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Analysis of Natural Food Colorants by

Electrospray and Atmospheric


Pressure Chemical Ionization LC/MS

Application

Food

Author deionized water. Each colorant was filtered


through a 0.2-µm filter. A 10-µL portion was
Masahiko Takino injected into the system, which consisted of an
Yokogawa Analytical Systems, Inc. Agilent 1100 Series binary pump, thermostatted
USA column compartment, vacuum degasser, autosam-
pler, and LC/MSD. The LC/MSD used either an ESI
Originally published April 2000 or APCI source. Complete system control and data
handling were done on the Agilent ChemStation
Abstract for LC/MS. Operating conditions were optimized
for each sample.
Four commercial natural colorants – red cabbage,
Monascus, paprika, and lac – were analyzed using ESI
and APCI-LC/MS. The MS data provided molecular Results and Discussion
weight information and some structural information for
the major components in these pigments. Red Cabbage Colorant

Figure 1 shows the structure of seven major pig-


Introduction ments of red cabbage. The pigments share the
Many kinds of natural colors are used in beverages, basic cyanidin 3-diglucoside structure with differ-
jellies, and candies. In many countries, food regula- ing R1 and R2 groups. Figure 2 shows the total ion
tions have been recently revised to cover natural chromatogram (TIC) and extracted ion chro-
colorants to the same degree as synthetic ones. matograms (EICs) of red cabbage pigments.
Accordingly, it has become necessary to develop Although every major pigment can be chromato-
reliable analytical methods for various natural col- graphically separated using 10% formic acid in the
orants in food. In this study, LC/MS methods using mobile phase, the high acid concentration reduces
electrospray ionization (ESI) or atmospheric pres- sensitivity. Therefore, 1% formic acid was used in
sure chemical ionization (APCI) were developed to this study. The EICs show the separation of the
identify major pigments in four natural colorants: pigments based on their main ion (base peak).
red cabbage, paprika, Monascus, and lac.
Figure 3 shows the mass spectra of the seven
major pigments in red cabbage colorant. For these
Experimental pigments, the singly charged molecular ion is

Paprika and Monascus colorants were dissolved in


acetone; the other colorants were dissolved in
Figure 1. The structure of major pigments in red cabbage colorant.

Figure 2. Total and extracted ion chromatograms of red cabbage colorant.

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LC Conditions
Column: Inertsil ODS3, 250 mm × 2.1 mm, 5 µm
Mobile phase: A = 1% formic acid
B = acetonitrile
Gradient: Start with 5% B
At 30 min 50% B
Flow rate: 0.2 mL/min
Column temperature: 40 °C
Injection volume: 10 µL
MS Conditions
Source: ESI
Ion mode: Positive
Vcap voltage: 4000 V
Nebulizer: 50 psig
Drying gas flow: 10 L/min
Drying gas temp: 350 °C
Corona: 4 µA
Vaporizer temperature: 350 °C
Scan range: 100–1200 amu
Step size: 0.1
Peak width: 0.15 min
Time filter: On
Fragmentor: 200 V

observed rather than the more typical [M+H]+ ion,


because the cyanidin group already has a positive
charge on an oxygen. In-source collision-induced
dissociation (CID) can be used to generate frag-
ment ions to provide structural confirmation.
Using CID, mass spectra of these pigments show
common fragments corresponding to the loss of a
glucose, as well as cyanidin (m/z 287) and cyanidin
3-glucoside (m/z 449) ions.

Monascus Colorant

Monascus contains six major pigments; their struc-


tures are shown in Figure 4. Four pigments were
identified from the mass spectra of major peaks in
the TIC. See Figure 5.

Figure 3. Mass spectra of major pigments in red cabbage


colorant.

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Figure 4. The major pigments of Monascus colorant.

