Method for the Determination of Beta Carotene in Supplements and Raw Materials by Reversed-Phase Liquid Chromatography : Single Laboratory Validation

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SCHIERLE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 87, NO. 5, 2004
DIETARY SUPPLEMENTS
Method for the Determination of b-Carotene in Supplements and
Raw Materials by Reversed-Phase Liquid Chromatography:
Single Laboratory Validation
JOSEPH SCHIERLE, BERND PIETSCH, ALAN CERESA, and CHRISTIAN FIZET
DSM Nutritional Products Ltd, Research and Development, Analytical Research Center, PO Box 3255, CH-4002 Basel,
Switzerland
EDWARD H. WAYSEK
Caravan Products Co. Inc., 100 Adams Dr, Totowa, NJ 07512
A single laboratory validation (SLV) study was
conducted for a liquid chromatography (LC)
b-carotene is generally regarded as the most commercially
important and widely used carotenoid. It is used as a food
method for the determination of total and
coloring agent, an antioxidant, and an important and safe
all-trans-b-carotene in a variety of dietary
pro-vitamin A source (1–3). b-carotene is currently incorporated
supplements, including multivitamin tablets,
in a wide variety of dietary supplements, including multivitamin,
softgels, capsules, and beadlet raw materials.
vitamin A, and antioxidant formulations. Recently, additional
Extraction variants were developed for the different
carotenoids, including lutein and lycopene, have been the subject
types of supplements tested based upon the
of nutritional studies and are now also incorporated, both in
supplement type and level of b-carotene. Water
combination products and in dietary supplements available to the
dispersible formulations such as powders,
general public. Spectrophotometry is still a common technique
emulsions, tablets, and capsules were
for the analysis of b-carotene in commercial product forms.
enzymatically digested with protease and extracted
Schierle et al. (4) reported a spectrophotometric procedure for the
with dichloromethane–ethanol. Oily suspensions
determination of total b-carotene in food additives with varying
were directly dissolved in dichloromethane–
cis-/trans-ratios using an isobestic wavelength. However,
ethanol. After appropriate dilution or
spectrophotometric methods cannot differentiate between
concentration, the extracts were chromatographed
all-trans-b-carotene and cis-isomers of b-carotene, which may
by using either a reversed-phase C18 column or, in
be formed during processing (5–12). Spectrophotometric
products containing high amounts of a-carotene, a
analytical procedures are also not capable of determining
reversed-phase C30 column. The LC systems
b-carotene in combination products containing other carotenoids
provided linear responses in the range of 0.1–50 mg
such as a-carotene, lutein, or lycopene. b-carotene and carotenoids
b-carotene/mL. The main geometrical isomers of
in general have been intensively studied by liquid chromatography
b-carotene (all-trans, 9-cis, 13-cis, and 15-cis) were
(LC), and procedures have been reported for the separation of
well separated from each other and from other
b-carotene cis-/trans-isomers (13–18) and for b-carotene in
carotenoids such as a-carotene, cryptoxanthin, lutein,
supplements and foods (19, 20). Thus, a chromatographic
lycopene, and zeaxanthin. Duplicate determinations
procedure capable of separating all-trans-b-carotene from the
of total b-carotene performed by 2 technicians in 8
corresponding cis-isomers and from other commercially used
different test materials on 5 different days resulted
carotenoids was deemed necessary to properly determine the
in relative standard deviations of 1.2–4.4%.
b-carotene content in dietary supplements.
Recoveries determined for supplements and
beadlet raw material spiked with b-carotene levels
METHOD
of 10 mg to 100 mg/test portion and 0.2–40%,
Test and Negative Control Materials
respectively, ranged from 97.5 to 102.1%. On the
basis of the accuracy, precision, and recovery
(a) Test materials.—Dietary supplements test materials
results from the SLV study, the method is
used in the study were chosen to represent the range of dietary
suggested for a collaborative study on the
supplements containing b-carotene currently available to the
determination of total and all-trans-b-carotene in
general public and included the following (1000 IU
dietary supplements.
b-carotene = 0.6 mg b-carotene; 21):
(1) Multivitamin tablets; claim: vitamin A 3500 IU, 29%
as b-carotene.
Received January 30, 2004. Accepted by AP July 28, 2004.
(2) Softgels with b-carotene in soybean oil; claim:
Corresponding author's e-mail: Joseph.Schierle@dsm.com.
b-carotene 25 000 IU.

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SCHIERLE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 87, NO. 5, 2004 1071
Figure 1. The predominant geometrical isomers of b-carotene: all-trans-b-carotene, 9-cis-b-carotene,
13-cis-b-carotene, and 15-cis-b-carotene.
(3) Capsules with b-carotene in fish oil; no claim for
Nutritional Products (Basel, Switzerland). Test and negative
b-carotene.
control materials were stored at 5°C in the dark (refrigerator).
(4) Softgels with b-carotene from carrot oil extract; claim:
Principle
vitamin A (as b-carotene) 10 000 IU.
(5) Tablets with b-carotene; claim: 25 mg b-carotene.
Water-dispersible formulations such as powders,
(6) Capsules with b-carotene; claim 6 mg b-carotene.
emulsions, tablets, and capsules are digested with protease
and extracted with dichloromethane and alcohol. Oily
(7) Softgels with vitamin A (as b-carotene) and vitamin E;
suspensions are dissolved directly in dichloromethane and
claim: vitamin A 25 000 IU (as b-carotene).
alcohol. The extract is chromatographed on a C18 isocratic
(8) Beadlets raw material with pure b-carotene; claim:
LC system that separates the predominant geometrical
20% b-carotene.
isomers of b-carotene from each other (Figure 1) and from
(b) Negative control materials.—
other carotenoids such as all-trans-a-carotene, lycopene,
(1) Placebo multivitamin tablets, Reference No.
