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Journal ArticleDOI

Determination of β-carotene in commercial foods : Interlaboratory study

01 Sep 1997-Journal of AOAC International (AOAC International)-Vol. 80, Iss: 5, pp 1057-1064
TL;DR: In this article, two sample preparation procedures for determination of total β-carotene and (all-E)-β-Carotene were tested in a collaborative study involving 14 laboratories and using 4 commercial products containing supplemented or natural β-CAROTene.
Abstract: Two sample preparation procedures for determination of total β-carotene and (all-E)-β-carotene were tested in a collaborative study involving 14 laboratories and using 4 commercial products containing supplemented or natural β-carotene. One assay used classical sample preparation, consisting of saponification, extraction, washing, and drying of the extract and redissolution of residue for liquid chromatography (LC). The other assay used simple extraction without saponification. LC conditions were left for the analysts to decide as long as a clear separation of lycopene and α-carotene from β- carotene was achieved. Mean contents of test samples ranged from 0.3 to 18 mg/100 g for total β-carotene and from 0.2 to 16 mg/100 g for (all-E)-β-carotene. Repeatability relative standard deviations (RSD r ) for total β-carotene ranged from 2.9 to 5.6% and relative reproducibility standard deviation (RSD R ) ranged from 6.5 to 15%. Corresponding values for (all-E)-β-carotene were 3.3-5.1% for RSD r and 8.4-14% for RSD R . Excluding the (Z)-isomers from quantitation of β-carotene can result in significant underestimation of the effective α-carotene content especially if the LC systems used is high quality.

