Single Laboratory Validation of a Method for the Determination of

β-Carotene in Supplements and Raw Materials by Reversed-Phase

High Performance Chromatography
Abstract/Summary

A single laboratory validation study (SLV) was conducted for an HPLC method for the
determination of total and all-trans-β-carotene in a variety of dietary supplements including
multivitamin tablets, softgels, capsules, and beadletted raw materials. Extraction variants were
developed for the different types of supplements tested based upon the supplement type and level
of β-carotene. The procedure consisted of enzymatic digestion, extraction with ethanol:
dichloromethane, dilution/concentration as appropriate, and HPLC analysis of the final sample
solution using either a reverse phase C18 column or in products containing high amounts of α-
carotene, a C30 column. The systems were linear in the range of 0.1-50 µg β-carotene/ ml. The
main mono-cis-β-carotene isomers were well separated from all-trans-β-carotene as well as from
α-carotene, lycopene, lutein, and zeaxanthin. Double determinations performed with eight
different test materials by two analysts on five different days resulted in relative standard
deviations of 1.2 to 4.4%. Recoveries determined for supplements and beadletted raw material
spiked with β-carotene levels of 10µg to 100 mg/test portion and 0.2 to 40%, respectively, ranged
from 97.5 to 102.1%. On the basis of the accuracy, precision, and recovery results for this SLV
study, it is recommended that this method be collaboratively studied for the determination of β-
carotene in dietary supplements.

Introduction

Beta-carotene is generally regarded as the most commercially important and widely utilized
carotenoid. It is used as a food coloring agent, an antioxidant, and an important and safe pro-
vitamin A source (1-3). β-carotene is currently incorporated in a wide variety of dietary
supplements including multivitamins, vitamin A, and antioxidant formulations. Recently,
additional carotenoids, including lutein and lycopene, have been the subjects of nutritional studies
and are now also incorporated, both in combination products and in dietary supplements available
to the general public. Spectrophotometric analytical procedures are not capable of accurately
determining β-carotene in such combination products and in products containing α-carotene.
Spectrophotometric methods alone also cannot sufficiently differentiate between all-trans-β-
carotene and cis-isomers of β-carotene, which may be formed during processing (4-11) and are
reported to have different biological activities (12-15). Schierle et al. Have reported a
spectrophotometric procedure for the determination of total β-carotene in food additives with
varying cis-/trans- ratios using an isobestic wavelength (16). β-carotene and carotenoids in
general have been intensively studied using high performance liquid chromatography (HPLC)
and procedures reported for the separation of β-carotene cis-/trans- isomers (17-22). Thus, a
chromatographic procedure capable of separating all-trans-β-carotene from other commercially
used carotenoids was deemed necessary to properly determine its content in dietary supplements.

A. Principle

Water dispersible formulations such as powders, emulsions, tablets, and capsules are digested
with protease and extracted with dichloromethane and alcohol. Oily suspensions are dissolved
directly in dichloromethane and alcohol. The extract is chromatographed on a C18 isocratic
HPLC system that separates the predominant geometrical isomers of β-carotene from each other

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and from other carotenoids such as all-trans-α-carotene, lycopene, cryptoxanthin, lutein, and
zeaxanthin. In the case of products with relatively high α-carotene content, the cis- isomers of α-
carotene can interfere. In this case, the extract is also chromatographed with a more selective but
longer running C30 reversed phase HPLC system that avoids this interference

Study Design:

Applicable to the determination of 0.01 mg or greater concentrations of total β-carotene in tablets,

capsules, and more than 0.2% of β-carotene in oily or water-dispersible raw materials in the
presence of other carotenoids such as lycopene, α-carotene, and xanthophylls.

Test Sample Preparation:

Source of materials: the dietary supplement test materials used in this study were obtained from
commercial sources and provided by AOAC International. β-carotene raw materials (beadlet
product form) and reference materials were provided by Roche Viatamins Ltd., Kaiseraugst,
Switzerland.

Test Materials:

Test materials were chosen to represent the range of dietary supplements containing
β-carotene currently available to the general public and included the following:

1. Multivitamin tablets, Claim: Vitamin A 3500 IU, 29% as β-carotene.

2. Softgels with β-carotene in soybean oil, Claim: β-carotene 25,000 IU
3. Capsules with β-carotene in fish oil, no claim for β-carotene
4. Softgels with β-carotene from carrot oil extract, Claim: Vitamin A (as
β-carotene) 10000 IU
5. Tablets with β-carotene, Claim: 6 mg β-carotene
6. Capsules with β-carotene, Claim 25 mg β-carotene
7. Softgels with Vitamin A (as β-carotene) and Vit E, Claim: Vitamin A 25000 IU (as β-
carotene).
8. Beadlets raw material with pure β-carotene, Claim: 20% β-carotene

Test materials were stored at 5°C in the dark (refrigerator).

Negative Control Materials

The negative control materials for this study were the following:

1. Placebo Multivitamin tablets, Reference No. 81.16.03.32 (Hoffmann-La Roche),

composed according to formula No. 80.23 (see Attachment No.2)
2. Lycopene 10% WS Placebo beadlets, Lot UT02071003 (Hoffmann-La Roche),
containing fish gelatin, saccharose, vitamin C palmitate, maize oil, DL-α-tocopherol,
water

Negative control materials were stored at 5°C in the dark (refrigerator).

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B. Apparatus

(a) Balances. –– Balance with readability of 0.01 mg, precision (std dev.) of ± 0.015 mg,
capacity of 205 g (AT261 DeltaRange, Mettler-Toledo, CH-8606 Nänikon-Uster,
Switzerland); balance with readability of 0.01g, precision (std dev.) of ± 0.005 g, capacity
of 2100 g (PM2000, Mettler-Toledo, CH-8606 Nänikon-Uster, Switzerland)

(b) Spectrophotometer. –– Dual beam, wavelength range of 190-900 nm, 1.5 nm fixed
spectral bandwidth, wavelength accuracy of 0.07 nm (at 541.92 nm), wavelength
reproducibility of 0.01 nm (Cary 50 Scan, Varian, D-64289 Darmstadt, Germany)

(c) Ultrasonic water bath. –– 150 W, 35 kHz, 4 liter (TUC-160, Telsonic, CH-9552
Bronschhofen, Switzerland)

(d) Rotary evaporator. –– 5-240 rpm, 20-100°C (Rotavapor R-114, Water bath R-480, Büchi
Labortechnik, CH-9230 Flawil, Switzerland) connected to a vacuum pump, absolute
minimum pressure of 10 mbar (PC 5, Vacuubrand, D-97866 Wertheim, Germany)

(e) Homogenizer. –– 750 W, 12 mm diameter dispersing aggregate (Polytron PT3100 (drive

unit), PT-DA-3012/2T (aggregate), Kinematica, CH-6014 Lucerne, Switzerland)

