbance in the vicinity of 1450 nm, the
wavelength associated with water absorption, as shown
in Fig. 9.3.3. From the above, it is clear that the further
0.0
1900.0
to the right the samples are plotted on the score plot,
230.0 500.0 1000.0 1500.0
nm the greater their ultrav iolet absorbance values will be.
Fig. 9.3.5 Absorption Spectra of Water and Ethanol Correspondingly, the further to the upper left the samples
(Blue Line: Water; Red Line: Ethanol) are plotted, the lower their alcohol content will be. Put
Abs. another way, the further to the right a sample is plotted on
1.5
the score plot, the greater the amount of organic matter
(e.g., protein) it will contain; the further to the upper left
1.0
a sample is plotted, the lower its alcohol content will be.
As for the low-malt beers B1 - B6, their positions in the
lower left region are probably due to the fact that their
0.5 absorbance values are not that high in the ultraviolet
region (comparable to those of non-alcoholic beer), while
several of them have alcohol content comparable to that of
0.0
230.0 250.0 300.0 350.0 400.0 beer.
nm
Fig. 9.3.6 Absorption Spectra of Samples Diluted 1:5 (Thick Line: Beers;
Thin Line: Low-Malt Beers; Dotted Line: Non-Alcoholic Beers)
157
Score
QSummary
1.2
C3 We were able to con f i r m t he possibilit y i n t h is
1
Non-alcoholic
investigation of determining the differences in alcohol
0.8
beer and protein content in beers by examining their
0.6
C1 C4 absorption spectra. Further, by applying multivariate
C2
0.4 Beer A2
analysis to the acquired measurement data, we were able
PC-2 (5 %)
0.2 B4
A1
to classify the groups according to the type of beer, and
0
B5
A3 thereby gain an understanding of their characteristics.
-0.2 B3 A4 Comparative investigation of many products is required
-0.4 in the research and development of food products, but an
Low-malt beer
-0.6
B6 understanding of the degree of similarity among samples
B1
-0.8 B2 is possible using principal component analysis (PCA). The
-1
results obtained in this study suggest that a combination
-4 -3 -2 -1 0 1 2 3 4 5 of spectral analysis and multivariate analysis can be
PC-1 (94 %) effective in the development of food products, including
beer.
Fig. 9.3.7 Score Plot
1) The Unscrambler®, a multivariate analysis software application,
Abs. was used for conducting analysis. The Unscrambler is a trademark
1.5 or registered trademark of CAMO.
Regarding the present analysis, principal component analysis
was conducted using mean centering of the acquired data.
2) A score plot involves the projection of each sample point expressed
1.0
in multidimensional space on two loading vectors, expressed as a
two-dimensional graph.
For a description of "loading vector," refer to the following Note 3.
0.5 3) A loading plot refers to the plotting via two-dimensional
coordinates of substances corresponding to the loading
vectors of the rst principal component and second principal
component (or another combination of principal components).
0.0 Here, the loading vector refers to the vector obtained by
230.0 250.0 300.0 350.0 400.0
nm performing eigenvalue calculation for data matrix.
Fig. 9.3.8 Absorption Spectra of A1 and A3, C1 and C2 (Red Line:

A1; Blue Line: A3; Black Line: C1; Green Line: C2)
Loading
1400 – 1480 nm
230 – 300 nm
0.1
PC-2 (5 %)
- 0.1
- 0.1 0 0.1
PC-1 (94 %)
Fig. 9.3.9 Loading Plot
158
9.4 Detection of Allergenic Substances (1) í0&(
Q Explanation QAnalytical Conditions for PCR Products

Japan is the world’s earliest adopter of a display system Instrument : MCE-202 "MultiNA"
for foods containing allergens. In April, 2001 it become Analysis Mode : DNA-500 on-chip mode
mandatory that food labels include likely allergens,
and in June, 2008, two additional food articles, prawn
and crab, were added to that list of likely allergens.
The dissemination of allergen-related information to Sample
the consumer using this label display is meant to help
avert the possibility of allergic responses and harm to DNA extraction
health beforehand. Therefore, if specific substances Ion-exchange resin kit
are included or mixed in among the other ingredients,
even at ultra small quantities, the label is required to
inform the consumer to that fact. Among the allergenic DNA Purification
substances required to be mentioned on the label, those
that can be detected by qualitative PCR are wheat, Purity and concentration verification
buckwheat, peanuts, prawn and crab. Here we conducted BioSpec-nano
amplification of allergen-related genes by PCR using
extracted DNA as the template, according the method
specified by The Japanese Ministry of Health, Labour PCR
and Welfare (Ministry of Health, Labour and Welfare Primer specific for each sample
Notification "Regarding the testing method for foods
containing allergenic substances," No. 0724, Publication
No. 1 issued by the Dept. of Food Safety, July 24, 2009). PCR Products
Here we introduce the detection of these substances

using the MCE-202 MultiNA Microchip Electrophoresis Analysis of PCR products
System for DNA/RNA analysis. MCE-202 MultiNA
Q Experimental Procedure Detection of Allergenic Substance

The DNA extraction and PCR were conducted according
to the method and conditions specified in the above-
mentioned Ministry of Health, Labour and Welfare Fig. 9.4.1 Experimental Procedure for Detection of Allergenic
Noti cation. We extracted the DNA from food products Substances
containing the wheat, buckwheat, peanuts, prawn and
crab, respectively. Ion-exchange resins were used for
carrying the extractions. The DNA purity verification
and quantitation were conducted using the Shimadzu
BioSpec-nano Life Sciences Spectrophotometer.
QReagents/Kits
· DNA-500 Kit
(Shimadzu) P/N: 292-27910-91
· SYBR® Gold nucleic acid gel stain
(Invitrogen) S-11494
· 25 bp DNA Ladder
(Invitrogen) 10597-011
· QIAGEN Genomic-tip 20/G
(QIAGEN) 10223
159
9.4 Detection of Allergenic Substances (2) í0&(
QResults
The results of analysis of the PCR amplification products same samples as those analyzed with the MultiNA are
of DNA derived from wheat, buckwheat, peanuts, prawn shown in [Reference]. The sizes of the PCR amplification
and crab, respectively, using the MultiNA are shown products are imprecise due to lack of objectivity in
in Fig. 9.4.2. The PCR amplification products derived interpreting the gel electrophoresis results. However,
from the wheat, buckwheat, peanuts, prawn and crab the results obtained using the MultiNA consist of an
substances were all clearly detected using the MultiNA. electropherogram in addition to a gel image, ensuring a
(The estimated sizes shown in the figure were obtained high level of accuracy. Particularly noteworthy is that the
in this experiment. The sizes of PCR-amplified DNA amplification products of wheat and buckwheat are very
indicated in The Ministry of Health, Labour and Welfare near each other. Compared to agarose gel electrophoresis,
Notification are wheat: 141 bp, buckwheat: 127 bp, the MultiNA’s excellent resolution and sensitivity allow
SHDQXWV ES SUDZQ ES DQG FUDE ES7KH these to be clearly detected.
results of analysis by agarose gel electrophoresis of the
Buckwheat
Buckwheat
Peanuts
Peanuts
Ladder
Wheat
Prawn
Wheat
Prawn
Crab
Crab
Gel Image Electropherogram
Fig. 9.4.2 Analytical Results for PCR Products from Allergenic Substances
Buckwheat
Peanuts
Marker
Marker
Wheat
Prawn
Crab
[Reference]
Agarose Gel Electrophoresis of PCR Products from Allergenic Substances
160
9.5 Detection of Food Poisoning-Related Genes (1) í0&(

In recent years, genetic detection methods have become (For PCR)
widely adopted for the identification of the causative Reagent Kit : Ampdirect® Plus Enzyme Set
agents of food poisoning, allergies, and infectious Shimadzu P/N: 241-08890-92
diseases such as inf luenza. The most widespread (For MultiNA)
conventional gene-level detection method involves Instrument : MCE-202 ''MultiNA''
amplification of specific genes using PCR (Polymerase Analysis Mode : DNA-500 on-chip mode
C h a i n Re a c t io n), fol lowe d by d e t e c t io n of t he Reagent Kits : DNA-500 Kit
Shimadzu P/N: 292-27910-91
amplification products and sizes using electrophoresis.
6<%5® Gold nucleic acid gel stain
The conventional agarose gel electrophoresis technique Invitrogen S-11494
is a labor-intensive series of processes from preparation 25 bp DNA Ladder
of the gel to obtaining results. Fur ther more, size Invitrogen 10597-011
measurement involves visual comparison with the sizes
of known bands, which often leads to variation in results
due to the reliance on individual objectivity. Here we QExperimental Procedure
introduce the analysis of genes related to food poisoning The samples consisted of DNA extracted from the respective
using the MCE-202 MultiNA Microchip Electrophoresis strains, and re ned.
System for DNA/RNA Analysis. This system offers high- PCR was conducted using Shimadzu’s Ampdirect ®
speed, automated analysis, higher detection sensitivity Plus gene ampli cation reagents, and the obtained PCR
than agarose gel electrophoresis, and automatically ampli cation products were analyzed using the MultiNA
calculated size estimation using pretreatment and (Fig. 9.5.1).
electrophoretic parallel processing.
QFood Poisoning-Related Genes Sample

Here, the 10 types of genes related to food poisoning
shown in Table 9.5.1 were selected as targets. DNA extraction
Phenol-chloroform extraction
Table 9.5.1 Food Poisoning-Related Genes
Thermostable direct hemolysin-related hemolysin (trh 1&2) DNA refinement

gene of Vibro parahaemolyticus
Staphylococcus aureus enterotoxin A gene PCR
Staphylococcus aureus toxic shock syndrome toxin-1 gene Ampdirect® Plus
invA gene of Salmonella sp.
LT gene of enterotoxigenic E. coli
PCR Products
STh gene of enterotoxigenic E. coli
STp gene of enterotoxigenic E. coli
Analysis of PCR products
VT1 genes of enterohemorrhagic E. coli
MCE-202 ''MultiNA''
VT2 genes of enterohemorrhagic E. coli
VT genes of enterohemorrhagic E. coli
Detection
Fig. 9.5.1 Experimental Procedure of Food Poisoning-Related Genes
161
9.5 Detection of Food Poisoning-Related Genes (2) í0&(
Q Detection of Food Poisoning-Related Genes

The results of analysis of the target region for the 10 electropherograms. The estimated size and concentration
types of genes related to food poisoning based on the values for the ampli cation products were calculated from
procedure of Fig. 9.5.1 are shown in Fig. 9.5.2. All of the the calibration curve for the standard sample (ladder) and
food poisoning-related genes and the targeted regions expressed as numeric values, allowing simple and reliable
were detected. The results of analysis using the MCE-202 evaluation of the target ampli cation products.
MultiNA were obtained as electrophoretic images and
Gel Images
L 1 2 3 4 5 6 7 8 9 10
Lane : Ladder
Lane 1 : Vibrio parahaemolyticus (250 bp)
Lane 2 : Staphylococcus aureus EA (423 bp)
Lane 3 : Staphylococcus aureus TSST-1 (228 bp)
Lane 4 : Salmonella (378 bp)
Lane 5 : E. coli LT (263 bp)
Lane 6 : E. coli STh (131 bp)
Lane 7 : E. coli STp (123 bp)
Lane 8 : E. coli VT1 (349 bp)
Lane 9 : E. coli VT2 (404 bp)
Lane 10 : E. coli VT1&2 (171 bp)
Electropherograms
10
9
8
7
6
5
4
3
2
1
L
Fig. 9.5.2 Analytical Results of Food Poisoning-Related Genes
162
9.6 4XDOLWDWLYH$QDO\VLVRI*HQHWLFDOO\0RGL¿HG&RUQí0&(
Q Explanation Table 9.6.1 Genetically Modi ed Corn Samples