81.16.03.32 (DSM Formula No. 80.23) containing vitamin A
acetate, vitamin D3, vitamin E, ascorbic acid, thiamin
mononitrate, vitamin B2, nicotinamide, pyridoxine HCl,
Table 1. Dilutions and concentrations
vitamin B12, folic acid, pantothenic acid, biotin, vitamin K1,
Final concentration,
iron fumarate, magnesium oxide, potassium iodide, zinc
Solution to be diluted, mg/mL
Dilution
mg/mL
sulfate monohydrate, manganese sulfate monohydrate, copper
sulfate anhydrous, chromium chloride hexahydrate, sodium
100
1 + 1
50
selenite anhydrous, sodium molybdate dihydrate, calcium
phosphate anhydrous, potassium chloride, silicon dioxide,
100
1 + 4
20
cellulose, stearic acid, magnesium stearate,
100
1 + 9
10
n-vinyl-2-pyrollidone. Coated with Sepifilm LP014 and
50
1 + 9
5
Sepifilm 6097 Red (Seppic, Inc., Fairfield, NJ).
20
1 + 9
2
(2) Lycopene 10% WS (water soluble) placebo beadlets,
10
1 + 9
1
Lot UT02071003 32, containing fish gelatin, saccharose,
5
1 + 9
0.5
vitamin C palmitate, maize oil, DL-a-tocopherol, water.
2
1 + 9
0.2
Test materials for dietary supplements were obtained from
commercial sources and provided by AOAC
1
1 + 9
0.1
INTERNATIONAL.
b-carotene
beadlets, reference materials,
0.5
1 + 9
0.05
and the negative control materials were provided by DSM

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SCHIERLE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 87, NO. 5, 2004
Table 2. Method precision for measurement of total and all-trans-b-carotene in test materials Nos. 1–8a
Total b-carotene, mg/g
All-trans-b-carotene, mg/g
Extraction
LC
Mean value,
Mean value,
Test material No.
variant
system
n = 10
RSD, %
n = 10
RSD, %
Multivitamin tablets (1)
c,2
A
0.494
2.4
0.428
3.5
Softgels with b-carotene in soybean oil (2)
c,3
A
47.1
1.5
45.2
1.4
Capsules with b-carotene in fish oil (3)
c,1b
B
0.0520
3.1
0.0310
4.6
Softgels with b-carotene from carrot oil extract (4)
c,3
B
9.64
2.2
6.62
2.4
Tablets with b-carotene (5)
d,3b
A
49.3
2.6
39.2
3.0
Capsules with b-carotene (6a)
d,2b
A
23.7
10.4
22.8
10.4
Capsules with b-carotene (6b)
c,3
A
26.2
1.2
25.1
1.2
Capsules with b-carotene (7)
c,3
B
32.2
4.4
23
5.4
Beadlets raw material with pure b-carotene (8)
b
A
201
1.3
160
2.0
a
Analyses were performed in duplicate by 2 technicians on 5 different days.
b
Only for validation purpose (analytical method prescribes other variants for these test materials).
cryptoxanthin, lutein, and zeaxanthin. In the case of products
(e) Homogenizer.—750 W, 12 mm diameter dispersing
with relatively high a-carotene content, the cis-isomers of
aggregate [Polytron PT3100 (drive unit), PT-DA-3012/2T
a-carotene can interfere. In this case, the extract is also
(aggregate); Kinematica, Lucerne, Switzerland].
chromatographed with a more selective but longer running
(f) Syringe.—Disposable, 2 mL (Henke-Sass, Wolf
C30 reversed-phase LC system that avoids this interference.
GmbH,
Tuttlingen, Germany).
(g) Filter.—Disposable, 0.45 mm pore size, 25 mm
Scope
diameter, for organic solvents (Chromafil Type O-45/25;
The method is suitable for the determination of all-trans
Machery-Nagel, Düren, Germany); 0.45 mm pore size, for
and total b-carotene. The term total b-carotene comprises
organic solvents (Millipore, Bedford, MA; Type DVPP, 5 mm
all-trans-b-carotene and all other compounds identified as
diameter).
cis-isomers of b-carotene. The method is applicable to the
(h) LC system.—Consisting of online degasser (154,
determination of 0.01 mg or greater of total b-carotene in
Gastorr; Omnilab, Mettmenstetten, Switzerland); gradient
tablets, capsules, and >0.2% b-carotene in oily or
pump (L6200; Merck-VWR, Dietikon, Switzerland);
water-dispersible raw materials in the presence of other
autosampler with cooling unit (AS-1559; Jasco, available
carotenoids such as lycopene, a-carotene, and xanthophylls.
from Omnilab); column thermostat (Jetstream 2 Plus;
Thermotechnic Products, A-2103 Langenzersdorf, Austria);
Apparatus
UV-Vis detector (Jasco PU-2070 Plus, available from
Omnilab); integrator (ATLASä Chromatography Data
(a) Balances.—With readability of 0.01 mg, precision
System; Thermo LabSystems, Manchester, UK, combined
[standard deviation (SD)] of ±0.015 mg, capacity 205 g
with Microsoftâ Windows 2000 Terminal Server, Citrix
(AT261 DeltaRange; Mettler-Toledo, N@nikon-Uster,
MetaFrame).