(f) Syringe. –– Disposable, 2 mL (Henke-Sass.Wolff)

(g) Filter.–– Disposable, 0.45 µm pore size, 25 mm diameter, for organic solvents
(Chromafil Type O-45/25, Machery-Nagel, D-52313, Düren, Germany)

(h) HPLC. –– Consisting of online degasser (154, Gastorr, available from Omnilab, CH-8932
Mettmenstetten, Switzerland), gradient pump (L6200, Merck-VWR, CH-8953 Dietikon,
Switzerland), autosampler with cooling unit (AS-1559, Jasco, available from Omnilab,
CH-8932 Mettmenstetten, Switzerland), column thermostat (Jetstream 2 plus,
Thermotechnic products, A-2103 Langenzersdorf, Austria), UV/VIS-detector (Jasco PU-
2070plus, available from Omnilab, CH-8932 Mettmenstetten, Switzerland), integrator
(ATLASTM Chromatography Data System, Thermo LabSystems, Manchaster, UK,
combined with a Microsoft Windows 2000 Terminal Server, Citrix MetaFrame)

C. Reagents:

(a) n-Hexane. –– C6H14, FW: 86.18, CAS 110-54-3, purity ≥ 99% (GC), grade: pro analysis,
available from Merck in Darmstadt, Germany

(b) Cyclohexane. –– C6H12, FW: 84.16, CAS 110-82-7, purity: 99.5% (GC), grade: pro
analysis, available from Fluka, Buchs, Switzerland

(c) Tetrahydrofuran. –– C4H8O, FW: 72.11, CAS 109-99-9, purity ≥ 99.5% (GC), grade: pro
analysis,), available from Fluka, Buchs, Switzerland, stabilized with 0.025% BHT. Store
at 5° C and discard 1 month after opening.

(d) Ethanol. –– C2H6O, FW: 46.07, CAS 64-17-5, purity ≥ 99.5% (GC), grade: pro analysis,
available from Merck in Darmstadt, Germany.

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(e) Dichloromethane. –– CH2Cl2, FW: 84.93, CAS 75-09-2, purity ≥ 99.5% (GC), grade: pro
analysis, available from Merck in Darmstadt, Germany. Discard 1 month after opening.

(f) Protex 6L. –– Bacterial alkaline protease enzyme preparation in water, Synonyms:
subtilisin, IUB 3.4.21.62, CAS 9014-01-1. EINECS 2327522, available from Genencor
International, Inc. (Palo Alto CA 94304; www.genencor.com).

(g) Methanol. –– CH4O, FW: 32.04, CAS 67-56-1, purity: 99.8% (GC), grade: pro analysis,
available from Merck in Darmstadt, Germany.

(h) Butylated hydroxytoluene. –– (BHT, 2,6-Di-tert-butyl-p-cresol), C15H240, FW: 220.36,

CAS 128-37-0, purity: ≥ 99%, grade: purum, available from Fluka, Buchs, Switzerland

(i) N-Ethyldiisopropylamine. –– N8H19N, FW: 129.25, CAS 7087-68-5, purity: ≥ 98% (GC),
grade: purum, available from Fluka, Buchs, Switzerland

(j) 2-Propanol. –– C3H8O, FW: 60.10, CAS 67-63-0, purity: ≥ 98% (GC), grade: pro
analysis, available from Fluka, Buchs, Switzerland

(k) Ammonium acetate. –– C4H7O2N, FW: 77.08, CAS 631-61-8, purity: ≥ 98% (NT), grade
pro analysis, available from Fluka, Buchs, Switzerland

(l) Acetonitrile. –– C2H3N, FW: 41.05, CAS 75-05-08, purity: ≥ 99.9% (GC), grade: pro LC,
available from Merck in Darmstadt, Germany

(m) Tert-Butyl methyl ether. –– C5H12O, FW: 88.15, CAS 1634-04-4, purity: ≥ 99.5% (GC),
grade: purissimun, available from Fluka, Buchs, Switzerland

(n) Water. –– H2O, FW: 18.01, CAS 7732-18-5, distilled or de-mineralized

(o) L(+)Ascorbic acid. –– (Vitamin C), C6H8O6, FW: 176.13, CAS 50-81-7, purity: ≥ 99.7%
(Iodometry), grade: pro analysis, available from Merck in Darmstadt, Germany

(p) Suplex PKB-100 HPLC column, 5 µm, 250 x 4.6 mm, Catalog No. 58934, available from
Supelco in Bellefonte, USA

(q) YMC C30 HPLC column, 5 µm, 250 x 4.6 mm, product code: CT99S052546WT,
available e.g. from Stagroma in Wallisellen, Switzerland

(r) Ammonium acetate solution. –– 0.2%. Dissolve 0.50 g ammonium acetate in 250 mL
water. Store the solution at 5° C for no longer than 1 month.

(s) Mobile solvent for HPLC system A. –– In a 1 L volumetric flask, dissolve 50 mg

butylated hydroxytoluene (BHT) in 20 mL 2-propanol and add 0.2 ml N-
ethyldiisopropylamine, 25 mL 0.2% ammonium acetate solution (r), 455 mL acetonitrile,
and approximately 450 mL methanol. Mixture cools and contracts. Warm to room
temperature and dilute to volume with methanol. Discard after 2 days.

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(t) Mobile solvent 1 for HPLC system B. –– In a 1 L volumetric flask, dissolve 100 mg
vitamin C in ca 100 ml methanol (sonicate), add 100 mL tert-Butyl methyl ether and
adjust to volume with methanol. Discard after 2 days.

(u) Reference standard β-carotene. –– β-carotene, C40H56, FW 536.9, CAS 7235-40-7, purity
≥ 95 % (HPLC), available under product code 11045800 from Dr. Ehrenstorfer GmbH,
Bgm.-Schlosser-Str. 6A, D-86199 Augsburg, Germany. Store under argon or nitrogen in
cool dark place. The compound deteriorates in the presence of oxygen.

D. Calibration

(a) Standard solution. – 3 µg/mL β-carotene. Weigh with an accuracy of 0.01 mg

approximately 6 mg all-trans-β-carotene reference material into a 100 mL volumetric
flask, dissolve in about 20 mL tetrahydrofuran by treating in an ultrasonic bath for about
30 s. Dilute to volume with tetrahydrofuran (60 µg/mL). From this standard stock
solution, pipet exactly aliquots of 5 mL into two 100 mL volumetric flasks. Dilute to
volume with cyclohexane in one flask (standard measuring solution, 3 µg/mL β-carotene
in cyclohexane-tetrahydrofuran (95+5)). Pipet 5 mL tetrahydrofuran into the other flask
and dilute to volume with ethanol (standard working solution, 3 µg/mL β-carotene in
ethanol-tetrahydrofuran (9+1)). Store standard stock, measuring and working solutions at
5° C and use on day of preparation.