Lepidoptera Resistant Maize
The "JAS (Japanese Agricultural Standard) Analysis
Inspection Handbook, Manual for Inspection and IRMM*2 CRM*3 0210DL]H/LQH*92VWDQGDUG(50%)D
Analysis of Genetically Modified Foods," including IRMM CRM 0210DL]H/LQH*92VWDQGDUG(50%)G
standard methods for genetically modified agricultural IRMM CRM 0210DL]H/LQH*92VWDQGDUG(50%)I
products and processed goods, is open to the public on
the web site of the Ministry of Agriculture, Forestry (*1) Ministry of Agriculture, Forestry and Fisheries Food Label
and Fisheries and the Food and Agricultural Materials System Q&A and guideline, etc.
Inspection Center, an independent agency.(*1) Inspections http://www.maff.go.jp/j/jas/hyoji/qa.html
for monitoring food labeling of genetically modified Food and Agricultural Materials Inspection Center "Manual
agricultural products are conducted by the independent for Inspection and Analysis of Genetically Modi ed Foods"
Food and Agricultural Materials Inspection Center <Revised 2nd Edition>
http://www.famic.go.jp/technical_information/jashandbook/
based on the JAS Analysis Inspection Handbook. In
index.html
addition, the handbook is quoted in several places in the (*2) Institute for Reference Materials and Measurements (IRMM)
"Inspection Method for Food Produced by Recombinant (*3) Certi ed Reference Materials (CRM)
DNA Technology" based on the Ministry of Health,
Labour and Welfare noti cation, attesting to the fact that
this analysis method has become one of the standards for
inspection of genetically modi ed agricultural products
in Japan. Furthermore, not limited to compliance through Specimen
government control, the analysis method is widely used
as a voluntary inspection system throughout enterprises
involved in food processing and distribution to ensure DNA extraction
obser vance of the food labeling system. Here we
introduce an example of detection through qualitative
inspection of genetically modified corn as specified in DNA purification
the JAS analysis inspection handbook, using the MCE-
202 "MultiNA" microchip electrophoresis DNA/RNA
analyzer. PCR
Q Experimental Procedure PCR products

Three certified standards consisting of genetically
modi ed corn powder (MON 810 maize line) were used Analysis of PCR products-MultiNA
(Table 9.6.1). The conditions used from DNA extraction
to PCR were in accordance with the "JAS Analysis
Inspection Handbook, Manual for Inspection and
Detection
Analysis of Genetically Modi ed Foods, Basic Operation
Volume."(*1) The DNA was extracted from 1 g taken from
each of the samples using the Qiagen DNeasy Plant Maxi
kit. The extracted DNA concentration was measured, and Fig. 9.6.1 Experimental Procedure for Genetically Modi ed Corn
based on this measurement, the solution was diluted to
10 ng/μL for use as a PCR template. In PCR, 2 types of
primer pairs were used for detection of the endogenous
corn gene SSIIb, and for detection of the recombinant
MON 810 maize line. PCR was conducted from 3 points
of each sample extract. In addition, PCR was also
conducted on a positive control (standard plasmid DNA
for corn added as a template) and negative controls (one
without template DNA, the other without primer). The
obtained PCR products were analyzed using the MultiNA.
163
9.6 4XDOLWDWLYH$QDO\VLVRI*HQHWLFDOO\0RGL¿HG&RUQí0&(
QReagents/Kits QResults
· DNA-500 Kit From Fig. 9.6.2, it is clear that the endogenous corn gene
(Shimadzu) 66,,EESZDVGHWHFWHGLQDOORIWKHVDPSOHV7KLV
· SYBR® Gold nucleic acid gel stain confirms that the DNA extraction and PCR were achieved
(Invitrogen) without problem. On the other hand, only in the samples
· 25 bp DNA Ladder containing the MON 810 maize line could the band (M
ES EH LGHQWLILHG ,Q DGGLWLRQ WKH SUHVHQFH
(Invitrogen)
absence of assumed DNA amplification products was
· DNeasy Plant Maxi Kit confirmed in the positive and negative controls, indicating
(Qiagen) that the results were accurate. Both an electropherogram
and a gel image were obtained in the analysis results
using the MultiNA. Therefore, the amplification products
QAnalytical Conditions for PCR Products of interest were reliably and easily identified by the
Instrument : MCE-202 "MultiNA" presence/absence of peaks and their sizes verified two-
Analysis Mode : DNA-500 on-chip mode dimensionally.
Gel Image
SSIIb Primer M810 Primer

L 1 2 3 4 5 6 7 8 9 10 11 12 1 2 3 4 5 6 7 8 9 10 11 12
L : Ladder Marker (25bp DNA Ladder)

1-3 : MON 810 0% added sample
10 : Negative control (without template DNA)
11 : Negative control (without primer)
12 : Positive control (template is plasmid DNA
with detection target sequence)
Electropherogram
LM UM
SSIIb Primer
M810 Primer
Fig. 9.6.2 Analytical Results for PCR Products from Genetically Modi ed Corn
164
9.7 Detection of Mold and Yeast Genes (1) í0&(
Q Explanation Table 9.7.1 Molds and Yeast Samples

Mold and yeast exist throughout the environment and Molds : Eurotium chevalieri
there are many beneficial varieties of each in food Penicillium digitatum
VRXUFHVVXFKDVPLVRIHUPHQWHGVR\EHDQSDVWHFKHHVH Yeast : Saccharomyces cerevisiae
bread, wine, and sake, etc., and those that are mildly
WR H[WUHPHO\ WR[LF 3RO\PHUDVH &KDLQ 5HDFWLRQ 3&5 (1) The Internal Transcribed Spacer (ITS) regions refer to the 2
technology has been used in recent years as one of the regions between the 3 ribosomal RNA genes (rDNA), 18 S,
PROHFXODU ELRORJLFDO WHFKQLTXHV IRU GHWHFWLQJ IXQJL 5.8 S and 28 S, (ITS 1 between 18 S and 5.8 S, ITS 2 between
5.8 S and 28 S). It is known that there are differences in the
such as molds and yeasts. When PCR is performed, a base sequences in these ITS regions among different kinds
FRPSOLFDWHGSUHWUHDWPHQWRSHUDWLRQLVW\SLFDOO\UHTXLUHG of bacteria.
to extract the DNA from fungus samples like molds and (2) The Japanese Pharmacopoeia is book containing standard
yeasts. Here we introduce an example of direct mold and criteria for pharmaceutical products intended to provide
\HDVWDQDO\VLVXVLQJWKH0&(´0XOWL1$µ'1$51$ guidelines for establishing standard properties and quality
of pharmaceutical products.
analyzer based on microchip electrophoresis technology,
in which the PCR reagent “Ampdirect®3OXVµLVXVHGWR
enable the PCR reaction without the need for any PCR
pre-processing operations.
Samples consisted of two types of mold and one type
of yeast, as shown in Table 9.7.1. The genus Eurotium
is a type of mold which grows in dried foods, such as Bacterial Culture PCR Reagent PCR
dried goods, bread, filled buns, and jam, etc. The genus
PenicilliumDOVRUHIHUUHGWRDV´EOXHPROGµLVDJHQXV Fig. 9.7.1 Method of Direct PCR
of mold that occurs in many types of food, such as citrus
fruits, grains, and dairy products. There are various types
of Penicillium, ranging from beneficial varieties that
are used in foods such as in the production of cheese,
to harmful types such as toxic mold. Saccharomyces Culture Specimen
cerevisiae is budding yeast, and includes such varieties
as baker’s, wine, and sake yeast. For the PCR reaction
reagent, we used Shimadzu’s “Ampdirect® Plus Enzyme Pretreatment, PCR
6HWµ IRU JHQH DPSOLILFDWLRQ DQG WKH 3&5 UHDFWLRQ Ampdirect ® Plus
conditions used are listed in the included instruction
manual. The mold and yeast that had been cultured in
agar medium were attached to a micropipette tip, then
suspended in the PCR reagent solution, and PCR was PCR Products
conducted (Fig. 3ULPHUV,76SULPHUVIRUIXQJL
GHVLJQHG IRU TXLFN LGHQWLILFDWLRQ RI PLFURRUJDQLVPV
Analysis of PCR Products
E\ JHQHWLF DQDO\VLV DV GHVFULEHG LQ WKH -DSDQHVH
Pharmacopoeia ZHUH XVHG IRU GHWHFWLRQ RI ,76
MultiNA
regions when performing PCR. The PCR products were
DQDO\]HGXVLQJWKH0XOWL1$)LJ
Detection
Fig. 9.7.2 Analytical Procedure for Mold and Yeast Genes
165
9.7 Detection of Mold and Yeast Genes (2) í0&(
QReagents/Kits QDetection of Mold and Yeast Genes

· Ampdirect® Plus Enzyme Set The analysis results for the mold and yeast gene ITS
(Shimadzu Corp.) P/N 241-08890-92 regions obtained according to the procedures of Fig. 9.7.1
· DNA-500 Kit and Fig. 9.7.2 are shown in Fig. 9.7.3. The ampli cation
(Shimadzu Corp.) P/N 292-27910-91 products speci c to the genes of the respective molds and
· SYBR® Gold nucleic acid gel stain yeast were clearly detected (The size estimations shown
(Invitrogen) S11494 in the electropherogram in this figure were obtained
· 25 bp DNA Ladder through actual experimentation). The measurement
(Invitrogen) 10597-011 results using the MultiNA were obtained as a gel image
and electropherogram. In addition, the estimated size
values for the amplification products were calculated
QAnalytical Conditions for PCR Products using a calibration curve generated using a standard
Instrument : MCE-202 "MultiNA" sample (Ladder) with known sizes, and the estimated
Analysis Mode : DNA-500 Pre-mix mode concentration values were calculated based on a high
molecular internal standard marker (Upper Marker) of
known concentration used as a standard. Thus, evaluation
of the target amplif ication products are detected
more reliably and easier than when using agarose gel
electrophoresis.
Gel Image Electropherogram
L 1 2 3
3
(x-axis of electropherogram) Migration Time Index (%)
L : Ladder Marker (25 bp DNA Ladder)

1 : Eurotium chevalieri (Product Length 229 bp)
2 : Penicillium digitatum (Product Length 261 bp)
3 : Saccharomyces cerevisiae (Product Length 448 bp)
Fig. 9.7.3 Analytical Results for Mold and Yeast Genes
166
9.8 Detection of Irradiated Foods (1) í*&06

Food irradiation is a technology in which foods that Instrument : GCMS-QP2010 Ultra
are to be stored for long periods of time are exposed -GC-
to X-rays or gamma rays to kill any microorganisms Column : Rxi-5MS (30 m × 0.25 mm I.D. GI ƫP
adhering to the surface of the food. This technology : &PLQ&PLQ - 300 °C
Column Temp.
has been used worldwide for such foods as spices PLQ
and meats, but in Japan, except for the purpose of
Carrier Gas : +HFPVHFN3D
preventing the germination of potatoes, its use has not
been approved. 2-alkylcyclobutanones are formed in Carrier Gas Mode : Constant Linear Velocity Mode
the fats contained in food as a result of irradiation. The Injection Temp. : 250 °C
alkyl groups formed in the 2-alkylcyclobutanones differ Injection Method : 6SOLWOHVV,QMHFWLRQPLQ
depending on the fatty acid composition in the fat; Injection Volume : ƫ/
2-dodecylcyclobutanone (2-DCB) derived from palmitic -MS-
acid and 2-tetradecylcyclobutanone (2-TCB) derived from I.F. Temp. : 280 °C
stearic acid are known to be principle products generated I.S. Temp. : 230 °C
due to irradiation. Since these are not detected in non-
Ionization Method : EI
irradiated foods, they are used as radiation detection
indicators in the European Standards (EN1785) and in one Mode : SCAN/SIM
of the detection methods for irradiated food established Scan Range : m/z 95-115
by the Japan's Ministry of Health, Labour and Welfare Scan Interval : 0.3 sec
(Alkylcyclobutanone Method; Notice No. 0330 Article 3 Monitor Ion : m/z 98, 112
of the Pharmaceutical and Food Safety Bureau, Ministry SIM Interval : 0.2 sec
of Health, Labour and Welfare, March 30, 2010). Here we
introduce an example of analysis of 2-alkylcyclobutanone 2-DCB
in foods from which fat can be extracted (beef, pork,
chicken, salmon, and Camembert cheese, etc.) according O
to the Alkylcyclobutanone Method. 2-TCB
Q Materials and Method