Switzerland); with readability of 0.01 g, precision (SD) of
(i) Suplex PKB-100 LC column.—5 mm, 250 ´ 4.6 mm,
± 0.005 g, capacity 2100 g (PM2000; Mettler-Toledo).
Cat. No. 58934 (Supelco, Bellefonte, PA).
(b) Spectrophotometer.—Dual beam, wavelength range of
(j) YMC-Pack C30 LC column.—5 mm, 250 ´ 4.6 mm,
190–900 nm, 1.5 nm fixed spectral bandwidth, wavelength
Product Code CT99S052546WT, available, e.g., from
accuracy of 0.07 nm (at 541.92 nm), wavelength
Stagroma (Wallisellen, Switzerland).
reproducibility of 0.01 nm (Cary 50 Scan; Varian, Darmstadt,
Germany).
Reagents
(c) Ultrasonic water bath.—150 W, 35 kHz, 4 L
(TUC-160;
Telsonic,
Bronschhofen, Switzerland).
All chemicals and solvents were analytical grade unless
(d) Rotary evaporator.—5–240 rpm, 20°–100°C
otherwise specified.
(Rotavapor R-114, water bath R-480; Büchi Labortechnik,
(a) Chemicals.—Butylated
hydroxytoluene (BHT,
Flawil, Switzerland), connected to a vacuum pump with
2,6-di-tert-butyl-p-cresol): Purity ³99% (Fluka purum grade);
absolute minimum pressure of 10 mbar (PC 5; Vacuubrand,
N-ethyldiisopropylamine: purity ³98% (GC; Fluka purum
Wertheim, Germany).
grade).

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SCHIERLE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 87, NO. 5, 2004 1073
Table 3. Isomerization of b-carotene during extraction procedure
Percentage of b-carotene isomers, %a
Direct
After extraction
Sample
All-trans
9-cis
13-cis
15-cis
All-trans
9-cis
13-cis
15-cis
Almost pure all-trans-b-carotene
97.2
0.7
2.2
—
97.4
0.8
1.9
—
Heat-isomerization b-carotene
73.0
1.1
23.4
2.5
72.6
1.6
24.0
1.8
a
Total b-carotene = 100%.
(b) Solvents.—Acetonitrile: purity ³99.9% (Merck LC
to volume with ethanol [standard working solution, 3 mg/mL
grade); tert-butyl methyl ether (tBME): purity ³99.5% (Fluka
b-carotene in ethanol–tetrahydrofuran (9 + 1)]. Store standard
puriss grade); water: demineralized.
stock, measuring and working solutions at 5°C and use only
(c) Protex 6L.—Bacterial alkaline protease enzyme
on day of preparation.
preparation in water. Synonyms: subtilisin, IUB 3.4.21.62,
(b) Spectrophotometry.—Measure absorption of the
CAS 9014-01-1. EINECS 2327522, available from Genencor
standard measuring solution against cyclohexane at the
International, Inc. (Palo Alto, CA; www.genencor.com).
maximum, ca 456 nm. Calculate concentration of b-carotene,
(d) Ammonium acetate solution 0.2%.—Dissolve 0.50 g
Cstd, as follows:
ammonium acetate in 250 mL water. Store solution at 5°C for
no longer than 1 month.
Cstd (mg/L) = E ´ 10 000/2500
(e) Mobile phase A.—In 1 L volumetric flask, dissolve
here E is the absorption at the maximum, 2500 is the E
50 mg BHT in 20 mL 2-propanol and add 0.2 mL
1%,1cm of
pure all-trans-b-carotene in cyclohexane, and 10 000 is the
N-ethyldiisopropylamine, 25 mL 0.2% ammonium acetate
factor to convert % to mg/L.
solution, 455 mL acetonitrile, and ca 450 mL methanol.
Calculate the apparent E
Mixture cools and contracts. Let warm to room temperature
1%,1cm of the reference substance
(ap E
and dilute to volume with methanol. Discard after 2 days.
1%,1cm) used to prepare the standard solutions as follows:
(f) Mobile phase B.—In 1 L volumetric flask, dissolve
Apparent E1%,1cm = E ´ 10 000 ´ 2/W
100 mg vitamin C in ca 100 mL methanol (sonicate), add
100 mL tBME, and dilute to volume with methanol. Discard
where E is the absorption at the maximum, 10 000 is the factor
after 2 days.
to convert mg/L to %, 2 is the theoretical volume (in L) in hich
(g) b-carotene reference substance.—b-carotene, C
the reference substance is dissolved, and W is the weight of
40H56,
FW 536.9, CAS 7235-40-7, purity ³95% (LC), available
under Product Code 11045800 from Dr. Ehrenstorfer GmbH
(Augsburg, Germany). Store under argon or nitrogen in a cool
dark place. The compound deteriorates in the presence of
oxygen.
(h) Other carotenoids reference substances.—All-trans-lutein,
all-trans-zeaxanthin,
all-trans-b-cryptoxanthin,
all-trans-lycopene,
all-trans-a-carotene, all-trans-b-carotene¢, and 9-cis-, 13-cis-,
15-cis-, 9,9¢-di-cis, 9,15-di-cis-, 13,15-di-cis-b-carotene were
synthesized at DSM Nutritional Products.
Calibration
Protect solutions from direct sun or UV light.