(b) Spectrophotometry. –– Measure the absorption of the standard measuring solution (a)
against cyclohexane at the maximum, ca 456 nm. Calculate the β-carotene concentration,
C, as follows:
C (mg/L) = E x 10 000/2500,

where E is the absorption at the maximum, 2500 is the E1%,1cm –value of pure all-trans-β -carotene
in cyclohexane, and 10 000 is the factor to convert % to mg/L.

Calculate the apparent E1%,1cm of the reference substance (ap E1%,1cm ) used to prepare the standard
solution as

apE1%,1cm = E x 10 000 x 2/W,

where W is the weight of the reference substance (mg). The apparent E1%,1cm must exceed 2375
for a spectrophotometric purity >95%.

(c) PLC of standard solution. –– Immediately after the preparation of the standard working
solution, (a), inject at least 6 aliquots of 20 µL into the HPLC system. Calculate the
response factor of all-trans-β-carotene from the averaged total peak areas of the
chromatograms and the spectrophotometrically measured β-carotene concentration as

RFtrans [AU x L/mg] = Astd/Cstd,

where RFtrans is the response factor of all-trans-β-carotene, Astd is the mean total peak area of the
standard chromatograms [Area Units, AU], and Cstd is the spectrophotometrically measured β-
carotene concentration of the standard measuring solution [mg/L]. The peak area of all-trans-β-
carotene must exceed 95% of the total peak area (i.e., chromatographic purity is >95%).

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(d) Control of the HPLC response. –– Control solutions are solutions of heat isomerized β-
carotene, concentrations of which have been found to be stable at 5° C at least 3 months
in the dark. It is not necessary to re-calibrate the HPLC if the previous response factor
has been shown to be valid by use of control solutions.

(1) Preparation of the HPLC control solution. –– Dissolve about 3 mg of β-carotene

reference substance and 1 g of BHT in 50 mL of tetrahydrofuran. Add 200 mL
ethanol and reflux for 2 hr in a water bath at 80° C. Cool, dilute to 500 mL with
ethanol, and transfer the solution to a dispenser bottle. Mix well, leave overnight
at room temperature, and dispense the solution into a large number of HPLC
vials. Carefully seal the vials immediately after filling with Teflon/silicone septa,
and store at ca 5° C in the dark.

(2) Use of the HPLC control solution. –– Measure the initial β-carotene content of
the control solution, (1), when the HPLC system is calibrated. Inject in parallel
with the standard solution, at least 6 aliquots of 20 µL into the HPLC system.
Calculate the mean β-carotene content of the control solution from the resulting
chromatograms using the newly determined response factor. Subsequently inject
the control solution together with each series of test extracts. The response factor
is regarded as constant as long as the measured β-carotene content of the control
solution corresponds to the initial value within ±2%. As long as the response
factor remains within these limits, the original response factor can be used for
calculations. But the HPLC system must be re-calibrated if the measured β-
carotene content exceeds the tolerance.

(3) Linearity of the HPLC response. – Dissolve 10 mg all-trans-β-carotene in 5 mL

dichloromethane in a 100 mL volumetric flask. Add 50 mL ethanol and reflux at
80° C in a water bath for 2 hr. Add ca. 40 ml dichloromethane, bring to room
temperature and adjust to volume with dichloromethane. Dilute this solution (100
µg/mL) with this mixed solvent as shown in the table below to obtain β-carotene
concentrations of 100 µg/mL to 0.05 µg/mL.

Table 1: β-Carotene Linearity – Dilutions & Concentrations

Solution to be diluted (µg/mL) Dilution Final concentration (µg/mL)

100 1+1 50
100 1+4 20
100 1+9 10
50 1+9 5
20 1+9 2
10 1+9 1
5 1+9 0.5
2 1+9 0.2
1 1+9 0.1
0.5 1+9 0.05

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Inject these solutions into the HPLC and calculate the β-carotene content as in D.b. The response
must be linear for β-carotene concentrations of 0.1 to 50 µg/mL with a correlation coefficient, R2
> 0.995, and the back calculated concentration is within the target of ± 10% of theoretical.

Optimization of the Method

The following factors were investigated in order to optimize and adapt the method for the wide
variety of dietary supplements tested:

1. Enzymatic digestion with Protex 6L

2. HPLC response factors for 9-cis-, 13-cis-, and 15-cis-β-carotene
3. Extraction efficacy
4. Stability and isomeric ratios of β-carotene during the autoinjector queue time
5. Isomerization of β-carotene during the analysis
6. Effect of BHT
7. Optimization of the C30 HPLC column for the separation of α- and β-carotene isomers
8. Batch-to-batch variation of Suplex PKB-100 C18 and YMC C30 HPLC Columns
9. Selectivity – separation of all-trans-β-carotene from cis-β-carotene isomers, α-carotene,
and other carotenoids.

E. Sample Preparation:

Sample preparation was dependent on the physical form of the material, the claimed content of β-
carotene, and the weight of the dosage form.

(a) Mean weight per dose: Weigh 20 tablets or capsules and calculate the mean
weight of the dosage form.

(b) Number of tablets or capsules per assa: For content uniformity tests one tablet or
the contents of one capsule was taken for each assay and 10 assays performed in
parallel. Report individual results. For the determination of average β-carotene
content the equivalent of content of three tablets or capsules was taken for each
assay and two assays performed in parallel. The averaged result of the two assays
was reported.

(c) Preparation of test sample: Mix suspensions or emulsions until homogeneous.

Crush tablets to a powder by grinding between the two halves of a folded
weighing paper with a pestle. Empty capsules containing powder formulations
and extract the contents together with the capsule shells. Use capsules containing
liquid formulations as such.

F. Extraction

(a) Oily solutions or suspension: Accurately weigh a test portion equivalent to

approximately 20 mg β-carotene, add 250 mg BHT, and transfer into a 250 mL
volumetric flask with 120 mL dichloromethane. Add 100 mL ethanol and shake.
Mixture cools and contracts. Allow extract to stand in the dark until room
temperature is reached, ca 2 hrs., dilute to volume with dichloromethane, and
shake vigorously. Dilute an aliquot in a ratio of 1+9 with dichloromethane-

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 ethanol (1+1) in a volumetric flask, e.g., 5mL to 50 mL, and inject 20 µL into the
HPLC system.