O
Using commercially-available ground pork as samples, Fig. 9.8.1 Structures of 2-DCB and 2-TCB
one sample was irradiated with 1 kGy of cobalt-60 as
Mix 3 g of sample and 6 g of anhydrous
the Ƣ-ray source under room temperature, and the other Sample sodium sulfate.
was not irradiated. The sample preparation procedure Let stand approximately 30 minutes.
Soxhlet Extraction
is shown in Fig. 9.8.2. SIM measurement is required
Add approximately 120 mL of hexane,
for quantitation in this GC/MS test method, and SCAN reflux and extract for 6 hours.
Add anhydrous sodium sulfate to the lipid
measurement is required for qualitative identification. extract, and let stand overnight.
Here, the measurement was conducted in a single analysis Evaporation
using the FASST SCAN/SIM mode, and quantitation of Re-Suspension 100 mL

2-DCB and 2-TCB in the sample was conducted from
Determination of Lipid Content
the SIM measurement results using the internal standard
method. Apply the lipid extract (approximately
Florisil® Column Chromatography 200 mg of lipid) to the column.
Dispose of the first eluent (150 mL of hexane).
Collect the second eluent.
(150 mL of diethylether/hexane (1/99))
Evaporation
Re-Suspension 200 μL of IS
(0.5 μg/mL 2-cyclohexylcyclohexanone)
GC/MS 1 μL Injection
167
9.8 Detection of Irradiated Foods (2) í*&06
QSample Analysis
The SIM chromatograms of the irradiated and non-irradiated 2) The relative ion intensity ratio of m/z 112 with respect to m/z
samples are shown in Fig. 9.8.3 and Fig. 9.8.4, respectively, 98 is within ± 20 % of the relative intensity ratio of a standard
and the mass spectra of the target analytes in the irradiated solution with a concentration level near that concentration.
samples are shown in Fig. 9.8.5. In the irradiated samples, 3) When SCAN measurement is conducted in the vicinity of the
both 2-DCB and 2-TCB peaks were detected, and their retention time within the range of m/z 95 to m/z 115, m/z 98
concentrations in fat were 0.077 μg/g and 0.151 μg/g, and m/z 112 are the main ions (total intensity of the 2 ions is
respectively. The peaks were not detected in the non-irradiated at least 50 %).
samples. According to the Alkylcyclobutanone Method, 4) Even if the above items are satis ed, the quantitative values
if the following 4 conditions are satisfied, the test result is must be greater than the concentration calculated using S/N
considered to be positive for irradiation of food products. ratio = 3 of the standard solution.
1) Peaks with S/N ratio greater than 3 are recognized at The irradiated food measurement results obtained here
m/z 98 and m/z 112 at the same retention times as in the satisfy all of the above criteria, and therefore the results were
standard solution. “positive” for irradiation.
(× 100,000) (× 100,000)
6.0 98.00 4.0 98.00
112.00 112.00
3.5
5.0
3.0
4.0
2.5
3.0 2.0
1.5
2.0
1.0
1.0
0.5
15.25 15.50 15.75 16.00 17.25 17.50 17.75 18.00

A B
Fig. 9.8.3 SIM Chromatogram of Non-Irradiated Sample
(× 1,000,000) (× 100,000)
98.00 8.0 98.00
112.00 112.00
1.00 7.0
6.0
0.75
5.0 2-TCB
4.0
0.50
3.0
2-DCB
2.0
0.25
1.0
15.25 15.50 15.75 16.00 17.25 17.50 17.75 18.00

A B
Fig. 9.8.4 SIM Chromatogram of Irradiated Sample
% %
100 98 2-DCB 100 98 2-TCB
75 75
50 50
112
25 112 25
99 99
0 0
95.0 97.5 100.0 102.5 105.0 107.5 110.0 112.5 97.5 100.0 102.5 105.0 107.5 110.0 112.5 115.0
A B
Fig. 9.8.5 Mass Spectra of 2-DCB and 2-TCB (Irradiated Sample)

[Reference]
Notice No. 0330 Article 3 of the Pharmaceutical and Food Safety Bureau, Ministry of Health, Labour and Welfare of
Japan, March 30, 2010 “Detection Method for Irradiated Foods”
168
10. Food Containers Packing
Materials
10.1 Leach Test for Phenol in Rubber Nipples (1)í89
Q Explanation Q Preparation of Reagents

As food moves through the process starting from the The various reagents are prepared as described below.
raw material stage to the point of ingestion, it comes · Phenol standard solution
into contact with a wide variety of items, including Dissolve 1.0 g of phenol in water, and adjust the volume to 100 mL.
implements, containers and packaging materials. These Take 1 mL of this solution, and add water to bring the volume to
100 mL. Take 1 mL of this solution, and add water to bring the
points of physical contact can include equipment used for volume to 20 mL. (1 mL of this solution contains 5 μg of phenol.)
manufacture and processing, containers and packaging · Borate buffer solution
materials used for storage and transport, and cooking 1st solution: Dissolve 4.0 g of sodium hydroxide in water, and adjust
and eating utensils used in restaurants and in the home. the volume to 100 mL.
Because these implements, containers and packaging 2nd solution: Dissolve 6.2 g of boric acid in water, and adjust the
volume to 100 mL.
materials are made of various types of materials, such as Mix equal volumes of the 1st solution and the 2nd solution.
rubber, glass, and metals, there is always the possibility · 4-aminoantipyrine reagent
that their constituent substances, as well as impurities, can Dissolve 1.36 g of 4-aminoantipyrine in water, and adjust the volume
be taken into the body via the ingested food. Therefore, to 1000 mL.
it is necessary to secure the safety of implements, · Potassium hexacyanoferrate (Ⅲ)
Dissolve 8.6 g of potassium hexacyanoferrate (Ⅲ) in water, and adjust
containers, and packaging materials as provided for in the volume to 1000 mL using 1.8 mL of aqueous ammonia (28 to 30 %
the Japan's Food Sanitation Act, "Standards and Criteria concentration) and water.
for Food and Food Additives, etc., Chapter 3: Apparatus
and Containers and Packaging." One example of a rubber
Table 10.1.1 Target Analytes and Analytical Instruments
implement is the rubber nursing nipple, which is inserted
directly in an infant's mouth. Since it is possible that Implement /
Packaging and
*HQHUDO6SHFLÀF Target Analyte Analytical
toxic phenol can leach out of the rubber nipple when it is Container
Standard Substances Instrument Note 1)
inserted into an infant's mouth, a leach quantity standard Glass,

has been established in the Food Sanitation Act, and earthenware, < Cd, Pb AA/ICP
and enamel
testing is specified to be conducted using an UV-VIS Cd, Pb AA/ICP
spectrophotometer. Here we introduce the phenol leach Metal cans <
Phenol UV-VIS
test for quantitation of phenol leached from a rubber Epichlorohydrin GC
Vinyl chloride GC
nipple according to the Food Sanitation Act. Cd, Pb, Zn AA/ICP
General standard
Phenol UV-VIS
Rubber
Cd, Pb, Zn AA/ICP
Rubber nipples
QStandard for Implements, Containers and Phenol UV-VIS
General standard Cd, Pb AA/ICP
Packaging for Foods Phenol plastic
Table 10.1.1 lists the target analytes and analytical Melamine plastic Phenol UV-VIS
instruments to be used for measurement with respect to Urea plastic
Dibutyltin
the various types of implements and packaging materials. compounds
GC/MS
Both general standards and specific standards are Polyvinyl chloride Cresol phosphate
HPLC
esters
speci ed for rubber and plastic implements, or packaging Vinyl chloride GC
and containers. Phenol is indicated as an analyte for metal Volatile substances
Polystyrene GC, GC/MS
cans, for rubber in the general standard as well as the Plastics
(5 types)
Vinylidene chloride GC
nursing nipple speci c standard, and for phenol plastic, Polyvinylidene chloride
Ba AA/ICP
melamine plastic and urea plastic speci c standard. In all Polyethylene
Sb, Ge AA/ICP
of these cases, an UV-VIS spectrophotometer is speci ed terephthalate
Polymethyl Methyl
for conducting measurement. methacrylate methacrylate
GC
Nylon Caprolactam GC
Amines GC
Polycarbonate Bisphenol A,
HPLC
diphenyl carbonate
Polylactate Total lactate HPLC
Note 1: AA stands for Atomic Absorption spectrometer,
ICP for inductively coupled plasma emission spectrometer,
UV-VIS for ultraviolet-visible spectrophotometer,
GC for gas chromatograph, GC/MS for gas chromatograph - mass
spectrometer, and HPLC for liquid chromatograph.
169
10.1 Leach Test for Phenol in Rubber Nipples (2)í89
Q Pretreatment and Analytical Conditions QResults

Using a ratio of 1 g of sample to 20 mL of water, After preparing test solutions as described above for
immerse the sample in water. Cover the receptacle with four types of commercially available rubber nipples,
a glass plate and set aside for 24 hours, maintaining a in addition to the standard solution, measurement was
temperature of 40 °C. Use this leach solution as the test conducted using the UV-VIS spectrophotometer. The
solution. Add 3 mL of the borate buffer solution to 20 mL absorbance spectra obtained for each test solution and the
of the test solution, and after shaking it well to thoroughly standard solution are shown in Fig. 10.1.1, and the phenol
mix the solutions, add 5 mL of the 4-aminoantipyrine contents indicated by the absorbance values at 510 nm
solution and 2.5 mL of the potassium hexacyanoferrate are shown in Table 10.1.3. Extremely low absorbance
(Ⅲ) solution. Add water to bring the volume to 100 mL, values were obtained with all of the rubber nipples.
and after shaking well to mix the solutions, set the With absorbance values lower than that of the speci ed
solution aside for 10 minutes at ambient temperature. value for leached phenol, the test solutions here showed
Separately, take 20 mL (5 μg/mL) of the phenol standard almost no leaching of phenol, thereby confirming that
solution, and perform the same procedure as that for the speci ed standard was satis ed. Here we introduced
the test solution. Here we used the UV-1800 UV-VIS the phenol leach test for rubber nipples, however, it
spectrophotometer, and conducted measurement using the must be noted that some of the phenol leach conditions
analytical conditions shown in Table 10.1.2. are different for types of rubber other than that used in
nipples, as well as for metal cans and plastics. In this way,
Table 10.1.2 Analytical Conditions an UV-VIS spectrophotometer can be used for conducting
the leach test of phenol from implements, containers, and
Photometric Value Absorbance
Slit Width 1.0 nm
packaging material used for food.
Wavelength Range 300 to 700 nm
Scan Speed Medium
Sampling Pitch 1 nm
Fig. 10.1.1 Spectra of Standard Solution and Sample Solutions
Table 10.1.3 Results

Sample Name Absorbance (510 nm)
Company A: isoprene rubber 0.003
Company A: silicone rubber 0.002
Company B: isoprene rubber 0.002
Company B: silicone rubber 0.002
Standard solution 0.146
[Reference]
Author: Yoko Kawamura, "Standards and Criteria for Apparatuses, Containers and Packaging, March 2006 Revision"
Chuohoki Publishing
170
Food Containers Packing Materials
10.2 Analysis of Epichlorohydrin Dissolved from Metal Food Cans

í*&
Residual organic solvents in food packaging materials are Instrument : GC-2010 Plus AF
receiving attention due to the heightened concern for food Column : 5W[:$;PðPP,'GI ƫP
safety and public health. Speci c standards and speci cations Column Temp. : &PLQ&PLQ&
as well as testing methods are established for each type Carrier Gas : He 74 cm/sec
of material used in food packaging materials in Japan's &RQVWDQW/LQHDU9HORFLW\0RGH
"Food Sanitation Act-Section 3: Implements, Containers, Injection Temp. : 220 °C
and Packaging in the Standards and Criteria for Food and Injection Method : Split Injection
Food Additives, etc." The inside surface of metal food cans Split Ratio : 1:10
is typically coated with synthetic resin to prevent direct Injection Volume : ƫ/
contact of the food with the metal surface of the can. Various Detector : FID
types of resins are used for this coating material, including Detector Temp. : 220 °C
epoxy resin, phenolic resin, and polyvinyl chloride. Separate
standards have been established for testing of phenol and
formaldehyde, volatile residues, epichlorohydrin, and vinyl Sample wash Wash with water.
chloride; all of these standards rely on dissolution testing
followed by gas chromatographic analysis. Here we introduce
Fill sample with n-pentane.
an example of analysis of epichlorohydrin dissolved from a Dissolution
Heat at 25°C for 1 hour.
resin coating on the inside of a can used for canned foods.
QOverview of Epichlorohydrin Analysis Method