(a) Standard solutions.—3 mg/mL b-carotene. Weigh with
an accuracy of 0.01 mg ca 6 mg all-trans-b-carotene reference
material into 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 mg/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,
Figure 2. LC system A chromatogram of an isomeric
3 mg/mL b-carotene in cyclohexane–tetrahydrofuran (95 +
mixture of b-carotene as present in many supplements.
5)]. Pipet 5 mL tetrahydrofuran into the other flask and dilute
BCA: b-carotene.

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SCHIERLE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 87, NO. 5, 2004
Figure 3. Chromatogram of test material No. 4 (Nature's Plus, Octa-carotene) obtained with LC system A. ACA:
a-carotene; BCA: b-carotene.
Figure 4. Chromatogram of test sample No. 4 (Nature's Plus, Octa-carotene) obtained with LC system B. ACA:
a-carotene; BCA: b-carotene.

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SCHIERLE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 87, NO. 5, 2004 1075
Table 4. Total and all-trans-b-carotene in test materials Nos. 1–8 measured by LC systems A and B
Total-b-carotene, mg/dose
All-trans-b-carotene, mg/dose
LC
LC
LC
LC
Test material (No.)
system A system B
Difference, %a
system A system B Difference, %a
0.62
Multivitamin tablets (1)
0.725
0.743
2.5
0.625
9
0.5
Softgels with b-carotene in soybean oil (2)
15.9
16.00
0.8
15.2
15.3
0.9
0.0
0.037 0.0
Capsules with b-carotene in fish oil (3)
599
0.0599
0.0
0
364
–1.5
Softgels with b-carotene from carrot oil extract (4)
6.83
6.31
–7.7
4.85
4.36
–10.0
Tablets with b-carotene (5)
24.6
25.1
2.4
19.5
19.5
–0.4
Capsules with b-carotene (6)
6.82
6.81
–0.0
6.53
6.53
0.0
Capsules with b-carotene, claim 25 mg b-carotene
(7)
20.7
20.9
1.2
14.9
15.0
0.6
157
Beadlets raw material with pure b-carotene (8)b
199
205
3.2

158
0.7
a
Results fo rLC system A = 100%.
b
Unit is g/kg instead of mg/dose.
the reference substance (in mg). The apparent E1%,1cm must
(2) Use of the LC control solution.—Measure the initial
exceed 2375 for a spectrophotometric purity >95%.
total b-carotene content of the control solution when the LC
(c) LC of standard solution.—Inject at least 6 aliquots of
system is calibrated. Inject in parallel with the standard
20 mL of the standard working solution, (a), immediately after
solution, at least 6 aliquots of 20 mL into the LC system.
preparation into the LC system. Calculate the response factor
Calculate the mean b-carotene content of the control solution
of all-trans-b-carotene from the averaged total peak areas of
from the resulting chromatograms using the newly determined
the chromatograms and the spectrophotometrically measured
response factor. Subsequently inject the control solution
b-carotene concentration as
together with each series of test extracts. The response factor
is regarded as constant as long as the measured total
b-carotene content of the control solution corresponds to the
RFtrans [AU ´ L/mg] = Astd/Cstd
initial value within ±2%. As long as the response factor
remains within these limits, the original response factor can be
where RFtrans is the response factor of all-trans-b-carotene,
used for calculations. However, the LC system must be
Astd is the mean total peak area of the standard chromatograms
recalibrated if the measured total b-carotene content exceeds
(Area Units, AU), and Cstd is the spectrophotometrically
the tolerance.
measured b-carotene concentration of the standard measuring
(3) Linearity of the LC response.—Dissolve 10 mg
solution [mg/L]. The peak area of all-trans-b-carotene must
all-trans-b-carotene in 5 mL dichloromethane in 100 mL
exceed 95% of the total peak area (i.e., chromatographic
volumetric flask. Add 50 mL ethanol and reflux at 80°C in
purity is >95%).
water bath for 2 h. Add ca 40 mL dichloromethane, bring to
(d) LC response of control solutions.—Control solutions
room temperature, and dilute to volume with
are solutions of heat isomerized b-carotene, concentrations of
dichloromethane. Dilute solution (100 mg/mL) with mixed
which have been found to be stable at 5°C for at least 3
solvent as shown in Table 1 to obtain b-carotene
months in the dark. It is not necessary to recalibrate the LC
concentrations of 100–0.05 mg/mL.
system if the previous response factor has been shown to be
Inject solutions into the LC system and calculate the
valid by use of control solutions.
b-carotene content as in Calculations. The response must be
(1) Preparation of the LC control solution.—Dissolve
linear for b-carotene concentrations of 0.1–50 mg/mL with a
about 3 mg b-carotene reference substance and 1 g BHT in
determination coefficient R2 > 0.995, and the back calculated
50 mL tetrahydrofuran. Add 200 mL ethanol and reflux for 2 h
concentration is within the target of ±10% of theoretical.
in a water bath at 80°C. Cool, dilute to 500 mL with ethanol,
and transfer solution to a dispenser bottle. Mix well, leave
Sample Preparation
overnight at room temperature, and dispense solution into a
large number of LC vials. Carefully seal the vials immediately
Sample preparation is dependent on the physical form of
after filling with Teflon/silicone septa, and store at ca 5°C in
the material, the claimed content of b-carotene, and the weight
the dark.
of the dosage form.