(b) Powders, beadlets, or emulsion: Accurately weigh a test portion equivalent to

approximately 10 mg β-carotene into a 250 mL volumetric flask. Add 250 mg
BHT, 0.5 mL Protex 6L, and 15 mL water. Tilt gently to wet the entire contents
and place in an ultrasonic bath at ca 50° C for 30 min, swirling about 15 min.
Add 100 mL ethanol to the warm suspension and shake vigorously. Add 135 mL
of dichloromethane and shake again. Mixture cools and contracts. Allow extract
to stand in the dark until room temperature is reached, ca 2 hrs., dilute to volume
with dichloromethane, shake vigorously, and let solids settle. Dilute an aliquot of
the supernatant in a ratio of 1+9 with dichloromethane-ethanol (1+1) in a
volumetric flask, e.g., 5 mL to 50 mL. Filter the solution through a 0.45 µm
membrane, if necessary, and inject 20 µL into the HPLC system.

(c) Tablets and capsules with mass of test portion below 5 : Accurately weigh a test
portion of 1-3 tablets or capsules, intact or broken as in E.c, into a 250 mL
volumetric flask. Add 250 mg BHT, 0.5 mL Protex 6L, and 15 mL water. Tilt
gently to wet the entire contents and place in an ultrasonic bath at ca 50° C for 30
min, swirling at about 15 min. Add 100 mL ethanol to the warm suspension and
shake vigorously. Add 120 mL of dichloromethane and shake again. If clumps
form, disperse them with a homogenizer, rinse with 15 mL dichloromethane,
combining the rinsings with the contents of the volumetric flask. Allow extract to
stand in the dark until room temperature is reached, ca 2 hr, dilute to volume with
dichloromethane, shake vigorously, and let solids settle. Proceed as follows:

(1) β-carotene content of the test portion below 0.1 mg: Evaporate an aliquot, e.g.,
50 mL, of the supernatant under reduced pressure at 50° C using a rotary
evaporator and a 250 mL round bottom flask. Dissolve the dry residue in a
volume of ethanol-dichloromethane (1+1) such that the extract is concentrated by
a factor of 10, e.g., residue from 50 mL dissolved in 5 mL.

(2) β-carotene content of the test portion between 0.1 and 10 m: Use the supernatant
without dilution or concentration.

(3) β-carotene content of the test portion above 10 mg: Dilute an aliquot of the
supernatant with ethanol-dichloromethane (1+1) such that the β-carotene content
of the final solution is 1-10 µg/mL.

Filter the solution from (1), (2), or (3) through a 0.45 µm membrane, if
necessary, and inject 20 µL into the HPLC system.

(d) Tablets with mass of test portion above 5 g: Accurately weigh a test portion of 1-3
tablets, prepared as in E.c, into a tared 100 mL volumetric flask. Add 1 g BHT, 1 mL
Protex 6L and 40 mL of water. In the case of effervescent tablets, add the water slowly
and in small portions to avoid excessive foaming and proceed with the next step when all
gas has been released. Shaking and the addition of a few droplets of ethanol help to
repress foaming. Place in an ultrasonic bath at ca 50° C for 30 min, swirling at about 15
min. Add 50 mL ethanol to the warm suspension, cool to room temperature, and dilute to
volume with water. Weigh flask and contents and shake vigorously. Immediately pour 8-

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 12 g of the suspension into a tared 100 mL volumetric flask using a funnel. Weigh the
transferred aliquot of the suspension. Add 35 mL of ethanol and shake. Add 35 mL of
dichloromethane and shake again. If clumps form, homogenize with a rotation
homogenizer, rinse with 15 mL dichloromethane, combining the rinsings with the
contents of the volumetric flask. Allow extract to stand in the dark until room
temperature is reached, ca 2 hr, dilute to volume with dichloromethane, shake vigorously,
and let solids settle. Proceed as follows:

(1) β-carotene content of the test portion below 0.5 mg: Evaporate an aliquot, e.g.,
50 mL, of the supernatant under reduced pressure at 50° C using a rotary
evaporator and a 250 mL round bottom flask. Dissolve the dry residue in a
volume of ethanol-dichloromethane (1+1) such that the extract is concentrated by
a factor of 10, e.g., residue from 50 mL dissolved in 5 mL.

(2) β-carotene content of the test portion between 0.5 and 50 mg: Use the
supernatant without dilution or concentration.

(3) β-carotene content of the test portion above 50 mg: Dilute an aliquot of the
supernatant with ethanol-dichloromethane (1+1) such that the β-carotene content
of the final solution is 1-10 µg/mL.

Filter the solution from (1), (2), or (3) through a 0.45 µm membrane, if necessary, and inject 20
µL into the HPLC system.

G. Chromatography

Test solutions are first injected into HPLC-system A involving a C18 column. In this system, cis-
isomers of α-carotene can interfere with the trans- and cis-isomers of β-carotene. If in
chromatograms of HPLC-system A, the peak area of all-trans-α-carotene exceeds the total peak
area of β-carotene isomers by more than 5%, the test solution is analyzed by the more selective
HPLC-system B.

(a) HPLC system A

(1) Conditions:
Column. –– Suplex pKb-100, Supelco, 5 µm, 250 x 4.6 mm.
Column temperature. –– 30° C.
Autosampler temperature. –– 15° C.
Mobile phase. –– See C.s.
Flow rate. –– 0.6 mL/min.
Pressure. –– Approximately 33 bar.
Injection volume. –– 20 µL.
Detection. –– 448 nm.
Run time. –– Approximately 30 min.

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Retention times:
all-trans-β-carotene: ca 20-25 min.
Approximate retention times relative to all-E-β-carotene:
all-trans-Lutein: 0.30
all-trans-Zeaxanthin: 0.32
all-trans-β-Cryptoxanthin: 0.58
all-trans-Lycopene: 0.67
non-identified cis-Lycopene: 0.69, 0.75, 0.79
9’-cis-α-Carotene: 0.91
all-trans-α-Carotene: 0.93
9-cis-α-Carotene: 0.98
13-cis and 13’-cis-α-Carotene: 1.03
non-identified cis-α-Carotene: 1.08
all-trans-β-Carotene: 1.00
9-cis-β-Carotene: 1.07
13-cis-β-Carotene: 1.17
15-cis-β-Carotene: 1.21

Other minor cis-isomers of β-carotene elute in the range of the trans- and mono-cis- isomers
of β-carotene (e.g. with relative retention times of 1.02 for 13,15-di-cis-β-carotene, 1.11 for
9,9’-di-cis-β-carotene, and 1.12 for 9,15-di-cis-β-carotene).
(b) HPLC system B
(1) Conditions:
Column. –– YMC-Pack C30, 5 µm, 250 x 4.6 mm
Column temperature. –– 30°C.
Autosampler temperature. –– 15°C.
Mobile phase. –– Step gradient using solvent 1 and solvent 2
Solvent 1: Methanol - tert.-Butyl methyl ether (9+1) incl. 200 ppm vitamin C, see
C.t.
Solvent 2: Tert.-Butyl methyl ether
0 - ca 57 min*: 100% solvent 1
ca 57- 60 min*: 100% solvent 2 (flush)
ca 60-65 min: 100% solvent 1
Flow rate. –– 0 - ca 57 min*: 0.9 mL/min
ca 57-60 min*: 2.0 mL/min
ca 60-65 min: 0.9 mL/min

Pressure. –– Approximately 40-80 bar.