GC/FID
This test method provides for GC/FID measurement of
epichlorohydrin, one of the raw material monomers of epoxy
resin used as an internal-surface coating in metal food cans. Fig. 10.2.1 Preparation of Metal Food Can
(Cans that are used for dried foods or solid foods that make
little direct contact with the can's inside surface, and foods QAnalysis of Standard Solution and Test Solution
other than oily and fatty foods are exempt from this testing. The chromatograms of the epichlorohydrin standard
However, cans that are manufactured with the intention of solut ion a nd t he t e st solut ion obt a i ne d u si ng a
storing sterilized or pasteurized foods for a long period of commercially-available metal food can are shown in
time, regardless of the type of food, are subject to this testing.) Fig. 10.2.2. The epichlorohydrin peak in the test solution
In the dissolution test, n-pentane is used as the dissolving chromatogram has a smaller peak area than that of the
solution, and the peak area value of epichlorohydrin in the test standard solution, con rming that the concentration of the
solution is checked to ensure that it is not greater than the peak sample is smaller than the reference value.
area of epichlorohydrin standard solution (0.5 μg/mL).
QAnalytical Method
Sa mple pre pa r at ion wa s conduct e d a ccord i ng t o
that specified in the "Food Sanitation Act-Section 3:
Implements, Containers, and Packaging in the Standards
and Criteria for Food and Food Additives, etc." Using Standard solution
a commercially-available food can as the sample, the
Epichlorohydrin
n-pentane dissolved solution was heated in the can at 25 °C
for 1 hour to allow dissolving, and the obtained dissolved
Test solution
solution was used as the test solution. Since epichlorohydrin
and n-pentane evaporate easily, the can was covered with
aluminum foil and polyvinyl chloride wrapping lm, and
the lm was xed in place with a rubber band to seal in the
solution. A RESTEK Rtx-WAX (30 mL, 0.53 mm ID, 1 μm 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min
lm thickness) was used. The carrier gas owrate was set
so that epichlorohydrin would elute in about 7 minutes. The Fig. 10.2.2 Chromatograms of Epichlorohydrin Standard
sample preparation ow chart is shown in Fig. 10.2.1. Solution and Test Solution
[Reference]
March 31, 2006 Ministry of Health, Labour and Welfare Noti cation No. 201
Food Sanitation Act - Section 3: Implements, Containers, and Packaging in the Standards and Criteria for Food and Food Additives, etc.
171
10.3 Analysis of Triethylamine and Tributylamine in Polycarbonate
Plastics (1) í*&
Speci c standards and criteria as well as testing methods Instrument : GC-2010 Plus AF+FTD-2010 Plus
were established for each type of material used in food Column : Rtx-1PðPP,'GI ƫP
packaging materials in the Japan's "Food Sanitation Act Column Temp. : 150 °C (5 min) - 20 °C/min - 250 °C (5 min)
- Section 3: Implements and Container and Packaging Carrier Gas : He 32.5 cm/sec
in the Standards and Criteria for Food and Food &RQVWDQW/LQHDU9HORFLW\0RGH
Additives, etc." Polycarbonate is a transparent plastic Injection Temp. : 200 °C
material which possesses excellent mechanical strength, Injection Method : Split Injection
especially impact resistance and heat resistance. It is Split Ratio : 1:15
used in a wide variety of utensils in the food industry:
Detector : FTD
nursing bottles, tableware, chopsticks, mugs, and drip
Detector Temp. : 250 °C
coffee makers, to name a few. Individual standards have
been established for testing of specific polycarbonate
constituents, including bisphenol A, diphenyl carbonate,
amines (triethylamine and tributylamine). In addition,
testing procedures have been speci ed for leach testing Sample 1 g
for bisphenol A and residues after evaporation. Here we
introduce an example of analysis of amines (triethylamine Dichloromethane 20 mL
and tributylamine), two of the principle ingredients in
polycarbonate plastic. Dissolve
Acetone 100 mL
QOverview of Triethylamine and Tributylamine
Analysis in Polycarbonate Plastics Centrifugation 3000 rpm for 10 min
This test method consists of dissolving the polycarbonate
a nd ext ra ct i ng a ny exist i ng t r iet hyla m i ne a nd
tributylamine. Measurement of these constituents is then
conducted by Gas Chromatography with a nitrogen-speci c Vacuum concentration Concentrate to about 1 mL
Flame Thermionic Detector (GC-FTD). The test method
consists of verifying that the total content of triethylamine
Add dichloromethane
and tributylamine in the sample does not exceed 1 μg/g.
Test solution 2 mL
QSample Preparation
Sample pretreatment was conducted according to
that specified in the "Food Sanitation Act - Section Fig. 10.3.1 Preparation of Polycarbonate Plastic Sample
3: Implements and Container and Packaging in the (Analysis of Amines)
Standards and Criteria for Food and Food Additives,
etc." A commercially available garlic press made of
polycarbonate was used for the sample. About 1 g of
sample was accurately weighed and transferred to a
200 mL conical ask, and 20 mL of dichloromethane was
then added to the ask. After sample dissolution, 100 mL
acetone was added while mixing well, and centrifugation
at 3000 rpm was conducted for 10 minutes to separate the
phases. After concentrating the supernatant to 1 mL using
a vacuum concentrator, dichloromethane was added to
bring the total volume to 2 mL. This was used as the test
solution. The sample preparation procedure is shown in
the ow chart of Fig. 10.3.1.
172
10.3 Analysis of Triethylamine and Tributylamine in

Polycarbonate Plastics (2) í*&
QCalibration Curves Area (×10,000)
A stock solution of 0.1 mg/mL of triethylamine and 3.0 R = 0.99935
tributylamine in dichloromethane was prepared. Serial 2.5
dilutions of the stock solution were made to produce 2.0
standard solutions (0.2, 0.4, 0.6, 0.8, 1.0 μg/mL). 1.5
Measurement of each solution was conducted by GC 1.0
using a 1 μL injection volume, and the calibration curves
0.5
were generated from the peak areas of triethylamine and
0.0
tributylamine. 0.00 0.25 0.50 0.75 Concentration
Triethylamine μg/mL
Area (×10,000)
QAnalysis of Standard Solution and Sample
Solution R = 0.99985
1.5
T h e c h r o m a t o g r a m s of t h e t r i e t h y l a m i n e a n d
tributylamine standard solution (0.2 μg/mL) and the 1.0
test solution of the processed polycarbonate garlic press
sample are shown in Fig. 10.3.3. Although triethylamine 0.5
was detected in the chromatogram of the test solution,

0.0
the content was less than one-third that of the speci ed 0.00 0.25 0.50 0.75 Concentration
standard value. Tributylamine μg/mL
Fig. 10.3.2 Calibration Curves of Triethylamine and Tributylamine

(0.2 to 1.0 μg/mL)
■ Peaks
1. Triethylamine
2. Tributylamine
2 Standard solution
1
Test solution
2.5 5.0 7.5 10.0 12.5 min
Fig. 10.3.3 Chromatograms of Standard Solution (Triethylamine, Tributylamine 0.2 μg/mL each) and Test Solution
Note:
Consumption of the alkali source of the FTD when using dichloromethane as the sample solvent is slightly faster compared to that
when other (hydrocarbon) solvents are used. Details are described in the FTD instruction manual.
[Reference]
March 31, 2006 Ministry of Health, Labour and Welfare Noti cation No. 201
Food Sanitation Act - Section 3: Implements and Container and Packaging in the Standards and Criteria for Food and
Food Additives, etc.
173
10.4 Analysis of Vinylidene Chloride in Polyvinylidene
Chloride Plastics í*&
Q Explanation QAnalysis of Standard Solution and Sample
Specific standards and specifications as well as testing Solution
methods are established for each type of material used in T he ch rom at og r a m s of t he v i nyl ide ne ch lor ide
food packaging materials in Japan's "Food Sanitation Act- standard solution and the test solution prepared from
Section 3: Implements, Containers, and Packaging in the commercially-available household wrapping film, of
Standards and Criteria for Food and Food Additives, etc." which the principle ingredient is polyvinylidene chloride,
Polyvinylidene chloride is a polymer that is transparent, are shown in Fig. 10.4.1. The vinylidene chloride peak in
displays excellent water resistance, chemical resistance, the test solution chromatogram has a smaller peak area
and gas-barrier properties, and is resistant to temperatures than that of the standard solution, confirming that it is
in the range of 140 to 170 °C. In addition to household smaller than the reference value.
wrapping film, it is used as a film for wrapping foods
that have been heated to high temperatures. Vinylidene
chloride is the monomer of polyvinylidene chloride, and QAnalytical Conditions
long-term oral exposure is said to adversely affect the Instruments : TurboMatrix HS-40 + GC-2010PlusAF
liver and kidney. Due to the possibility that vinylidene Column : CP-PoraBOND Q FUSED SILICA
chloride may persist in products, polyvinylidene chloride
PðPP,'GI ƫP
material testing has been established as a separate
standard in the Food Sanitation Act. Here we introduce an Column Temp. : Ý&PLQÝ&PLQÝ&PLQ
example of analysis of vinylidene chloride in plastic Injection Temp. : Ý&
Carrier Gas : He 30 cm/sec
Detector : FID
QAnalytical Method Detector Temp. : Ý&
The vinylidene chloride test method is for measuring Injection Volume : 0.5 mL
vinylidene chloride monomer present in polyvinylidene Sample : Ý&PLQ
chloride by GC/FID using the headspace method. Sample Thermostatting
pretreatment is conducted according to that specified
in the "Food Sanitation Act-Section 3: Implements,
Containers, and Packaging in the Standards and Criteria
for Food and Food Additives, etc." Commercially-
available polyvinylidene chloride household wrapping
lm was used as the sample, and the vinylidene chloride
in the sample was analyzed. A porous polymer PLOT
(porous layer open tubular) CP-PoraBOND Q column
was used. The carrier gas flowrate was set so that the Standard solution
Vinylidene chloride
vinylidene chloride would elute at about 9 minutes.
Following is an overview of the analytical procedure.
(1) Test Solution Preparation
Test solution
Cut and weigh out a 0.5 g fragment of sample, and transfer it
to a headspace vial. Next, add 2.5 mL N,N-dimethylacetamide,
seal the vial, and use this as the test solution.
(2) Standard Solution Preparation
Transfer 50 μL vinylidene chloride standard solution (60 μg/mL) 2.5 5.0 7.5 10.0 12.5 15.0 17.5 min
to a vial containing 2.5 mL N,N-dimethylacetamide, and seal the
vial. Use this as the standard solution.
(3) Measurement Fig. 10.4.1 Chromatograms of Vinylidene Chloride Standard
Heat the sealed vials of test solution and standard solution for Solution and Test Solution
1 hour at 90 °C, and introduce 0.5 mL of the respective gas
phases into the gas chromatograph. For the gas chromatograph
column, use a 3 μm styrene-divinylbenzene porous polymer-
coated column, and conduct analysis by GC/FID.
(4) Determination [Reference]
Compare the detection times of test solution peak and the
March 31, 2006 Ministry of Health, Labour and Welfare
vinylidene chloride standard solution peak. If the retention
times correspond, compare their respective peak areas. Noti cation No. 201
Verify that the test solution vinylidene chloride peak area Food Sanitation Act - Section 3: Implements, Containers,
is not greater than that of the vinylidene chloride standard and Packaging in the Standards and Criteria for Food and
solution peak area (6 μg/g or less in the material). Food Additives, etc.
174
10.5 Analysis of Vinyl Chloride in Polyvinyl Chloride Plastics