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SCHIERLE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 87, NO. 5, 2004
Table 5. Peak area ratios of b-carotene isomers in eluents of LC-systems A and B of the present method compared
with earlier LC system (25)
Eluent of LC system published earliera 445
Eluent of LC system A,
nm, 0.8 mL/min
448 nm, 0.6 mL/min
Sample
Peak areasb
cis/trans ratio
Peak areasb
cis/trans ratio
All-trans-b-carotene
1209 ± 21
—
902 ± 15
—
9-cis-b-carotene
1241 ± 4
1.03
925 ± 7
1.03
13-cis-b-carotene
375 ± 19
0.31
281 ± 9
0.31
15-cis-b-carotene
774 ± 25
0.64
573 ± 8
0.64
Eluent of LC system published earliera
Eluent of LC system B,
445 nm, 0.8 mL/min
445 nm 0.4 mL/minc
Peak areasb
cis/trans ratio
Peak areasb
cis/trans ratio
All-trans-b-carotene
546 ± 2
—
1083 ± 3
—
9-cis-b-carotene
560 ± 4
1.03
1115 ± 1
1.03
13-cis-b-carotene
447 ± 1
0.82
881 ± 4
0.81
15-cis-b-carotene
390 ± 2
0.71
774 ± 1
0.72
a
Methanol–THF (99 + 1), 100 ppm vitamin C (25).
b
Mean and standard deviation from 4 injections.
c
Flow rate was sent to a 0.4 mL/min for better separation of isomers.
(a) Mean weight per dose.—Weigh 20 tablets or capsules
50 mL. If necessary, filter the solutions through a 0.45 mm
and calculate the mean weight, WD. Use the mean weight to
membrane. Inject 20 mL into LC system.
calculate the b-carotene content of test material.
(b) Powders, beadlets, or emulsions.—Accurately weigh
(b) Number of tablets or capsules per assay.—For content
test portion equivalent to about 10 mg b-carotene into 250 mL
uniformity tests, take 1 tablet or the content of 1 capsule for
volumetric flask. Add 250 mg BHT, 0.5 mL Protex 6L, and
each assay and perform 10 assays in parallel. Report
15 mL water. Sway to wet contents and place in ultrasonic
individual results. For determination of the mean b-carotene
bath at ca 50°C for 30 min, swirling after about 15 min. Add
content, take equivalent of content of 3 tablets or capsules for
100 mL ethanol to the warm suspension and shake vigorously.
each assay and conduct 2 assays in parallel. Report the
Add 135 mL dichloromethane, and shake again. Mixture
averaged result of the 2 assays.
cools and contracts. Let stand in dark until room temperature
(c) Preparation of test sample.—Homogenize
is reached (ca 2 h), dilute to volume with dichloromethane,
suspensions or emulsions by intensive stirring, e.g., with a
shake vigorously, and let solids settle. Dilute an aliquot of the
glass rod. Crush tablets to a powder by grinding a tablet placed
supernatant in a ratio of 1 + 9 with dichloromethane–ethanol
between the 2 halves of a folded weighing paper with a pestle.
(1 + 1) in a volumetric flask, e.g., 5 to 50 mL. If necessary
Empty capsules containing powder formulations and extract
filter the solutions through 0.45 mm membrane. Inject 20 mL
powder together with capsule shells. Use capsules containing
into LC system.
liquid formulations as such.
(c) Tablets and capsules with test portion mass
Extraction
<5 g.—Accurately weigh test portions of 1–3 tablets or
capsules, intact or broken as in Sample Preparation (c), into
Protect extracts and solutions from direct sun or UV light.
250 mL volumetric flask. Add 250 mg BHT, 0.5 mL Protex
(a) Oily solutions or suspensions.—Accurately weigh test
6L, and 15 mL water. Sway to wet contents and place in
portion equivalent to ca 20 mg b-carotene, add 250 mg BHT,
ultrasonic bath at ca 50°C for 30 min, swirling after about
and rinse into 250 mL volumetric flask with 120 mL
15 min. Add 100 mL ethanol to the warm suspension and
dichloromethane. Add 100 mL ethanol, and shake. Mixture
shake vigorously. Add 120 mL dichloromethane and shake
cools and contracts. Let stand in dark until room temperature
again. If clumps form, homogenize with rotation
is reached (ca 2 h), dilute to volume with dichloromethane,
homogenizer, rinse with 15 mL dichloromethane, combining
and shake vigorously. Dilute aliquot in ratio of 1 + 9 with
rinse with contents of volumetric flask. Mixture cools and
dichloromethane–ethanol (1 + 1) in volumetric flask, e.g., 5 to
contracts. Let stand in dark until room temperature is reached

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SCHIERLE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 87, NO. 5, 2004 1077
Table 6. Recovery of different extraction variants
Spiked total b-carotene,a mg/g
Recovered total b-carotene, mg/g
Extraction
Negative control
variant
material No.
Mean
RSD, % (n = 3)
Mean
RSD, % (n = 3)
Recovery, %b
b
2
2.05
0.5
2.04
1.5
99.7
b
2
398
0.5
396
0.3
99.5
c,1
1
0.00876
2.1
0.00854
7.6
97.5
c,2
1
1.023
0.5
0.999
1.2
97.6
c,3
1
99.5
0.5
100.5
1.1
101.1
d,1
1
0.00983
0.8
0.00996
4.3
101.3
d,2
1
0.995
0.7
0.983
2.3
98.8
d,3
1
99.5
0.5
101.6
2.0
102.1
a
Concentration determined after appropriate dilution of spiking solution.
b
Relative to amount of spiked total-b-carotene (= 100%).