Injection volume. –– 20 µL.
Detection. –– 445 nm
Run time. –– Approximately 55 -75 min

* adjust the start point of the flush to the retention time of 9-cis-β-carotene (see G, b,2)

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27
 (2) Retention times:
all-trans-β-carotene: ca 40-55 min
Approximate retention times relative to all-trans-β-carotene:
all-trans-Lutein: 0.22
all-trans-Zeaxanthin: 0.26
all-trans-β-Cryptoxanthin 0.51
13-cis-α-Carotene: 0.48
non-identified cis-β-Carotene: 0.50*
non-identified cis-α-Carotene: 0.50
13’-cis-α-Carotene: 0.53
15-cis-β-Carotene: 0.56
13,15-di-cis-β-carotene: 0.58
13-cis-β-Carotene: 0.61
9,15-di-cis-β-carotene: 0.64
non-identified cis-β-carotene: 0.73
all-trans-α-Carotene: 0.76
9-cis-α-Carotene: 0.79
9,9’-di-cis-β-Carotene: 0.91
9’-cis-α-Carotene: 0.95
all-trans-β-Carotene: 1.00
9-cis-β-Carotene: 1.12
Lycopene isomers: > 1.20

* this cis-isomer of β-carotene is only taken into account if total β-carotene exceeds total α-
carotene by far (e.g. by factor 10).

H. Calculations

Calculate the contents of all-trans- and total β-carotene in the test samples as follows:

Ctot [mg/g] = [(Atrans + A9cis + A13cis ·1.2 + A15cis · 1.4 + AXcis) · V] / (RFtrans · m)

Ctot [mg/dose] = [(Atrans + A9cis + A13cis · 1.2 + A15cis · 1.4 + AXcis) ·V ·WD] / (RFtrans · m · n)

Ctrans [mg/g] = (Atrans · V) / (RFtrans · m)

Ctrans [mg/dose] = (Atrans·V ·WD) / (RFtrans · m · n)

where:
Ctot = total β-carotene content [mg/g or mg/dose]
Ctrans = all-trans β-carotene content [mg/g or mg/dose]
Atrans = peak area of all-trans-β-carotene [Area units = AU]
A9cis = peak area of 9-cis-β-carotene [AU]
A13cis = peak area of 13-cis-β-carotene [AU]
A15cis = peak area of 15-cis-β-carotene [AU]
AXcis = sum of peak area of other cis-isomers of β-carotene [AU]

1.2, 1.4 are relative response factors (correction factors to compensate for lower specific
absorption of 13-cis- and 15-cis-β-carotene compared to all-trans-β-carotene at this wavelength)

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 WD = mean weight of a dose [g]
m = test portion amount [g]
n = number of tablets or capsules used as amount of test portion
RFtrans = response factor of all-trans-β-carotene [AU*L/mg]
V = theoretical volume in which the test portion is dissolved [L]
= [V1 * V3/V2]*[W1/W2]
where V1 = volume of the flask used for extraction with
dichloromethane
ethanol [L] (0.25 for (F,a)-(F,c) and 0.1 for (F,d))
V2 = volume aliquot which is diluted or evaporated [L]
V3 = volume to which aliquot V2 is diluted or in which the residue
after evaporation of aliquot V2 is dissolved [L]

W1 and W2 apply only to section (F,d): W1 is the weight of the aqueous alcohol suspension in the
first 100 mL volumetric flask and W2 is the weight of the aliquot of the aqueous alcohol
suspension transferred to the second 100 mL flask.

Linearity of HPLC Response

The linearity of the response of HPLC systems A and B was examined. Dilutions from a solution
of heat isomerized β-carotene were prepared as described in the method section(D,d,3) with a
total β-carotene range of ca. 0.005 to 100 µg/mL. All dilutions were prepared and measured in
duplicate. From the resulting acceptable chromatograms, the total peak areas of all detected β-
carotene isomers as well as the peak area of all-trans β-carotene were determined and averaged
for the duplicates. The relative response of the analyte, measured as mean peak areas of all-trans-
β-carotene and of total β-carotene, was plotted against the respective concentrations. Curves were
constructed using the least-squares linear regression method (Figures 1 & 2).
Table 2: HPLC response (peak areas) vs. the concentration of total
β-Carotene in HPLC-system A.

β-Carotene Concentration Peak Area (AU) Deviation of Mean

Peak Area from
Effective Regression Line (%)
Relative 1. Value 2. Value Mean RSD (%) n=2
(µg/mL)
100 97 32182381 32291361 32236871 0.24 1.59
50 48 16295572 16422594 16359083 0.55 0.10
20 19 6543809 6592811 6568310 0.53 -0.28
10 9.7 3285216 3329345 3307281 0.94 -0.98
5 4.8 1638588 1672344 1655466 1.44 -1.09
2 1.9 651545 665997 658771 1.55 -0.57
1 0.97 330864 332068 331466 0.26 -1.20
0.5 0.48 163804 165784 164794 0.85 -0.63
0.2 0.19 66220 66575 66398 0.38 -1.35
0.1 0.097 32680 32750 32715 0.15 0.11
0.05 0.048 17338 16763 17051 2.38 -3.96
0.02 0.019 7375 5732 6554 17.73 -0.05
0.01 0.0097 2977 2612 2795 9.24 17.19
0.005 0.0048 1456 1282 1369 8.99 19.61

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Figure 1. Linearity of Total β-Carotene Response

Table 3. HPLC response (peak areas) vs. the concentration of all-trans-β-Carotene in HPLC-
system A.

β-Carotene Concentration Peak Area (AU) Deviation of Mean

Peak Area from
Effective Regression Line (%)
Relative 1. Value 2. Value Mean RSD (%) n=2
(µg/mL)
100 75 26015719 26095924 26055822 0.22 2.09
50 38 13231707 13331267 13281487 0.53 0.14
20 15 5336427 5331992 5334210 0.06 -0.26
10 7.5 2694554 2721352 2707953 0.70 -1.77
5 3.8 1349767 1373339 1361553 1.22 -2.31
2 1.5 543258 549182 546220 0.77 -2.60
1 0.75 275279 275775 275527 0.13 -3.45
0.5 0.38 136518 137661 137090 0.59 -2.98
0.2 0.15 55010 55241 55126 0.30 -3.49
0.1 0.075 27620 27435 27528 0.48 -3.36
0.05 0.038 14151 13992 14072 0.80 -5.48
0.02 0.015 5797 5360 5579 5.54 -4.63
0.01 0.0075 2977 2508 2743 12.09 -3.00
0.005 0.0038 1456 1282 1369 8.99 -2.84

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Figure 2. Linearity of all-trans-β-Carotene Response

Table 4: HPLC response (peak areas) vs. the concentration of total-β-Carotene in HPLC-system
B.