í*&
Q Explanation QAnalysis of Standard Solution and Sample
Specific standards and specifications as well as testing Solution
methods are established for each type of material used in The chromatograms of the vinyl chloride standard
food packaging materials in Japan's "Food Sanitation Act- s olut ion a nd t he t e st s olut ion p r e p a r e d f r om a
Section 3: Implements, Containers, and Packaging in the commercially-available synthetic polymer glove, of which
Standards and Criteria for Food and Food Additives, etc." the principle ingredient is polyvinyl chloride, are shown
Polyvinyl chloride, a transparent plastic, can be easily in Fig. 10.5.1. The vinyl chloride peak in the test solution
mixed with plasticizers to obtain a desired degree of chromatogram has a smaller peak area than that in the
exibility depending on the compounding ratio. For that standard solution, con rming that it is smaller than the
reason, it is used in a variety of applications, including reference value.
food containers, wrapping films, and gloves. Vinyl
chloride is the monomer of polyvinyl chloride, and is
generated when polyvinyl chloride is subjected to thermal QAnalytical Conditions
decomposition. Due to its widely-reported carcinogenicity, Instruments : TurboMatrix HS-40 + GC-2010PlusAF
the concentration of vinyl chloride in materials is
Column : CP-PoraBOND Q FUSED SILICA
restricted in conjunction with material testing. Here we
introduce an example of analysis of vinyl chloride in PðPP,'GI ƫP
Column Temp. : Ý&PLQÝ&PLQÝ&PLQ
plastic with polyvinyl chloride as its principal ingredient.
Injection Temp. : Ý&
Carrier Gas : He 28.7 cm/sec
QAnalytical Method Detector : FID
The vinyl chloride test method is for measuring vinyl Detector Temp. : Ý&
chloride monomer present in polyvinyl chloride by GC/ Injection Volume : 0.5 mL
FID using the headspace method. Sample pretreatment Sample : Ý&PLQ
is conducted according to that specified in the "Food Thermostatting
Sanitation Act - Section 3: Implements, Containers, and
Packaging in the Standards and Criteria for Food and
Food Additives, etc." A commercially-available polyvinyl
chloride glove was used as the sample, and the vinyl
chloride in the sample was analyzed. A porous polymer
PLOT (porous layer open tubular) CP-PoraBOND Q
column was used. The carrier gas flowrate was set so
that the vinyl chloride would elute at about 5 minutes.
Following is an overview of the analytical procedure. Vinyl chloride
(1) Test Solution Preparation Standard solution

Cut and weigh out a 0.5 g fragment of sample, and transfer it to
a headspace vial. Next, add 2.5 mL N,N-dimethylacetamide,
seal the vial, and use this as the test solution. (If it is dif cult
to dissolve the sample, shake the sealed vial occasionally at Test solution
ambient temperature, and allow it to stand overnight.)
(2) Standard Solution Preparation
Transfer 50 μL vinyl chloride standard solution (10 μg/mL)
(cooled using methanol and dry ice) to a vial containing 2.5 2.5 5.0 7.5 10.0 12.5 min
mL N,N-dimethylacetamide, and immediately seal the vial.
Use this as the standard solution.
(3) Measurement Fig. 10.5.1 Chromatograms of Vinyl Chloride Standard Solution
Heat the sealed vials of test solution and standard solution for
1 hour at 90 °C, and introduce 0.5 mL of the respective gas and Test Solution
phases into the gas chromatograph. For the gas chromatograph
column, use a 3 μm styrene-divinylbenzene porous polymer-
coated column, and conduct analysis by GC/FID. [Reference]
(4) Determination
Compare the detection times of test solution peak and the March 31, 2006 Ministry of Health, Labour and Welfare
vinyl chloride standard solution peak. If the retention times Noti cation No. 201
correspond, compare their respective peak areas. Verify that Food Sanitation Act - Section 3: Implements, Containers,
the test solution vinyl chloride peak area is not greater than
that of the vinyl chloride standard solution peak area (1 μg/g and Packaging in the Standards and Criteria for Food and
or less in the material). Food Additives, etc.
175
10.6 Analysis of Methyl Methacrylate in Polymethyl Methacrylate
Plastics í*&
Due to the high level of concern for food safety, attention is Instrument : GC-2010 Plus AF
being focused on residual organic solvents in food packaging. Column : 5W[PðPP,'GI ƫP
Utensils and food packaging materials are subject to standards Column Temp. : &PLQ&PLQ&
controlled through material testing as specified in Japan's Carrier Gas : He 29.5 cm/sec
Food Sanitation Act-Section 3: Implements, Containers, and &RQVWDQW/LQHDU9HORFLW\0RGH
Packaging in the Standards and Criteria for Food and Food
Injection Temp. : 200 °C
Additives, etc. Polymethyl methacrylate is primarily used in
table articles such as chopsticks, cups, soy sauce dispensers, Injection Method : Split Injection
etc. A separate food packaging standard is provided for the Split Ratio : 1:10
monomer methyl methacrylate. Here we introduce an example Injection Volume : ƫ/
of analysis of methyl methacrylate in plastics in which Detector : FID
polymethyl methacrylate is the main ingredient. Detector Temp. : 200 °C
QOverview of Methyl Methacrylate Analysis QAnalysis of Standard Solution and Test Solution
in Polymethyl Methacrylate Plastics Fig. 10.6.2 shows the chromatograms obtained from
This standard covers mainly polymethyl methacrylate synthetic analysis of a methyl methacrylate standard solution
resins, in which the base polymer consists of at least 50 % (15 μg/mL) and the test solution prepared using a
methyl methacrylate. Polymethyl methacrylate is primarily commercially available chopstick made of polymethyl
used in table articles due to its high transparency, good weather
methacrylate synthetic resin. The peak area value of
resistance and excellent machinability. A separate leachate
testing standard is provided for methyl methacrylate, the methyl methacrylate in the chromatogram test solution is
monomer of polymethyl methacrylate. The methyl methacrylate clearly smaller than that in the standard solution, which
test method speci es the use of GC/FID to measure the amount con rms that the test value satis es the standard criterion.
of methyl methacrylate that leaches from the sample immersed
in a 20 % aqueous ethanol solution. In the leaching test, the
peak area value of methyl methacrylate in the test solution is
not to exceed the peak area value obtained from analysis of a
15 μg/mL methyl methacrylate standard solution.
QAnalytical Method Methyl methacrylate
Sample preparation was conducted according to that speci ed in

Standard solution
Japan's Food Sanitation Act-Section 3: Implements, Containers,
and Packaging in the Standards and Criteria for Food and
Food Additives, etc. Commercially available chopsticks made
of polymethyl methacrylate synthetic resin were used for the Test solution
testing. For the leaching test, a piece of a chopstick with a 1 cm2
surface area was immersed in 2 mL of 20 % aqueous ethanol 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min
solution and maintained at 60 °C for 30 minutes for leaching,
and the resulting solution was used as the test solution. A Restek
Rtx-1 capillary column (0.32 mm I.D., 30 m length, 5 μm lm Fig. 10.6.2 Chromatograms of Standard Solution (15 μg/mL)
thickness) was used, and analysis was conducted by GC/FID. The and Test Solution
preparation procedure is shown in the ow chart of Fig. 10.6.1.
Sample collection
20% aqueous ethanol solution
(2mL per 1cm2 surface area)
Leaching 60°C, let stand for 30min
GC/FID
Fig. 10.6.1 Preparation of Polymethyl Methacrylate Plastic Sample

[Reference]
Noti cation No. 201 of the Ministry of Health, Labour and Welfare, March 31, 2006
Food Sanitation Act-Section 3: Implements, Containers, and Packaging in the Standards and Criteria for Food and Food
Additives, etc.
176
10.7 Analysis of Heavy Elements in Tableware (1) í,&3$(6
Q Explanation
Kitchen utensils and containers regularly come in direct and lead in accordance with ISO. Table 10.7.1 shows the
contact with food articles, and therefore can contaminate revised speci cation standards according to material. The
food with toxic heavy metals, etc. through migration, thus inspection method is a migration test, in which the heavy
posing a health hazard. In particular, ceramic and glass metal is placed in contact with 4 % acetic acid, assuming
tableware may be decorated and coated with pigments and vinegar as the easiest dissolution food substance, for 24
glaze which contain toxic lead and cadmium, and physical hours at ambient temperature to allow migration into
injury due to migration of these toxic elements becomes the acid solution. Quantitative analysis of the solution
a problem. Based on this, the Ministry of Heath, Labour is conducted by atomic absorption spectrophotometry
and Welfare of Japan revised the Food Sanitation Act or ICP emission spectrometry. ICP-AES (ICP emission
"Standards for Foods and Additives" on July 31, 2008 with spectrometry) is a high-sensitivity technique that allows
respect to Noti cation No. 370 of the Ministry of Health, simultaneous analysis of multiple elements. Here we
Labour and Welfare (1959). The main points of revision introduce the analysis of commercial tableware using the
concerning tableware are (1) standardization according Shimadzu ICPE-9000 multi-type emission spectrometer.
to the material, capacity, and shape of containers, and (2)
strengthening of the standard for migration of cadmium
Table 10.7.1 Revised Speci cations According to Material Category

Material Classification Cd Pb
Sample which cannot be filled with liquid, or sample
0.7 ѥJFP2 ѥJFP2
whose depth is less than 2.5 cm
Sample whose depth is 2.5 cm or greater when filled with liquid
Glass Capacity less than 600 mL ѥJP/ ѥJP/
Capacity 600 mL or more,
Other than heat-cookware ѥJP/ ѥJP/
and less than 3 L
Capacity 3 L or more ѥJP/ ѥJP/
Heat-cookware ѥJP/ ѥJP/
Sample which cannot be filled with liquid, or sample
ѥJFP2 ѥJFP2
whose depth is less than 2.5 cm
Sample whose depth is 2.5 cm or greater when filled with liquid
Capacity less than 1.1 L ѥJP/ ѥJP/
Ceramic
Capacity 1.1 L or more,
and less than 3 L
Capacity 3 L or more ѥJP/ ѥJP/
Sample which cannot be filled with liquid, or sample whose depth is less than 2.5 cm
Other than heat-cookware ѥJFP2 ѥJFP2
Heat-cookware ѥJFP2 ѥJFP2
Porcelain Enamel Sample whose depth is 2.5 cm or greater when filled with liquid
Capacity 3 L or more ѥJFP2 ѥJFP2
Capacity less than 3 L
177
10.7 Analysis of Heavy Elements in Tableware (2) í,&3$(6
Q Samples QAnalytical Conditions

Commercial tableware (glass, ceramic) Instrument : ICPE-9000
RF Output : N:
QSample Preparation Plasma Gas Flowrate : /PLQ
(According to Standards for Foods and Additives) Auxiliary Gas Flowrate : /PLQ
After washing the sample well with water, fill it with Carrier Gas Flowrate : /PLQ
4 % acetic acid solution (v/v), and leave it at ambient Sample Introduction : Coaxial nebulizer
temperature in a dark place for 24 hours. Use this solution Spray Chamber : Cyclone chamber
Plasma Torch : Mini torch
as the test solution.
Observation Method : Axial
QResults
Fig. 10.7.1 shows the ICPE-9000 spectral profiles,
and Table 10.7.2 shows the quantitation results and
detection limit of this analysis. The detection limit in
this analysis is less than 1/100 of the standard value,
demonstrating high sensitivity.
Table 10.7.2 Quantitative Results for Tableware (Unit: μg/mL)

Element Name Cadmium (Cd) Lead (Pb)
Detection Limit 0.0001 0.002
Sample Name Sample Type
Sample 1 Glass cup with pattern design 0.0002 < 0.002
Sample 2 Earthenware cup < 0.0001 0.020
Sample 3 Earthenware saucer < 0.0001 < 0.002
Sample 4 Porcelain teacup with pattern design 0.0010 0.056
Sample 5 Small porcelain pot with pattern design 0.0027 0.221
Sample 6 Porcelain coffee cup < 0.0001 0.061
<: Detection limit (3ѫ) obtained from the standard deviation of N=10 repeat measurements using a calibration curve blank (4 % acetic acid solution (v/v))
Cd 214.438 nm Pb 220.353 nm
1000
700
Standard sample 0.01 ppm
Standard sample 0.25 ppm
600
750 Sample 5
Intensity
Intensity
Sample 5 500
Sample 4 Sample 6
500 Sample 4
400
Sample 2
300
250
200
Fig. 10.7.1 Spectral Pro les of Tableware
178
10.8 Analysis of Heavy Elements in a Cup í(';