(ca 2 h), dilute to volume with dichloromethane, shake
volume with dichloromethane, shake vigorously, and let
vigorously, and let solids settle. Proceed as follows:
solids settle. Proceed as follows:
(1) b-carotene content of the test portion <0.1 mg.—
(1) b-carotene content of the test portion
Evaporate an aliquot, e.g., 50 mL, of the supernatant under
<0.5 mg.—Evaporate an aliquot, e.g., 50 mL, of supernatant
reduced pressure at 50°C using rotary evaporator and 250 mL
under reduced pressure at 50°C using a rotary evaporator and
round-bottom flask. Dissolve dry residue in
250 mL round-bottom flask. Dissolve dry residue in
dichloromethane–ethanol (1 + 1) so that extract is
dichloromethane–ethanol (1 + 1) so that the extract is
concentrated by a factor of 10, e.g., dissolve residue from
concentrated by a factor of 10, e.g., dissolve residue from
50 mL in 5 mL.
50 mL in 5 mL.
(2) b-carotene content of the test portion between 0.1 and
(2) b-carotene content of the test portion between 0.5 and
10 mg.—Use supernatant without dilution or concentration.
50 mg.—Use supernatant without dilution or concentration.
(3) b-carotene content of test portion >10 mg.—Dilute
(3) b-carotene content of the test portion >50
aliquot of supernatant with dichloromethane–ethanol (1 + 1)
mg.—Dilute aliquot of supernatant with
so that b-carotene content of final solution is between 1 and
dichloromethane–ethanol (1 + 1) so that b-carotene content of
10 mg/mL. Filter solutions through 0.45 mm membrane, if
final solution is between 1 and 10 mg/mL. Filter the solutions
necessary, and inject 20 mL into LC system.
through 0.45 mm membrane, if necessary, and inject 20 mL
into LC system.
(d) Tablets with mass of test portion >5 g.—Accurately
weigh test portions of 1–3 tablets prepared as in Sample
Chromatography
Preparation (c), into weighed 100 mL volumetric flask. Add
1 g BHT, 1 mL Protex 6L, and 40 mL water. In the case of
Test solutions are first injected into LC system A involving
effervescent tablets, add water slowly and in small portions to
a C18 column. In this system, cis-isomers of a-carotene can
avoid foaming over and wait for the next step until all gas has
interfere with the trans- and cis-isomers of b-carotene. If in
been released. Shaking and adding a few droplets of ethanol
chromatograms of LC system A, the peak area of
help to repress foaming. Place in ultrasonic bath at ca 50°C for
all-trans-a-carotene exceeds the total peak area of b-carotene
30 min, swirling after about 15 min. Add 50 mL ethanol to the
isomers by >5%, the test solution is analyzed by the more
warm suspension, let cool to room temperature, and dilute to
selective LC system B.
volume with water. Weigh flask and contents. Shake
(a) LC system A.—(1) Conditions.—Column: Suplex
vigorously and immediately pour 8–12 g suspension into a
pKb-100; column temperature 30°C; mobile phase A (see
tared 100 mL volumetric flask using a funnel. Weigh the
Reagents); flow rate 0.6 mL/min; pressure ca 33 bar; run time
transferred aliquot of the suspension. Add 35 mL ethanol and
ca 30 min; injection volume 20 mL; autosampler temperature
shake. Add 35 mL dichloromethane and shake again. If
15°C; detection at 448 nm. (2) Retention
clumps form, homogenize with rotation homogenizer, rinse
times.—All-trans-b-carotene: ca 20–25 min. Retention times
with 15 mL dichloromethane, combining rinse with contents
relative to all-trans-b-carotene: all-trans-lutein 0.30;
of volumetric flask. Mixture cools and contracts. Let stand in
all-trans-zeaxanthin 0.32; all-trans-b-cryptoxanthin 0.58;
dark until room temperature is reached (ca 2 h), dilute to
all-trans-lycopene 0.67; nonidentified cis-lycopenes 0.69,

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1078
SCHIERLE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 87, NO. 5, 2004
0.75, 0.79; 9¢-cis-a-carotene 0.91; all-trans-a-carotene 0.93;
all-trans-b-carotene (AU ´ L / mg); V is the theoretical
9-cis-a-carotene 0.98; 13-cis and 13¢-cis-a-carotene 1.03;
volume in which the test portion is dissolved (L); V1 is the
nonidentified cis-a-carotene 1.08; all-trans-b-carotene 1.00;
volume of the flask used for extraction with
9-cis-b-carotene 1.07; 13-cis-b-carotene 1.17;
dichloromethane–ethanol (L); V2 is the volume aliquot which
15-cis-b-carotene 1.21. Other minor cis-isomers of b-carotene
is diluted or evaporated (L); V3 is the volume to which aliquot
eluted in the range of the trans- and mono-cis-isomers of
V2 is diluted or in which the residue after evaporation of
b-carotene (e.g., with relative retention times of 1.02 for
aliquot V2 is dissolved (L). W1 and W2 apply only to
13,15-di-cis-b-carotene, 1.11 for 9,9¢-di-cis-b-carotene, and
extraction (d). W1 is the weight of the aqueous alcohol
1.12 for 9,15-di-cis-b-carotene).
suspension in the first 100 mL volumetric flask, and W2 is the
(b) LC system B.—(1) Conditions.—Column YMC-Pack
weight of the aliquot of the aqueous alcohol suspension
C30; column temperature 30°C; mobile phase B (see
transferred to the second 100 mL flask.
Reagents); flow rate 0.9 mL/min (use 2 mL/min during flush);
pressure ca 80 bar; total run time ca 65 min; injection volume
Procedures Used for Optimization and Validation
20 mL; autosampler temperature 15°C; detection at 445 nm.