β-Carotene Concentration Peak Area (AU) Deviation of Mean

Peak Area from
Effective Regression Line (%)
Relative 1. Value 2. Value Mean RSD (%) n=2
(µg/mL)
100 100 28756736 28719439 28738088 0.09 1.26
50 50 14477016 14673413 14575215 0.95 -0.18
20 20 5741770 5766178 5753974 0.30 1.14
10 10 2924930 2906636 2915783 0.44 -0.20
5 5.0 1441165 1459068 1450117 0.87 0.33
2 2.0 581480 579465 580473 0.25 0.26
1 1.0 289171 288312 288742 0.21 0.78
0.5 0.50 143245 141851 142548 0.69 2.07
0.2 0.20 58764 56545 57655 2.72 0.94
0.1 0.10 28488 27636 28062 2.15 3.70
0.05 0.050 12776 12479 12628 1.66 15.22
0.02 0.020 5681 3969 4825 25.09 20.62
0.01 0.010 1830 2467 2149 20.96 35.44
0.005 0.0050 0 847 424 141.42 243.55

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Table 5: HPLC response (peak areas) vs. the concentration of all-trans-β-Carotene in HPLC-
system B.

β-Carotene Concentration Peak Area (AU) Deviation of Mean

Peak Area from
Effective Regression Line (%)
Relative 1. Value 2. Value Mean RSD (%) n=2
(µg/mL)
100 77 22969943 22918213 22944078 0.16 1.52
50 38 11577243 11751362 11664303 1.06 -0.16
20 15 4617349 4583143 4600246 0.53 1.26
10 7.7 2358842 2339258 2349050 0.59 -0.85
5 3.8 1167349 1173364 1170357 0.36 -0.49
2 1.5 474781 468365 471573 0.96 -1.22
1 0.77 234908 234463 234686 0.13 -0.75
0.5 0.38 116123 115227 115675 0.55 0.68
0.2 0.15 47717 46781 47249 1.40 -1.41
0.1 0.077 23584 23337 23461 0.74 -0.72
0.05 0.038 10569 10615 10592 0.31 9.95
0.02 0.015 4852 3334 4093 26.22 13.81
0.01 0.0077 1384 2025 1705 26.59 36.65
0.005 0.0038 0 847 424 141.42 174.99

Table 6. Variation of the total β-carotene contents measured in the test materials on 5 different
days

Total β-Carotene
Test (mg/g)
Extraction HPLC Mean
Material RSD (%)
Variant System Value
No.
Day 1 Day 2 Day 3 Day 4 Day 5

0.490 0.492 0.481 0.486 0.506

1 F,c,2 A 0.494 2.4
0.486 0.479 0.514 0.495 0.508
46.2 47.1 47.3 47.0 46.4
2 F,c,3 A 47.1 1.5
46.9 48.8 47.5 47.0 46.6
0.0529 0.0519 0.0506 0.0512 0.0523
3 F,c,1* B 0.0520 3.1
0.0542 0.0537 0.0516 0.0486 0.0528
9.52 9.64 9.41 9.81 9.64
4 F,c,3 B 9.64 2.2
9.51 10.06 9.37 9.84 9.58
48.8 50.0 49.5 49.8 50.5
5 F,d,3* A 49.3 2.6
50.9 48.4 50.5 48.4 46.6**
24.9 24.2 26.8 20.7 25.0
6a F,d,2* A 23.7 10.4
19.3 21.5 24.7 26.3 24.0
26.5 26.3 26.0 26.0 25.8
6b F,c,3 A 26.2 1.2
26.4 26.8 26.0 25.9 26.1
33.6 34.4 31.2 33.0 31.8
7 F,c,3 B 32.2 4.4
32.2 33.0 32.0 32.2 29.3**
198 195 203 204 201
8 F,b A 201 1.3
200 201 201 202 202

* Only for validation purpose (proposed method prescribes other variants for these test materials)
** Outliers according to Grubbs test (P>90%). RSD% = 1.9% for No.5 and 3.0% for No.7, if
outliers are excluded. ,

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Table 7. Variation of the all-trans-β-carotene contents measured in the test materials on 5
different days

all-trans-β-Carotene
Test (mg/g)
Extraction HPLC Mean
Material RSD (%)
Variant System Value
No.
Day 1 Day 2 Day 3 Day 4 Day 5

0.423 0.427 0.412 0.427 0.428

1 F,c,2 A 0.428 3.5
0.412 0.418 0.463 0.438 0.430
44.3 45.1 45.1 45.0 44.9
2 F,c,3 A 45.2 1.4
44.9 46.7 45.4 45.2 45.1
0.0327 0.0323 0.0296 0.0302 0.0303
3 F,c,1* B 0.0310 4.6
0.0329 0.0323 0.0304 0.0287 0.0307
6.59 6.67 6.44 6.74 6.61
4 F,c,3 B 6.62 2.4
6.53 6.99 6.45 6.65 6.57
37.9 39.7 39.4 39.4 39.5
5 F,d,3* A 39.2 3.0
39.7 38.5 40.2 38.3 36.2**
23.9 23.2 25.7 19.8 24.0
6a F,d,2* A 22.8 10.4
18.5 20.6 23.6 25.2 23.1
25.4 25.2 24.9 24.9 24.8
6b F,c,3 A 25.1 1.2
25.3 25.6 24.8 24.7 25.2
24.1 24.6 22.1 23.8 22.7
7 F,c,3 B 23.0 5.4
23.1 23.5 22.7 23.1 20.1**
155 156 164 164 160
8 F,b A 160 2.0
157 160 162 162 161
* Only for validation purpose (proposed method prescribes other variants for these test materials)
** Outliers according to Grubbs test (P>90%). RSD% = 1.9% for No.5 and 3.0% for No.7, if
outliers are excluded. ,

Precision
On five different days, two test portions from each of the eight test materials were analyzed
according to the method. One technician performed the work on the first two days, another on
days 3 to 5. The test portions consisted of 3 tablets or capsules (test materials No. 1-7) or 100 mg
(test material No. 8). The extraction variants and HPLC-systems used are shown in table 11a-b.
The precision of extraction variant F,a, was not examined as it should be equal or better than
variant F,b.

On the first day of the precision tests, it was realized that the aqueously suspended capsule shells
of test material No. 6 agglutinated when ethanol was added. The clumps formed could not be
dispersed by use of a homogenizer (Polytron), and it was not possible to take a homogeneous
aliquot from this suspension for further extraction, as prescribed in extraction variant F,d (No 6a).
It seemed very probable that this variant could not work with this test material and it was decided
to also extract this material following variant F,c (No. 6b).