Lead, a harmful element, has recently been detected Instrument : EDX-720
in toys, household articles, and everyday products, X-ray Tube : Rh target
and the safety of these articles is becoming a problem. Filter : )LOWHUIRU&G6E%D)LOWHU
Since solid, powder, and liquid samples can be analyzed IRU$V+J3E6H)LOWHUIRU&U
nondestructively, both quickly and easily by X-ray Voltage : 50 kV except for Cr, Cr: 30 kV
uorescence analysis, this method is used as a screening Current : Auto
technique for safety inspections of such items as toys Atmosphere : Air
and household articles. Here we introduce the results Measurement Diameter : PPƴ
of analysis of a dishware article containing harmful Measurement Time : 100 sec
elements. A polyethylene (PE) resin (plastic) spiked with
Dead Time : 40 %
8 harmful elements was used as a standard sample.
QAnalysis of Cup
The photographs of the cup (Fig. 10.8.1) indicate the
locations where measurement was conducted on the
yellow coating site. The qualitative results of Fig. 10.8.2 （1）（2）
indicate the presence of Pb in the yellow portion of the
cup. In addition, the quantitative analysis results for
the (1) yellow, (2) red, (3) green, and (4) white portions
of coating are shown in Table 10.8.1. The quantitative （3）（4）
analysis results indicate that aside from Pb, the elements
Cr and Cd are also present. Fig. 10.8.1 Photographs of the cup
Fig. 10.8.2 Qualitative Analysis Results for Yellow Coating of Cup
Table 10.8.1 Quantitative Analysis Results for Cup

Element Sb As Ba Cd Cr Pb Hg Se
Spectrum Sb KƠ As KƠ Ba KƠ Cd KƠ Cr KƠ Pb Lơ1 Hg LƠ Se KƠ
(1) Yellow color 743.9 166.1 72.5 11.7 113.9 25554.2 N. D. 72.0
(2) Red 218.2 22.3 112.0 1207.3 15.6 9094.6 N. D. 1327.0
(3) Green 106.1 101.2 91.2 N. D. 4741.8 11643.3 N. D. 38.2
(4) White N. D. N. D. 111.2 N. D. N. D. 80.2 N. D. N. D.
(Unit: ppm)
179
10.9 X-Ray CT Observation of Seals of Food Product Packaging (1)
í1',
Q Explanation
The various types of commercial food product packaging
are designed for ease of use and superior aesthetic design,
while at the same time trying to reduce environmental
impact and ensure or improve safety. Given these design
constraints, the seal portion of packaging is one of the
most technically important areas, where ensuring a
reliable tight seal is extremely important in terms of
safety and quality assurance. Since X-ray CT systems are
able to nondestructively observe the internal structure of
packaging, it is a popular method of observing the seal
status. Therefore, this issue describes several examples
of observing the seal area. In this case, an SMX-100CT-
SV3 Shimadzu Microfocus X-Ray CT System, which is
suitable for objects with relatively low density, was used Fig. 10.9.1 SMX-100CT Shimadzu Microfocus X-Ray CT system
for observations (see Fig. 10.9.1).
QObservation of Plastic Bottle

Fig. 10.9.3 shows CT images of a plastic bottle before reconstructed from CT data. These gures show that even
and after it was opened (photos of the bottle exterior are after it had been opened once, the bottle was securely
shown in Fig. 10.9.2) and Fig. 10.9.4 shows 3D images sealed (in two locations) when the cap was closed.
After Opening
Fig. 10.9.2 Plastic Bottle and Cap
After Opening
Cap
Seal
Bottle
Fig. 10.9.3 CT Image of Plastic Bottle and Cap
180

í1',
After Opening
Fig. 10.9.4 3D Image of Plastic Bottle and Cap
QObservation of Food Packaging (1)