LC Conditions
Column: Inertsil ODS3, 250 mm × 2.1 mm, 5 µm
Mobile phase: A = 1% formic acid
B = acetonitrile
Gradient: Start with 50% B
At 10 min 90% B
Flow rate: 0.2 mL/min
Column temperature: 40 °C
Injection volume: 10 µL
MS Conditions
Source: ESI
Ion mode: Positive
Vcap voltage: 4000 V
Nebulizer: 50 psig
Drying gas flow: 10 L/min
Drying gas temp: 350 °C
Corona: 4 µA
Vaporizer temperature: 350 °C
Scan range: 100–1200 amu
Step size: 0.1
Peak width: 0.15 min
Time filter: On
Figure 5. The total ion chromatogram of Monascus colorant. Fragmentor: 100 V

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Figure 6. Mass spectra of major pigments in Monascus colorant.

Three major peaks with base peaks at m/z 439, The protonated molecular ions [M+H]+ were
467, and 495 were not identified. Figure 6 shows observed for every major pigment. See
the mass spectra of the identified pigments. Proto- Figure 9. However, with the exception of capsan-
nated molecular ions [M+H]+ were observed for the thin monoeicosanoate, the intensity of these ions
four identified pigments. was very low. Except for capsanthin monoeico-
sanoate, the pigments show fragment ions result-
Paprika Color ing from the loss of one or two fatty acid
fragments. A common fragment ion was observed
Capsanthin and the mono- and di- esters of cap- at m/z 567 in the mass spectra of these pigments.
santhin with fatty acids are known as the major
pigments in paprika colorant. See Figure 7. Two
monoesters and five diesters of capsanthin were
identified in the paprika colorant analyzed in this
study. See Figure 8.

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Figure 7. The structure of major pigments of paprika colorant.

Figure 8. The total ion chromatogram of paprika colorant.

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LC Conditions
Column: Inertsil ODS3, 250 mm × 2.1 mm, 5 µm
Mobile phase: A = acetone
B = methanol
Gradient: Start with 10% B
At 10 min 90% B
Flow rate: 0.2 mL/min
Column temperature: 40 °C
Injection volume: 10 µL
MS Conditions
Source: APCI
Ion mode: Positive
Vcap voltage: 4000 V
Nebulizer: 50 psig
Drying gas flow: 5 L/min
Drying gas temp: 350 °C
Corona: 4 µA
Vaporizer temperature: 350 °C
Scan range: 100–1200 amu
Step size: 0.1
Peak width: 0.15 min
Time filter: On

Lac Colorant

Figure 10 shows the structure of the major pigments


in lac colorant. Laccaic acids A, B, and C are
known as the major pigments in lac colorant.
These compounds have the same basic
anthraquinone structure but with different R
groups. Three major peaks were detected in the
TIC. See Figure 11. Although laccaic acids A, B,
and C were identified, A and B could not be sepa-
rated.

Figure 12 shows the mass spectra of two peaks,


laccaic acid C and a combination of laccaic acids A
and B. The deprotonated molecular ions were
observed at m/z 495, 536, and 538. Fragment ions
resulting from the loss of carbon dioxide were
observed at m/z 451, 492, and 494.

Figure 9. Mass spectra of major pigments in paprika colorant.

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Figure 10. The structure of major pigments of lac colorant. Figure 11. The total ion chromatogram of lac colorant.

LC Conditions
Column: Inertsil ODS3, 250 mm × 2.1 mm, 5 µm
Mobile phase: 30% acetonitrile in 5 mM dibutylamine,
isocratic
Flow rate: 0.2 mL/min
Column temperature: 40 °C
Injection volume: 10 µL
MS Conditions
Source: ESI
Ion mode: Negative
Vcap voltage: 4000 V
Nebulizer: 50 psig
Drying gas flow: 10 L/min
Drying gas temp: 350 °C
Scan range: 100–1200 amu
Step size: 0.1
Peak width: 0.15 min
Time filter: On
Fragmentor: 100 V

For More Information


Figure 12. Mass spectra of major pigments in lac colorant. For more information on our products and services,
visit our Web site at www.agilent.com/chem.

Conclusions
Four commercial natural colorants were analyzed
using ESI and APCI-LC/MS. The MS data provided
molecular weight information and some structural Agilent shall not be liable for errors contained herein or for incidental or consequential
damages in connection with the furnishing, performance, or use of this material.
information for the major pigments
Information, descriptions, and specifications in this publication are subject to change
without notice.

© Agilent Technologies, Inc. 2008

Printed in the USA


May 20, 2008
5989-8442EN

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