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Table 8: Recovery of β-carotene in beadletted raw material

Spiked Total β-Carotene* Recovered Total β-Carotene

(mg/g) (mg/g)
Extraction Recovery(
Variant %)**
Individual RSD (%) Individual RSD (%)
Mean Mean
Results (n = 3) Results (n = 3)

F,b 2.06 2.05 0.5 2.01 2.04 1.5 99.7

2.04 2.03
2.04 2.07

F,b 397 398 0.5 395 396 0.3 99.5

396 395
400 397

* Measured in diluted spiking solution

** Relative to the amount of spiked total β-Carotene (= 100%)

Table 9: Recovery of β-carotene with different assay variants for supplement types

Spiked Total β-Carotene* Recovered Total β-Carotene

(mg/test portion) (mg/test portion)
Extraction Recovery(
Variant %)**
Individual RSD (%) Individual RSD (%)
Mean Mean
Results (n = 3) Results (n = 3)

F,c,1 0.00885 0.00876 2.1 0.00806 0.00854 7.6 97.5

0.00852 0.00814
0.00890 0.00941

F,c,2 1.028 1.023 0.5 0.997 0.999 1.2 97.6

1.023 1.012
1.019 0.988

F,c,3 99.4 99.5 0.5 101.6 100.5 1.1 101.1

99.0 99.4
100.0 100.6

F,d,1 0.00978 0.00983 0.8 0.00995 0.00996 4.3 101.3

0.00980 0.00954
0.00992 0.01039

F,d,2 1.003 0.995 0.7 0.957 0.983 2.3 98.8

0.994 0.993
0.989 0.999

F,d,3 99.4 99.5 0.5 99.7 101.6 2.0 102.1

99.0 103.9
100.0 101.2

* Measured in diluted spiking solution

** Relative to the amount of spiked total β-Carotene (= 100%)

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Recovery

Negative control materials were fortified, in triplicate, with a mixture of β-carotene isomers
generated by heat isomerization and analyzed according to the method. For a positive control, the
solutions used to spike the negative control materials (spiking solutions) were diluted, in
triplicate, with ethanol-dichloromethane (1+1) and analyzed directly by HPLC. In addition,
triplicate unfortified controls were analyzed. Only HPLC system A was used in this experiment.

For preparation of the spiking solutions, 2.5 g of β-carotene reference material were dissolved in
ca. 70 mL chloroform. The solution was enclosed in a pressure-resistant 100 mL glass vessel and
heated for 2 hours in a water bath at 80°C. The solution was then cooled and diluted with
chloroform to a volume of 250 mL (spiking solution 1, 10,000 µg/mL). 5 mL of this solution
were pipetted into a 500 mL volumetric flask and diluted to volume with dichloromethane
(spiking solution 2, 100 µg/mL). Spiking solution 2 was diluted in the same manner with ethanol-
dichloromethane (1+1) (spiking solution 3, 1 µg/mL).

In order to spike the negative control material No. 1 (placebo tablets) 10 mL of spiking solutions
1-3 were pipetted into the volumetric flasks for the first extraction step. In the case of negative
control material No. 2 (placebo beadlets), 4 mL of spiking solution 1 and 2 mL of spiking
solution 2 (containing 40 mg or 0.2 mg β-carotene) were used. The solutions were evaporated
under reduced pressure at 50°C using a rotary evaporator. Then, the dry residues were combined
with the negative control materials and the mixtures extracted as described in the attached
method.

Negative control material No.1 (placebo tablets) was spiked, in test portions of 3 tablets (for
variant F,c) and 6 tablets (for variant F,d) at total β-carotene concentrations of ca. 0.01 (= lower
range limit of the attached method), 1, and 100 mg per test portion (= ca 130% of highest β-
carotene concentration in test materials of this study). Negative control material No.2 (placebo
beadlets) was fortified at total β-carotene concentrations of ca. 0.2% (= lower range limit of the
attached method) and 40% (= ca 130% of highest β-carotene concentration in commercial
product forms). The extraction variants used are listed in tables 12a-b. The recovery of extraction
variant F,a, was not examined as it should be equal or better compared to variant F,b.

Results and Discussion

Seven dietary supplements containing β-carotene and one β-carotene beadletted raw material
were analyzed. The dietary supplements included multivitamin tablets, fish oil capsules, softgels,
and tablets containing β-carotene as the main ingredient. The label claims ranged from no β-
carotene claim for the fish oil capsules, 0.6 to 25 mg β-carotene/tablet or capsule for the dietary
supplements, and 20% β-carotene for the beadletted raw material. The sources of β-carotene
included synthetic material, carrot oil, and Dunaliella salina algae extract.

All of the factors studied in the optimization of the method section were found to be satisfactory.
Typical HPLC chromatograms for HPLC Systems A and B are shown in Figures 3-8. They
include the β-carotene control solution (Figures 3 & 4), a sample material suitable for HPLC
system A (Figures 5 & 6), and a sample material that necessitated the use of HPLC system B
(Figures 7 & 8).

As shown in tables 2-5, in the range of 0.1 – 50 µg/mL, the regression line showed a correlation
coefficient (R2) of 1.000 for total β-carotene as well as for all-trans-β-carotene and thus met the

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requirement (R2 ≥ 0.995). The instrumental detection limit, estimated as the concentration for
which the peak signal of all-trans-β-carotene is approximately three times the baseline noise, was
ca. 0.001 µg/mL for HPLC A and at ca. 0.01 µg/mL for HPLC system B.

The results of the precision experiment (tables 6 & 7) clearly show that the capsules of test
material No. 6 could not be extracted reproducibly by use of variant F,d. (see No. 6a). The high
standard deviation of 10.4% was due to the extraction problem mentioned above. Therefore,
extraction variant F,d should only be used for large tablets that are dispersible in water (e.g.
effervescent tablets).

The relative standard deviations (RSD%) obtained for the total and all-trans-β-carotene contents
of test materials No 1,2,3,4,6(b), met the requirements for repeatability precision (RSDr)
recommended by the AOAC (comp. AOAC requirements for single laboratory validation of
chemical methods for dietary supplements and other materials, Draft 2002-11-24). In the case of
test material No. 8 (beadlets raw material) the fluctuation of total-β-carotene contents (RSD% =
1.3%) was within the recommended limit (RSDr =1.5%), but the measured all-trans-β-carotene
contents varied slightly more (2.0%).

In contrast, the variation of the total and all-trans-β-carotene values obtained for test materials
No. 5 and No. 7 clearly exceeded the repeatability precision limit recommended for this range of
concentration (1.5-2%). In each of these cases, one outlier could be identified using the Grubbs
test. Exclusion of these outliers shifted the coefficients of variation calculated for test material
No. 5 into the recommended range while those obtained for test material No. 7 remained above.
However, it is apparent from the results in table 11 that the extraction variant used for the softgels
of test material No. 7 gave acceptable results when applied to other very similar as well as
different test materials. Compare data for No.2 and 4 (softgels) and No. 6b (capsules). This
suggests that test material inhomogeneity may also contribute to the relatively high variation
found for test material No. 7.