As an example of observing packaging seals, CT cross
section images and a 3D image of the seal between a
cylindrical paper container and its aluminum lid (see
diagram in Fig. 10.9.5) are shown in Fig. 10.9.6. These
images show that the paper is applied to the outside of Image Location
container to improve its strength. It also shows that it is
securely sealed although the aluminum lid is adhered
along the curved surface. Fig. 10.9.5 Image Location
Enlargement of
Adhesion Layer
CT Image 3D Image
Aluminum
Lid
Paper Container
Fig. 10.9.6 Images of Food Packaging
181
í1',
QObservation of Food Packaging (2)
In addition, the joint area was observed on two types
of containers (cylindrical) similar to the one above (see
diagram in Fig. 10.9.7). Image Location
The observation example in Fig. 10.9.8 shows how the
container is joined by gluing 3 or 4 layers of paper together. Fig. 10.9.7 Image Location
CT Image 3D Image
Multiple Paper Layers
Enlargement of Adhesion Layer
Adhesive
The example in Fig. 10.9.9 is packaging for food products how there is aluminum foil between layers of paper and
with a high moisture content. The images show in detail how the container surface is coated with polymer.
CT Image 3D Image
Enlargement of Adhesion Layer
Polymer
Aluminum Foil
Paper
Adhesive
182
INDEX
Item Page Item Page
1-Butanol 137 trans-Sabinenehydrate 79
1-Pentanol 86 (Z)-beta-Farnesene 81
1-Propanol 86 (Z)-Chlorfenvinphos 95, 96, 97
1,8-Cineole 79 њ-Acids 35, 37, 38
2-Butanone 86 њ-Cyclodextrin 68, 109, 110
2-Decanone 86 њ-EG 28
2-Dodecylcyclobutanone (2-DCB) 167, 168 ћ-Acids 35, 37
2-Furanmethanol 86 ћ-Alanine 16, 17
2-Heptanol 86 ћ-Carotene 39
2-Heptanone 86 ћ-Caryophyllene 79
2-Hexenal 84 ћ-Cyclodextrin 68, 69
2-Hexenyl Acetate 84 ќ-Caprolactone 86
2-Isopropyl-4-methylthiazole 84 ќ-Cyclodextrin 68, 69
2-Methoxy-3-isobutylpyrazine (MIBP) 87, 88 ѝ-Decalactone 86
2-Methylbutanal 86 ѝ-Octalactone 86
2-Methylbutanoic acid 84
2-Methyl-4-propyl-1,3-oxathiane 84 A
2-Nonanol 86 Acesulfame K 66, 67
2-Nonanone 86 Acetaldehyde 86
2-Pentadecanone 86 Acetamide 86
2-Pentenal 86 Acetic acid 11
2-Phenylethyl Isothiocyanate 42 Acetoin 86
2-Tetradecanol 86 Acetol 86
2-Tetradecylcyclobutanone (2-TCB) 167, 168 Acetone 137
2-Tridecanone 86 Acetonitrile 137
2-Undecanol 86 Acid Red 72, 73
2-Undecanone 86 Acrinathrin 95, 96, 97
2,4,6-Trichloroanisole 126, 127 Adenine 22
2,5-Dimethyl-3-ethylpyrazine 86 Adenosine 22
3-Hexenyl Acetate 84 Adhumulone 35, 36, 37, 38
3-MCPD 6, 7 Adlupulone 35, 36, 37
3-MCPD-Dioleoyl Ester 6, 7 ADP 23
3-MCPD-Dipalmitoyl Ester 6, 7 Aflatoxins 117, 118, 119, 120, 121
3-Methylbutanal 86 Aflatoxin B1 117, 118, 119, 120, 121
3-Methylbutanote 86 Aflatoxin B2 117, 118, 119, 120, 121
3-Monochloropropane-1,2-diol 6, 7 Aflatoxin B2a 119, 120, 121
3-Penten-2-one 86 Aflatoxin G1 117, 118, 119, 120, 121
5-Methyl-2- (1-methyl-1-sulfanylethyl) cyclohexanone 84 Aflatoxin G2 117, 118, 119, 120, 121
cis-Geraniol 84 Aflatoxin G2a 119, 120, 121
cis-Isoadhumulone 37, 38 Al 131, 133
cis-Isocohumulone 37, 38 Alanine 12, 13, 15, 16, 17
cis-Isohumulone 37, 38 Alcoholic Beverage 16, 17, 28
cis-Resveratrol 32 Aldrin 100
D-Isomenthone 79 Alkylcyclobutanone Method 167, 168
D-Limonene 84, 86 Allergenic Substance 159, 160
(E)-Chlorfenvinphos 95, 96, 97 Alliin 29
Iso-њ-Acids 35, 37, 38 Allura Red AC 72, 73
L-Menthone 79 Allyl Isothiocyanate 42
n-Decanoic acid 84 Alpha-aminobutyric acid 12, 13
Neo-Menthol 79 Alpha-Endosulfan 95, 96, 97
n-Propyl Isothiocyanate 42 Alpha-Pinene 81
p-Hydroxybenzoic acid 33 Alpha-Terpineol 81
p-Menthan-2-one 84 Amaranth 72, 73
trans-Geraniol 84 Amino Acids 12, 13, 14, 15, 16, 17
trans-Isoadhumulone 37, 38 AMP 23
trans-Isocohumulone 37, 38 Animal Hair 134, 135
trans-Isohumulone 37, 38 Antioxidants 62, 63, 64, 65
trans-Resveratrol 32 AOAC 90, 92 183
INDEX
Item Page Item Page
AOCS 4, 5 Camphene 81
Arabinose 26, 27 Camphor 81
Arginine 15, 16, 17 Capsaicin 41
Arsenic 111, 124, 125 Capsaicinoids 41
Artificial Colorings 72, 73 Captan 95, 96, 97
As 111, 124, 125, 179 Carbaryl 100
Asahipak NH2P-50 4D 25 Carbendazim 103, 104
Asahipak NH2P-50 4E 24, 28, 68, 69 Carbofuran 94
Ascorbic acid 64 Carbon disulfide 137
Asparagine 12, 13, 15, 16, 17 Care Food 148, 149
Aspartame 66, 67 Carvone 79
Aspartic acid (ASP) 12, 13, 15, 16, 17 Caryophyllene oxide 81
ATP 23, 141 Cat 134, 135
Azo Rubine 73 Catechin (C) 30, 31
Catechin Gallate (CG) 30
B Cd
B1(Brilliant Blue FCF) 72, 73 109, 110, 111, 112, 113, 114, 124, 169, 177, 178, 179
B2 (Indigo Carmine) 72, 73 CDP 23
Ba 131, 169, 179 Cellobiose 26, 27
Baby Foods 89, 90, 91, 92 Cereal 21, 103, 122
Beer 17, 24, 35, 37, 38, 156, 157, 158 Cheese 61, 65, 86
Benzaldehyde 84, 86 Chicken 134, 135, 142, 143
Benzoic acid 33, 58, 59, 60 Chloroform 137
Benzoyl Peroxide 82 Chlorogenic Acid 33, 34
Benzyl acetate 84 Chloromethoxymethane 137
Beta-Bisabolene 81 Chlorothalonil 95, 96, 97
Beta-Damascenone 84 Chlorpropham 95, 96, 97
Beta-Endosulfan 95, 96, 97 Chlorpyrifos 95, 96, 97
Beta-Ionone 84 Chlorpyrifos-methyl 95, 96, 97
Beta-Linalool 84 Chocolate 138, 150, 151
Beta-Pinene 81 Cineole 79
BHA 62, 63 Citric acid 11
BHT 62, 63 Citruline 15, 16, 17
Bifenthrin 95, 96, 97 Citrus Juice 51, 52
Bilobalide 46 Citrus Oil 93, 94
Bisdemethoxycurcumin 75, 76 Cl 132, 133
Black PN 73 CMP 23
Black Soybeans 40 Coenzyme Q10 57
Bornyl acetate 81 Cohumulone 35, 36, 37, 38
Brandy 140 Colupulone 35, 36, 37
Brilliant Blue FCF 72, 73 Contaminants 130, 131, 132, 133, 134, 135
Bromopropylate 95, 96, 97 Corn 129, 139, 163, 164
Brown Rice 113, 114 Cow 134, 135
Buckwheat 159, 160 CP-PoraBOND Q FUSED SILICA 174, 175
Bupirimate 95, 96, 97 Cr 107, 108, 131, 179
Buprofezin 95, 96, 97 Crab 160
Butanal 86 CTP 23
Butanoic acid 86 Cu 107, 108
Butter 3, 62, 63 Curcumin 74, 75, 76
Butyl acetate 84 Curry 77, 101, 102, 124, 125
Cyanidin-3-Glucoside 40
C Cyanocobalamin 20, 21
C
6 131 Cyclodextrin 68, 69, 109, 110
Ca 107, 108, 109, 131, 132, 133 Cyfluthrin 95, 96, 97
Cadmium 109, 110, 111, 113, 114, 124, 177, 178 Cyhalothrin 95, 96, 97
Caffeic Acid 33 Cymene 81
Caffeine 30, 31 Cypermethrin 95, 96, 97
184 Calcium carbonate 132, 133 Cyprodanil 92
INDEX
Item Page Item Page
Cyprodinil 95, 96, 97 Epigallocatechin (EGC) 30
Cys-Cys 15 Epigallocatechin Gallate (EGCG) 30
Cystine 16 Epigallocatechin 3-(3-”O -methyl) gallate (EGCG3M” e) 30
Cytidine 22 Epigallocatechin 3-(4-”O -methyl) gallate (EGCG4M” e) 30
Cytosine 22 Erythorbic acid 64
Erythritol 70, 71
D Erythrosine 72, 73
Daidzein 43 Essential Oil 79
Daidzin 43 ETAAS 109, 111, 112
DB-1 3 Ethanol 137, 157
Decanoic acid 84, 86 Ethion 95, 96, 97
Dehydroacetic Acid 60 Ethofenprox 95, 96, 97
Delta-Decalactone 84 Ethoprophos 95, 96, 97
Delta-Dodecalactone 84 Ethyl acetate 84, 86
Delta-Hexylvalerolactone 84 Ethyl butanoate 84
Deltamethrin 95, 96, 97 Ethyl butyrate 86
Delta-Undecalactone 84 Ethyl caproate 84
Demethoxycurcumin 75, 76 Ethyldecanoate 86
Deoxynivalenol 122, 123 Ethyl њ-D-Glucoside 28
Diarrhetic Shellfish Poison (DSP) 115, 116 Ethylhexanoate 86
Diazinon 90, 95, 96, 97 Ethyl Isothiocyanate 42
Dichlofluanid 95, 96, 97 Ethyloctanoate 86
Dicloran 95, 96, 97 Ethyl p-hydroxybenzoate 58
Dietary Supplement 45, 46, 47, 56, 107, 108 Eucalyptol 81
Dihydrocapsaicin 41 Eudesm-7 (11) -en-4-ol 84
Dihydrocarvone 79 Eurotium chevalieri 165, 166
Dimethoate 102
Dimethyl disulfide 86 F
Dimethylpyrazine 86 F 131
Dimethyl sulfide 137 Fast Green FCF(G3) 72, 73
Dimethyl sulfone 86 Fast Red E 73
Dinophysistoxin-1 (DTX-1) 115 Fat 8, 9
Diphenylamine 95, 96, 97 Fatty acid 1, 2, 3, 4, 5, 6, 7, 167
Discharge pipe packing 132, 133 Fe 107, 108, 131, 133
DNA 134, 135, 152, 153, 154, 155, 159, 160, 161 Fenarimol 95, 96, 97
163, 164, 165, 166 Fenazaquin 95, 96, 97
Dodecanoic acid 86 Fenitrothion 95, 96, 97
Dog 134, 135 Fenpropathrin 95, 96, 97
Dog Food 112 Fenthion 95, 96, 97
DSP 115, 116 Fenvalerate 95, 96, 97
Ferulic acid 33
E Fipronil 95, 96, 97
E. coli LT 162 Fish 1, 2, 141, 154
E. coli STh 162 Flavanones 51, 52
E. coli STp 162 Flavonoids 44, 45, 46
E. coli VT1 162 Flour 43, 82, 118, 121, 122, 123, 129, 139
E. coli VT2 162 Flusilazole 95, 96, 97
E. coli VT1&2 162 Folic acid 20, 21
EI-MRM 1, 2 Folpet 95, 96, 97
EI-SIM 1, 2 Food Product Packaging 180, 181, 182
Elaidic acid 3 Formic acid 11
Ellagic acid 34 Fructofuranosylnystose 25
Endosulfan sulfate 95, 96, 97 Fructo-oligosaccharide 25
Epicatechin (EC) 30 Fructose 25, 28
Epicatechin Gallate (ECG) 30 Fruit crops 103
Epicatechin 3-(3-”O -methyl) gallate (ECG3M” e) 30 Functional Beverage 71
Epicatechin 3-(4-”O -methyl) gallate (ECG4M” e) 30 Functional Drink 69
Epichlorohydrin 169, 171 Fungicide 103 185
INDEX
Item Page Item Page
G I
G3 (Fast Green FCF) 72, 73 IDP 23
GABA (ќ-Aminobutyric Acid) 15, 16, 17 IMP 23
Galactose 26, 27 Imtakt Scherzo SM-C18 20
Gallic Acid (GA) 30, 33 Indigo Carmine 72, 73
Gallocatechin (GC) 30 InertCap 17MS 87
Gallocatechin Gallate(GCG) 30 InertCap Pure-WAX 83, 85
Gamma-Butylbutyrolactone 84 Inosine 23
Gamma-Caprolactone 84 Inositol 70
Gamma-Dodecalactone 84 Iprodione 95, 96, 97
Gamma linolenic acid 3 Irradiated Food 167, 168
Gamma-n-Amylbutyrolactone 84 Isoamyl acetate 84
Gamma-Terpinene 81 Isoborneol 81
Garlic 29 Isobutyl alcohol 86
GDP 23 Isobutyl isovalerate 84
Genistein 43 Isobutyric acid 86
Genistin 43 Isoflavones 43
Geraniol 84 Isoleucine 12, 13, 15, 16, 17
Germacrene D 81 Isopropyl alcohol 137
Ginger 49, 50 Isorhamnetin 44, 45
Gingerol 49, 50 Isothiocyanates 42
Ginkgo Biloba 44, 45, 46, 47, 48 Isovaleraldehyde 86
Ginkgolic Acid 47, 48 ITP 23
Ginkgolide 46
Ginseng 53, 54 K
Ginsenoside 53, 54 K 107, 108, 124, 131,133
Glucose 24, 25, 26, 27, 28, 71 Kaempferol 44, 45
Glutamic Acid 12, 13, 15, 16, 17 Kestose 25
Glutamine 12, 13, 15, 16, 17 .LQHWH[ѥP&c 22
Glyceryl trioleate 4 .LQHWH[ѥP&c 18, 72
Glycine 12, 13, 15, 16, 17
Glycyrrhizic acid 66 L
GMP 23 Lactitol 70
Goat 134, 135 Lactose 26, 27
Green S 73 Leach Test 169, 170
Green Tea 30, 31, 64, 69 Lead 105, 106, 109, 110, 111, 115, 177, 178, 179
GTP 23 Leucine 12, 13, 15, 16, 17
Guanine 22 Limonene 79, 81, 93
Guanosine 22 Linalool 81, 84
Linalyl acetate 81
H Linoleic acid 3
HALO®C18 30, 31, 35, 37 Lupulone 35, 36, 37
Happ-Genzel 130, 132 Lutein 55, 56
Heptaose 24 Lycopene 39
Hesperetin 51 Lysine 12, 13, 15, 16, 17
Hesperidin 51, 52
Hexanal 86 M
Hexanoic acid 86 Magnesium 132, 133
Hexyl acetate 84 Magnesium silicate 132, 133
Hg 179 Malathion 90, 95, 96, 97
HILIC 28, 64, 68, 70 Malonic acid 11
Histidine 12, 13, 15, 16, 17 Malonyldaidzin 43
Hop 35, 36 Malonylgenistin 43
Horse 134, 135 Malonylglycitin 43
Horseradish 42, 69 Maltitol 70, 71
Humulone 35, 36, 37, 38 Maltoheptaose 24
Hydrosphere C18 10 Maltose 24, 26, 27, 28
186 Hypoxanthine 23 Maltotriose 24
INDEX
Item Page Item Page
Mannitol 70 Okadaic Aid (OA) 115, 116
Mannose 26, 27 Oleic acid 3
Melamine 111, 128, 129, 169 Oligosaccharide 24, 25, 68, 109
Menthol 79 OPA 14, 16, 29
Menthone 79 OPTIC-4 83, 84, 85, 86, 87
Menthoturan 79 Orange Ⅱ 73
Mepanipyrim 95, 96, 97 Orange G 73, 77
Metalaxyl 94, 95, 96, 97 Orange Juice 103, 104
Methanethiol 86 Orange Oil 93, 94
Methidathion 95, 96, 97 Organic Acid 11
Methionine 12, 13, 15, 16, 17 Organic Orange Oil 93, 94
Methyl cis-11,14,17-Icosatrienoate 1, 2 Organophosphorus Pesticides 89, 90
Methyldecanoate 86 Ornithine 16, 17
Methylhexanoate 86 Orotic acid 10
Methyl linolenate 1, 2 Oxadixyl 95, 96, 97
Methyl Methacrylate 169, 176
Methyl p-hydroxybenzoate 58 P
Mg 107, 108, 132, 133 P 131, 133
Milk 8, 9, 129 Palatinol 70
Minerals 107, 108, 132 Palmitic acid 3, 167
Mn 107, 108, 131 Palm Oil 6, 7
Mo 107, 108 Pantothenic acid 20, 21
Mold 61, 165, 166 Para Red 78
MonoTrap 83, 84, 85, 86, 87, 88 Parathion 90, 95, 96, 97
Mouse 134, 135 Parathion-methyl 95, 96, 97
Multivariate analysis 8, 9, 156, 157, 158 Patent Blue V 73
Mycyclobutanil 92, 95 Pb 105, 106, 107, 109, 110, 111
Myrcene 81 112, 113, 169,177, 178, 179
PCI-MRM 1, 2
N PCI-SIM 1, 2
Na 131, 133 PCR 134, 135, 152, 153, 154, 155, 159
NaAsO2 124 160, 161, 163, 164, 165,166
Natamycin 61 Peach Juice 34, 83, 84
Naringenin 51 Peanuts 159, 160
Naringin 51, 52 Pectenotoxin (PTX) 115, 116
Narirutin 51, 52 Pendimethalin 95, 96, 97
Nb 133 Penicillium digitatum 165, 166
Neohesperidin 51, 52 Pepper Sauce 41
Neotame 66 Permethrin 95, 96, 97
Neryl acetate 81 Pesticide 89, 90, 91, 92, 93, 94, 95, 96
New Coccine 72, 73 97, 98, 99, 100,101, 102, 103
Ni 131 Pet Food 111, 112
Niacin 18 PHBA butyl 58, 59
Nicotinamide 18, 20, 21 PHBA ethyl 58, 59
Nicotinic acid 20, 21 PHBA methyl 58, 59
NIES No.10 113, 114 PHBA propyl 58, 59
Nivalenol 122, 123 Phenol 169, 170
Nootkatone 84 Phenolic Acids 33, 34
Nordihydrocapsaicin 41 3KHQRPHQH[.LQHWH[ѥP&c 72
Norvaline 12, 13 Phenomenex LUNA NH2 64
Nucleobase 22, 23 Phenomenex Luna PFP (2) 66
Nucleoside 22, 23 Phenomenex Synergi Hydro-RP 11, 122
Nucleotide 22, 23 3KHQRPHQH[6\QHUJLѥP3RODU53 53
Nystose 25 Phenthoate 95, 96, 97
Phenylalanine 12, 13, 15, 16, 17
O Phenyl Isothiocyanate 42
Octanoic acid 86 Phloxine 72, 73
Octyl acetate 84 Phosalone 95, 96, 97 187
INDEX
Item Page Item Page
Phytoalexin 32 Ronnel 90
Pig 134, 135 Rose Bengale 72, 73
Pirimicarb 95 Rtx-5MS 58, 62, 80
Pirimiphos-methyl 95, 96, 97 Rtx-200MS 99, 100, 101, 102
Pizza 130, 131 Rtx-WAX 3, 171
PLS 8, 9 Rubber Nipple 169, 170
Polycarbonate 169, 172, 173 Rxi-5MS 126, 167
Polymethyl Methacrylate 176 Rxi-5Sil MS 95, 97, 99, 100, 101, 102
Polyvinyl Chloride 169, 171, 175
Polyvinylidene Chloride 169, 174 S
Pork 147, 167 S 131, 132, 133
Prawn 159, 160 Saccharin 66
Preservatives 58, 59, 60, 109 Saccharomyces cerevisiae 165, 166
Processed Foods 29, 33, 100, 101, 102 Salicylic acid 33
Procymidone 95, 96, 97, 98 Salmonella 161, 162
Profenofos 95, 96, 97 Saury 1, 2
Proline 12, 13, 15, 16, 17 Sb 169, 179
Propiconazole 95, 96, 97 SBS 132, 133
Propyl p-hydroxybenzoate 58 Se 107, 108, 179
Propyzamide 95, 96, 97 Serine 12, 13, 15, 16, 17
Protocatechuic acid 33 Sheep 134, 135
Pyridaben 95, 96, 97 Shim-pack Amino-Na 29
Pyridine 137 Shim-pack CLC-C8 47
Pyridoxine 18, 20, 21 Shim-pack CLC-SIL (M) 65
Pyrimethanil 95, 96, 97, 100 Shim-pack FC-ODS 19, 46, 57, 78, 120
Pyriproxyfen 95, 96, 97 Shim-pack GVP-ODS 40
Shim-pack ISA-07/S2504 26
Q Shim-pack VP-ODS
QuEChERS 89, 90, 91, 92, 95, 96, 97, 98, 99, 101 33, 39, 40, 41, 44, 55, 61, 75, 77, 82, 116
Quercetin 44, 45 Shim-pack XR-ODS
Quick-DB 99, 100, 101, 102 34, 42, 43, 49, 51, 55, 60, 74, 75, 128
QuinolineYellow S 73 Shim-pack XR-ODS Ⅱ 33, 117
Quinoxyfen 98 Shim-pack XR-ODS Ⅲ 22, 23, 32
Shim-pack XR-Phenyl 34
R Shim-pack XR-SIL 65
R2 (Amaranth) 72, 73 Shogaol 49, 50
R3 (Erythrosine) 72, 73 Shrimp 136, 137
R40 (Allura Red AC) 72, 73 Si 131, 132, 133
R102 (New Coccine) 72, 73 Sinapic acid 33
R104 (Phloxine) 72, 73 Sinigrin 42
R105 (Rose Bengal) 72, 73 Soft Drink 11, 15, 59, 60, 64, 67
R106 (Acid Red) 72, 73 Sorbic Acid 58, 59, 60
Rabbit 134, 135 Sorbitol 70, 71
Rat 134, 135 Soy 43
Red 2G 73 Soybean Flour 43
Red Pepper 41 Soybean Paste 43, 165
RESTEK Stabilwax 137 Soymilk 43
Resveratrol 32 Soy sauce 6, 59, 176
Retinol 19 SP-2560 2
Retinol acetate 19 Spice 41, 49
Retinol palmitate 19 Sr 133
Rhamnose 26, 27 Staphylococcus aureus EA 162
Riboflavin 18, 20, 21 Staphylococcus aureus TSST-1 162
Riboflavin phosphate 18 Starch 131, 139, 144
Ribose 26, 27 Stearic acid 3, 167
Rice 28, 113, 114, 146, 148, 152, 153 Stirophos 90
Rice gruel 148 Styrene butadiene styrene 132
188 Rice Variety 152, 153 Sucralose 66, 67
INDEX
Item Page Item Page
Sucrose 25, 26, 27, 28, 66, 139 Tryptophan 12, 13, 15, 16, 17
SudanⅠ 77, 78 TTP 23
Sudan Ⅱ 77, 78 Tuna 154, 155
Sudan Ⅲ 77, 78 Turmeric 74, 75
Sudan Ⅳ 77, 78 Tyrosine 12, 13, 15, 16, 17
Sudan Dyes 77, 78
Sudan Orange G-a 77 U
Sudan Orange G-b 77 92U 131
Sudan Red 7B 77 UDP 23
Sudan Red G 77 ULBON HR-20M 79
Sugar Alcohols 70, 71 UMP 23
Sugarless Candy 71 Unison UK-Amino 70
Sugarless Mint Tablet 71 Uracil 22
Sukiyaki 148 Uridine 22
Sunset Yellow FCF 72, 73 USP32 45, 48
SVR 8, 9 UTP 23
Sweeteners 66, 67, 70, 109
6\QHUJLѥP3RODU53c 53 V
Syringic acid 33 Valine 12, 13, 15, 16, 17
Syrup 25 Vanillic acid 33
Vegetable juice 12, 13
T Vegetable soup 148
Tap Water 132, 133 Vermicelli 144, 145
Tartrazine 72, 73 Vibrio parahaemolyticus 162
Tau-Fluvarlinate 95, 96, 97 Vinclozolin 95, 96, 97
Taurine 16 Vinyl Chloride 169, 171,175
TBHQ 62, 63 Vinylidene Chloride 169, 174
TCA 126, 127 Vitamin B12 21
TDP 23 Vitamin C 62
Tebuconazole 95, 96, 97
Tebufenpyrad 95, 96, 97 W
Tefluthrin 95, 96, 97 Water-Soluble Vitamins 18, 20, 21, 107
Terpenoids 46 Wheat 118, 129, 144, 159, 160
Terpinene-4-ol 79 Whey 10
Terpineol 84 Whiskey 140
Tetraconazole 95, 96, 97 Wine 17, 32, 34, 87, 88, 126, 127, 165
Tetradifon 95, 96, 97
Tetrahydrofuran 137 X
Theanine 16 Xylose 26, 27
Thiamine 18, 20, 21 Xylitol 70, 71
Threonine 12, 13, 15, 16, 17
Thymidine 22 Y
Thymine 22 Y4 (Tartrazine) 72, 73
Ti 132, 133 Y5 (Sunset Yellow FCF) 72, 73
TMP 23 Yeast 61, 165, 166
Tocopherols 65 Yessotoxin 115
Tomato 39 YMC-Triart C18 14, 16
Tolclofos-methyl 95, 96, 97 Yogurt 10
Toluene 86, 137
Tolylfluanid 95, 96, 97 Z
Trans Fatty Acid 4, 5 ZB-AAA 12
Triacetin 84 Zeaxanthin 55, 56
Triazophos 95, 96, 97 Zn 107, 108, 132, 133, 169
Tributylamine 172, 173 Zr 133
Trielaidin 4, 5
Triethylamine 172, 173
Trifluralin 95, 96, 97
Trimethylamine 136, 137 189
Company names, product/service names and logos used in this publication are trademarks and trade names of Shimadzu Corporation or its
affiliates, whether or not they are used with trademark symbol “TM” or “®”.
Third-party trademarks and trade names may be used in this publication to refer to either the entities or their products/services. Shimadzu
disclaims any proprietary interest in trademarks and trade names other than its own.
For Research Use Only. Not for use in diagnostic procedures.