(a) Selectivity.—The selectivity of the used LC systems
After elution of 9-cis-b-carotene, flush column with pure
was studied by chromatography of various synthetic
tBME at flow rate of 2 mL/min for 3 min and recondition
carotenoid isomers (all-trans-lutein, all-trans-zeaxanthin,
column with mobile phase B for 5 min. (2) Retention
all-trans-b-cryptoxanthin,
all-trans-lycopene, all-trans-a-carotene,
times.—All-trans-b-carotene: ca 40–55 min. Retention times
all-trans-b-carotene,
9-cis-,
13-cis-,
15-cis-, 9,9¢-di-cis, 9,15-di-cis-,
relative to all-trans-b-carotene: all-trans-lutein 0.22;
13,15-di-cis-b-carotene) and of iodine-isomerized a- and
all-trans-zeaxanthin 0.26; all-trans-b-cryptoxanthin 0.51;
b-carotene solutions. Peak identification was done by
13-cis-a-carotene 0.48; nonidentified cis-b-carotene 0.50;
co-chromatography with the mentioned compounds, by
nonidentified cis-a-carotene 0.50; 13¢-cis-a-carotene 0.53;
comparison of diode array detector spectra with published
15-cis-b-carotene 0.56; 13,15-di-cis-b-carotene 0.58;
data (22) and by comparison with published elution
13-cis-b-carotene 0.61; 9,15-di-cis-b-carotene 0.64;
profiles (23). In LC system A, peaks of various cis-isomers of
nonidentified
cis-b-carotene 0.73; all-trans-a-carotene 0.76;
a-carotene were additionally identified by injecting
9-cis-a-carotene 0.79; 9,9¢-di-cis-b-carotene 0.91;
unidentified peak fractions collected from the eluate of LC
9¢-cis-a-carotene 0.95; all-trans-b-carotene 1.00;
system B. For iodine isomerization, 10 mg crystalline
9-cis-b-carotene 1.12; lycopene isomers >1.20.
all-trans-a-carotene, 10 mg all-trans-b-carotene, and
Calculations
1000 mg BHT were dissolved in 5 mL dichloromethane. The
solution was diluted with cyclohexane to 100 mL. A 10 mL
Calculate contents of total and all-trans-b-carotene in test
volume of this stock solution was combined in a 100 mL
samples as follows:
volumetric flask with 40 mL cyclohexane and 2 mL freshly
prepared solution of 10 ppm iodine in cyclohexane (w/v). The
C
[mg / g] = (A
´ )
V / (RF
´ )
m
trans
trans
trans
mixture was incubated in a water bath at ca 65°C. After 15 min,
the solution was quickly cooled to ambient temperature and
C
[mg / dose] = (A
´ V´ W ) / (RF
´ m´ n
trans
trans
D
trans
)
diluted to volume with cyclohexane. A 50 mL volume of the
C
solution was evaporated at 50°C under vacuum with a rotary
tot [mg/g] =
[(A
evaporator. The dry residue was dissolved in 5 mL
trans + A9-cis + A13-cis ´ 1.2 + A15-cis
dichloromethane–ethanol (1 + 1) and filled into LC- vials.
´ 1.4 + AX-cis) ´ V]/(RFtrans ´ m)
(b) Linearity.—The linearity of the response of LC
C
systems A and B was examined. Dilutions from a solution of
tot [mg/dose] =
[(A
heat-isomerized b-carotene were prepared as described above
trans + A9-cis + A13-cis ´ 1.2 + A15-cis
in Calibration, with a total b-carotene range of ca
´ 1.4 + AX-cis) ´ V ´ WD]/(RFtrans ´ m ´ n)
0.005–100
mg/mL. All dilutions were prepared and measured
V = [V ´ V / V ]´ [W / W
in duplicate. From the resulting acceptable chromatograms,
1
3
2
1
2 ]
the total peak areas of all detected b-carotene isomers as well
where C
as the peak area of all-trans-b-carotene were determined and
tot and Ctrans are the total and all-trans-b-carotene
contents (mg/g or mg/dose); A
averaged for the duplicates. The relative response of the
trans, A9-cis, A13-cis, A15-cis are the
peak areas of all-trans-, 9-cis-, 13-cis-, 15-cis-b-carotene,
analyte, measured as mean peak areas of all-trans-b-carotene
respectively, in area units (AU); A
and of total b-carotene, was plotted against the respective
X-cis is the sum of the peak
area of other cis-isomers of b-carotene (AU); 1.2 and 1.4 are
concentrations. Curves were constructed using the
relative response factors (correction factors to compensate for
least-squares linear regression method.
lower specific absorption of 13-cis- and 15-cis-b-carotene
(c) Precision.—On 5 different days, 2 test portions from
compared to all-trans-b-carotene at the given wavelength);
each of the 8 test materials were analyzed according to the
WD is the mean weight of a dose (g); m is the test portion
method. One technician performed the work on the first
amount (g); n is the number of tablets or capsules used as
2 days, another on Days 3–5. The test portions consisted of 3
amount of test portion; RFtrans is the response factor of
tablets or capsules (test materials Nos. 1–7) or 100 mg (test

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SCHIERLE ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 87, NO. 5, 2004 1079
material No. 8). The extraction variants and LC systems used
extraction of stabilized carotenoids and fat-soluble vitamins
are shown in Table 2. The precision of extraction variant a was
from formulations based on gelatin, pectin, gummy arabicum,
not examined as it should be equal or better than variant b.