It should be noted that the present precision test was not performed under distinct repeatability
conditions, as two technicians and 5 working days were involved. For this reason, the relatively
low limits for repeatability precision may not be fully adequate to evaluate the present results.

As shown in table 8, recoveries of 99.7% and 99.5% were found for the analysis of total β-
carotene in beadletted raw materials spiked at β-carotene concentrations of 0.2% and 40%,
respectively.

The recoveries obtained for the analysis of β-carotene in supplements varied between 97.5 and
102.1% independently of the spiked β-carotene concentrations (10 µg – 100 mg/test portion) and
of the assay variants (table 9).

β-Carotene was not detected in the extracts of unfortified control materials.

The present method is suitable for the analysis of α-carotene in the presence of other carotenes
and xanthophylls (comp. 5.1.4. Selectivity). HPLC system B should be used for samples
containing isomer mixtures of α-carotene (e.g. palm oil, carrot oil, or heat treated preparations).
HPLC system A would be applicable if α-carotene is present in the all-trans configuration. The
calibration of α-carotene could be done with reference material of all-trans-β-carotene by use of
a relation factor (ca. 0.95) since α-and β-carotene are similar with respect to stability, polarity,
and chromatographic behaviour.
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For several years, HPLC system A of the present method has been used in analytical laboratories
of Roche Vitamins Ltd. for the routine analysis of lycopene in feed, food, and pharmaceutical
preparations. The system is able to separate the most relevant isomers of lycopene from those of
α-and β-carotene as well as from xanthophylls, such as all-trans-lutein, all-trans-zeaxanthin, and
all-trans-β-cryptoxanthin. To our knowledge, only a cis-isomer of β-cryptoxanthin coelutes with
(all-trans-) lycopene. This interference can be neglected in most supplements and raw materials,
since β-cryptoxanthin is usually present in low amounts compared to lycopene. Calibration can be
performed by use of reference material of all-trans-lycopene. Relative response factors for the
cis-isomers of lycopene are not established because the specific absorbance of the quantitatively
most important cis-isomer of lycopene, 5-cis-lycopene, corresponds well to that of the all-trans-
isomer. Whereas all-trans-, 9-cis-, and 13-cis-lycopene are well resolved from each other, 5-cis-
lycopene usually occurs as a shoulder on the peak of all-trans-lycopene.

Both HPLC systems of the present method are able to separate all-trans-lutein from all-trans-
zeaxanthin, and both of these xanthophylls from many other carotenols and carotenes. However,
with HPLC system A, a main cis-fraction of lutein coelutes with all-trans-zeaxanthin. Such an
interference also probably occurs in HPLC system B because of the relatively short retention
times, but this has to be experimentally confirmed. Some commercial raw materials contain
isomeric mixtures of lutein and zeaxanthin. Therefore, based on our current knowledge, we would
recommend to combine the sample preparation procedure of the present method with an HPLC
system showing a higher selectivity for the cis/trans isomers of lutein and zeaxanthin (e.g. an
isocratic normal-phase system as e.g. used at Roche Vitamins Ltd. and other suppliers for this
purpose). Calibration could be done with only all-trans-zeaxanthin using relative response factors
for all-trans-lutein and the cis-isomers of both xanthophylls. Pure reference material of all-trans-
lutein is very difficult to obtain.

Recommendation

On the basis of the accuracy, precision, and recovery results for this SLV study, it is
recommended that this method be collaboratively studied for the determination of β-carotene in
dietary supplements.

Acknowledgements

This study is the result of a contract between the Center for Food Safety and Applied Nutrition,
FDA, the Office of Dietary Supplements, NIH, and the AOAC International. The purpose of this
contract is to provide the FDA, as well as other government agencies and the dietary supplements
industry with AOAC® Official MethodSM, applicable to commercially available dietary
supplements and their raw materials.

References

1. Mathews-Roth, M.M., (1986) Biochemie 68, 875-884

2. Olson, J.A. (1986) J. Nutr. 166, 1127-1130
3. Temple, N.J. & Basu, T.K. (1988) Nutr. Res. 8, 685-701
4. Sweeny, J.P., & Marsh, A.C. (1970) J. Assoc. Off. Anal. Chem. 53, 937-940
5. Panalaks, C.A., Warthesen, J.J., & Taoukis, P.S. (1990) J. Inst. Can. Technol.
Aliment. 3, 145-151
6. Sweeny, J.P., & Marsh, A.C. (1971) J. Am. Diet. Assoc. 59, 238-243

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27
7. Tsukida, K., Saiki, K., & Sugiura, M. (1981) J. Nutr. Sci. Vitaminalo. 27, 51-561
8. Bushway, R.J. (1985) J. Liq. Chromatogr. 8, 1527-1547
9. Chandler, L.A., & Schwartz, S.J. (1987) J. Food Sci. 52, 669-672
10. Quackenbusch, F.W. (1987) J. Liq. Chromatogr.10, 643-653
11. Godoy, H.T., & Rodriguez-Amaya, D.B. (1994) J. Agric. Food Chem. 42, 1306-1313
12. Lowe, G.M., Bilton, R.F., Davies, I.G., Ford, T.C., & Young, A.J., (1999) Ann. Clin.
Biochem. 36, 323-332
13. Levin, G., Ben-Amotz, A., & Mokady, S. (1994) Comp. Biochem. Physiol. 107A, 203-
207
14. Stahl, W., Schwartz, W., Sundquist, A.R., & Sies, H. (1992) Arch. Biochem. Biophys.
294, 173-177
15. Stahl, W., Schwartz, W., & Sies, H. (1993) Am. Inst. Nutr., 847-851
16. Schierle, J., Schellenberger, T., Fizet, C., & Betz, R. (2002) Eur. Food Res. Technol. 215,
268-274
17. Marty, C. & Berset, C. (1990) J. Agric. Food Chem. 38, 1063-1067
18. Tsukida, K., Saiki, K., Takii, T., & Koyama, Y. (1982) J. Chromatogr. 245, 359-364
19. Vecchi, M., Englert, G., Maurer, R., & Meduna, V. (1981) Helv. Chim. Acta 64, 2746-
2758
20. Quackenbusch, F.W., & Smallidge, R.I., (1986) J. Assoc. Off. Anal. Chem. 69, 767-771
21. Ben-Amotz, A., Lers, A., & Avron, M., (1988) Plant Physiol. 86, 1286-1291
22. Schüep, W. & Schierle, J. (1997) J. AOAC International 80, 1057-1064

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