The contents of this publication are provided to you “as is” without warranty of any kind, and are subject to change without notice. Shimadzu
does not assume any responsibility or liability for any damage, whether direct or indirect, relating to the use of this publication.
www.shimadzu.com/an/ © Shimadzu Corporation, 2014

C180-E059C - Small-Shimadzu Analysis Guide Book

Uploaded by

Copyright:

Available Formats

C180-E059C - Small-Shimadzu Analysis Guide Book

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

C180-E059C - Small-Shimadzu Analysis Guide Book

Uploaded by

Copyright:

Available Formats

Shimadzu Analysis Guidebook

H O 1. Food Product Components 2. Food Additives

Q Pretreatment Method QAnalysis Results

Methyl cis -11,14,17- Icosatrienoate;(Z)20:3n-3

2.0 3.0 0.75

1.2 Analysis of Fatty Acids in Butter Using GC × GC/MS - GC/MS

Fig. 1.2.1 2-Dimensional Image of GC × GC-MS Analysis Results for Butter

used the latter method, which features easier operation. H2

1.3 Quantitative Analysis of Trans Fatty Acid by FTIR (2) - FTIR

Trielaidin 0.1 % content 0

Abs Trielaidin (10.0 % spiked)

1000 990 980 970 960 950 940 930 1/cm

Q Measurement of Trans Fatty Acid by

Q Explanation GC/MS following derivatization with phenylboronic acid (DGF

10 V [M+H-C16H32O2]+ 1.0 10 V [M+H-C18H34O2]+

QCalibration Curve QAnalytical Conditions

Fig. 1.4.3 Calibration Curves (1–1000 μg/L, n = 6)

1:604.40>331.10(+) 200 2:656.50>357.20(+)

vector regression method (SVR) were used to conduct

low-fat samples of Table 1.5.1 were used as standard samples to

standard samples. As for the support vector regression method,

content samples of Table 1.5.1 were used as veri cation samples

High fat 3 (1st) 9.5 8.87（6.6 %） 9.57（0.7%） 9.72（2.3 %）

QConclusion spectral transmission measurement of liquid samples

Q Explanation QAnalytical Conditions

Fig. 1.6.1 Structure of Orotic Acid 0 1 2 3 4 5 6 7 8 9 min

Fig. 1.6.3 Chromatogram of Yogurt (10 μL injected)

1.7 High Speed Analysis of Organic Acids - LC

Q Explanation QAnalysis of Soft Drinks

1 Alanine 8 Leucine 14 As paragine 23 Glutam ine

1.8 Analysis of Amino Acids Contained in Vegetable Juice (2) - GC/MS

1 Alani ne 8 Leucine 14 As paragi ne 23 Glutam i ne

QSimultaneous Determination of 22 Amino Acids )02&ѥ/

1.9 High Speed Analysis of Pre-Column Derivatized Amino

0.0 2.5 5.0 7.5 min

Fig. 1.9.2 Chromatogram of 22 Amino Acids (10 μmol/L Each, 1 μL Injection)

QLinearity and Repeatability QAnalysis of Actual Samples

Arg 0.45 Lys 0.56 0.0 2.5 5.0 7.5 min

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

1.10 High Speed Analysis of Pre-Column Derivatized Amino

Typical Analysis Cycle

the autosampler begins to prepare the next sample by adding Analysis

reagent and mixing the contents. Fig. 1.10.2 shows a ow diagram

QAnalysis of Alcoholic Beverages

Q Explanation QAnalytical Conditions

Fig. 1.11.1 Chromatogram of a Standard Mixture Solution of 6 Water-Soluble Vitamins

1.12 High-Sensitivity Analysis of Retinol Acetate and

QAnalysis of Standard Solution QAnalysis of Multivitamin Tablets

0.0 2.0 4.0 6.0 8.0 10.0 min

Fig. 1.12.2 Chromatogram of a Standard Mixture of Retinol Acetate

Fig. 1.13.2 Mass Spectra of Water-Soluble Vitamins

1.13 Analysis of Water-Soluble Vitamins with Multi-Mode

Fig. 1.13.3 Calibration Curves of Water-Soluble Vitamins

QQuantitative Analysis of Water-Soluble

Fig. 1.13.5 SIM Chromatograms of Extract from Cereal

analytical conditions. The system back pressure during

Fig. 1.14.1 Chromatogram of a Standard Mixture of Nucleobases

1.14 High Speed Analysis of Nucleobases, Nucleosides,

QSimultaneous Determination of 22 Amino Acids )02&ѥ/

Time Program : %PLQ¤PLQ 20 Water 4 mL

¤PLQ 10 Filtration (0.2 +m)

Injection Volume : ƫ/

Injection Volume : ƫ/ 0.0 0.5 1.0 min HPLC

Detection : 63'0$QP