poly- and oligosaccharides. In order to demonstrate its
On the first day of the precision tests, we realized that the
efficacy, an artificially cross-linked gelatin formulation
aqueously suspended capsule shells of test material No. 6
(b-Carotene 10%B, DSM Nutritional Products, Product Code
agglutinated when ethanol was added. The clumps could not
04 3483 3) was extracted following the prescribed procedure.
be dispersed with a homogenizer (Polytron), and it was not
The filtered residue of this extraction was then washed from
possible to take a homogeneous aliquot from this suspension
cortically adhering b-carotene with ethanol and re-extracted
for further extraction, as prescribed in extraction variant d
using a 3-fold concentration of enzyme and the stronger but
(No. 6a). It seemed very probable that this variant could not
more toxic solvent chloroform instead of dichloromethane. The
work with this test material, and we decided to extract this
second extraction released <0.01% b-carotene found after
material following variant c (No. 6b).
routine extraction of the sample. Thus, the prescribed extraction
(d) Recovery.—Negative control materials were fortified,
can be regarded as quantitative. Note: This extraction procedure
in triplicate, with a mixture of b-carotene isomers generated
does not quantitatively extract carotenoids from plant cells
by heat isomerization and analyzed according to the method.
having thick cellulose walls. For such matrixes, extraction by
For a positive control, the spiking solutions used to spike the
saponification would be preferable.
negative control materials were diluted, in triplicate, with
(b) Isomerization and oxidation during the analysis.—
dichloromethane–ethanol (1 + 1) and analyzed directly by LC.
Carotenoids are susceptible to light- or heat-induced
In addition, triplicate unfortified controls were analyzed. Only
geometrical isomerization (24). Thus, b-carotene could be
LC system A was used in this experiment.
isomerized during the analytical procedure. However, as
For preparation of spiking solutions, 2.5 g b-carotene
shown in Table 3, the isomeric ratios in a heat-treated solution
reference material was dissolved in ca 70 mL chloroform. The
of b-carotene and in an almost pure all-trans-b-carotene were
solution was enclosed in a pressure-resistant glass vessel and
barely affected when the solutions were subjected to the
heated in a water bath at 80°C. After 2 h, the solution was
extraction procedure and the following steps of the proposed
cooled to room temperature and diluted with chloroform to
method (extraction variant b). The conditions of the assay
250 mL (spiking solution 1, 10 000 mg/mL). Spiking solution
were appropriate to retain the original isomeric ratio present in
2 (100 mg/mL) was prepared by pipetting 5 mL spiking
a sample to a high degree. Further, the procedure obviously
solution 1 into 500 mL volumetric flask and diluting to
did not give rise to oxidation of b-carotene as the analysis of
volume with dichloromethane. Spiking solution 3 was
various test samples performed in absence and presence of
obtained by diluting 5 mL spiking solution 2 to 500 mL with
BHT gave well corresponding results. Nevertheless, BHT was
dichloromethane–ethanol (1 + 1). The concentration of
prescribed in the present method to prevent any potential
spiking solution 3 was observed to be unstable in pure
attack by oxidants possibly present in other samples.
dichloromethane.
Similarly, the autosampler temperature was set to 15°C even
Negative control material No. 1 (placebo tablets) was
though no significant degradation or isomerization of
spiked, in test portions of 3 tablets (for variant c) and 6 tablets
b-carotene was observed, when standard solutions,
(for variant d) at total b-carotene concentrations of ca 0.01 (=
heat-isomerized control solutions, or extracts of test materials
lower range limit of the present method), and 100 mg per test
stood for 20 h at 23°C in the autosampler rack.
portion (= ca 130% of highest b-carotene concentration in test
(c) Selectivity.—LC system A of the present method has
materials of this study). Negative control material No. 2
been used for many years in our laboratory for the routine
(placebo beadlets) was fortified at total b-carotene
analysis of b-carotene in food (18). The system is able to
concentrations of ca 0.2% (= lower range limit of the attached
separate the mono-cis-isomers of b-carotene from each other
method) and 40% (= ca 130% of highest b-carotene
and from all-trans-b-carotene as well as the b-carotene
concentration in commercial product forms). The recovery of
isomers from other carotenoids as lutein, zeaxanthin,
extraction variant a was not examined, as it should be equal to
b-cryptoxanthin, and lycopene (Figures 2 and 3). The present
or better than variant b.
optimization revealed that LC system A also separated
all-trans- and 9¢-cis-a-carotene from all the b-carotene
Results and Discussion
isomers detected in an iodine-isomerized preparation, but
9-cis-, 13-cis-, and 13¢-cis-a-carotene co-eluted with the
Optimization
b-carotene isomers. This interference may be negligible for
supplements and raw materials containing low total amounts
(a) Extraction efficacy.—In the present method,
of a-carotene or only trans-a-carotene but not for materials
b-carotene is released from water-dispersible formulations
with high amounts of isomerized a-carotene, e.g., palm or
such as powders and emulsions, or tablets and capsules, by
carrot oil. For such samples, the more selective LC system B
enzymatic digestion with protease followed by extraction with
was used, which provided a good separation of many
a mixture of ethanol and dichloromethane. This procedure has
cis-isomers of a-carotene from those of b-carotene (Figure 4).
been routinely used for more than 2 decades in the laboratories
This system was optimized starting from the conditions
of Roche Vitamins Ltd. (now DSM Nutritional Products) for
described by Emenhiser et al. (23) by increasing the flow rate

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