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SCHUEP
&
SCHIERLE:
JOURNAL
OF
AOAC
INTERNATIONAL
VOL.
80,
No.
5,1997
1057 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
FOOD COMPOSITION AND ADDITIVES
Determination of p-Carotene in Commercial Foods:
Interlaboratory Study
WILLY SCHUEPzyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA and JOSEPH SCHIERLE
F.
Hoffmann-La Roche Ltd., Vitamins and Fine Chemicals, CH-4070 Basel, Switzerland zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
Two sample preparation procedures for determina-
tion of total
B-carotene
and (all-£)-B-carotene were
tested in a collaborative study involving 14 labora-
tories and using 4 commercial products containing
supplemented or natural B-carotene. One assay
used classical sample preparation, consisting of
saponification, extraction, washing, and drying of
the extract and redissolution of residue for liquid
chromatography (LC). The other assay used simple
extraction without saponification. LC conditions
were left for the analysts to decide as long as a
clear separation of lycopene and cc-carotene from B-
carotene was achieved. Mean contents of test sam-
ples ranged from 0.3 to 18 mg/100 g for total B-caro-
tene and
from 0.2 to 16 mg/100 g for (all-£)-B-carotene.
Repeatability relative standard deviations (RSD
r
)
for
total
B-carotene
ranged from 2.9 to 5.6% and
relative reproducibility standard deviation
(RSDR)
ranged from 6.5 to 15%. Corresponding values for
(all-£)-B-carotene were
3.3-5.1
%
for RSD
r
and 8.4-
14%
for RSDR. Excluding the (Z)-isomers from
quantitation of
B-carotene
can result in significant
underestimation of the effective
B-carotene
content
especially if the LC systems used is high quality.
P
-Carotene is the most important provitamin A
source
and
may serve as an antioxidant in the body (1-3). It is also
an approved natural food colorant in many countries. In
fruits and vegetables, B-carotene may occur as a mixture of
geometrical isomers. All B-carotene isomers may undergo
(££)-isomerization during processing (4—11). Pure (all-£)-B-
carotene is
easily isomerized by light or heat to generate
a
mix-
ture that
contains (13-Z)- and (9-Z)-B-carotene as the main (Z)-
isomers (12, 13).
The most recent methods for determining B-carotene in
foodstuffs are based on adequate separation of B-carotene iso-
mers by
liquid chromatography
(LC;
14-27). Stationary phases
leading to
good separations are easily available. A more critical
step
seems to be sample preparation. B-Carotene easily can be
Received November
14,
1996.
Accepted by JL May
7,
1997.
Presented at the 5th Symposium
of
the AOAC
INTERNATIONAL
Europe
Section,
Paris,
France,
April
18-19,
1996.
extracted directly from plant sources such as most vegetables
and quantitated by LC if no other components interfere in the
chromatographic separation. More complex samples such as
foodstuffs need to be saponified first to simplify the matrix.
Unsaponifiable components are then extracted. The extract is
washed, dried, concentrated, and quantitated by LC.
The goal of this collaborative study was to test the sample
preparation steps of
2
assay procedures—direct extraction and
saponification—applied to 4 commercially available products
containing natural B-carotene from a plant source or supple-
mented with B-carotene.
Interlaboratory Study zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
Participants
Fourteen laboratories in 8 European countries participated
in this study. Collaborators were analysts in food and chemical
companies, universities, and commercial and governmental
laboratories. Each laboratory received 8 test samples (4 dupli-
cates) for determination of total B-carotene, (all-£)- B-carotene,
and standards for B-carotene, a-carotene, and lycopene. Sam-
ples were assigned laboratory codes at random.
Samples
Samples were chosen according to
3
criteria: type of matrix,
range of concentration, and availability in food stores. The
samples were
A,
margarine;
B,
vitamin drink; C, pudding pow-
der; and D, natural mixed vegetable. The B-carotene contents
were as follows: A, <0.3 mg/100 g; B, >2 mg/100 g; C, 2-
3 mg/100 g; and D, >10 mg/100 g.
Samples A and B were packed items, and units were dis-
patched in original containers after the homogeneity and uni-
formity of units were checked. Sample C was insufficiently ho-
mogeneous for direct analysis. Contents of individual selling
units were pooled and mixed in a Turbula mixer (Bachofen,
Maschinenfabrik, Basel, Switzerland). Sample D, a mixture of
air-dried vegetables, was ground to a uniform size with a
Retsch mill (Retsch GmbH, Haan, Germany) with a 0.75 mm
screen. Adequate portions of samples C and D were vacuum
sealed.
Participants were asked to perform 5 complete assays per
sample, including extraction and
LC,
and
to
provide one repre-
sentative chromatogram.
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1058
SCHUEP
&
SCHIERLE:
JOURNAL
OF
AOAC INTERNATIONAL VOL.
80,
No.
5,1997 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
METHOD zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
Reagents and Standard
Solutions
(a)zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA Reagents.—Butylated hydroxytoluene (BHT), dichlo-
romethane, ethanol, n-hexane, methanol, potassium hydroxide,
2-propanol, tert-butyl methyl ether, tetrahydrofuran (all Merck
pro analysis or equivalent). Mobile phases were prepared with
solvents and reagents of recognized analytical grade.
(b) Standard solutions.—P-Carotene stock solution was
prepared by dissolving an accurately weighed amount (ca
3 mg) of P-carotene standard in 20 mL dichloromethane fol-
lowed by a ca 30 s treatment in an ultrasonic bath. The solution
was diluted with
rc-hexane
to 100 mL, of which 10.0 mL was
diluted further with n-hexane to 100 mL, yielding a standard
solution of ca 3 iig P-carotene/mL in n-hexane-dichlo-
romethane (98 +
2).
The absorbance of the P-carotene standard
solution was measured at the wavelength of maximum absor-
bance (453 nm), and the concentration was calculated by using
an EJ J value of 2592 (27). A 20 mL portion of the standard
was pipetted into a round-bottomed flask, and the solvent was
removed with a rotary evaporator under partial vacuum at a
temperature not exceeding 50°C. The residue was dissolved in
20 mL of a solvent compatible with the reversed-phase LC
used.
a-Carotene
and lycopene standard solutions were pre-
pared for qualitative purposes by predissolving ca 0.3 mg oc-
carotene or lycopene in ca 10 mL tetrahydrofuran and diluting
to 100 mL with ethanol or another solvent compatible with the
LC system.
(c) Thermal isomerization of ^-carotene.—A solution of
almost pure (all-£)-P-carotene in ethanol-tetrahydrofuran (9 +
1)
containing 1000 ppm BHT was heated in closed LC vials in
a water bath at 80°C for 2 h.
Sample Preparation
Assay of samples A, B, and C involved saponification fol-
lowed by extraction with fert-butyl methyl ether, washing of
extract, concentration, dissolving in an adequate solvent, and
quantitation by LC. Sample D, a vegetable mix containing
natural P-carotene, was extracted without saponification. The
following critical details of the procedure had to be followed,
whereas uncritical points were left to the analyst.
(a)
Saponification
method
(samples
A,
B, and
C).—Five
g
of each test sample was weighed in a 250 mL round-bottom
flask, 10 mL water was added, and the sample was dispersed
before addition of 50 mL ethanol (60 mL for sample A), 1 g
BHT, and
10
mL
tert-butyl
methyl
ether.
After
the
mixture was
mixed vigorously, 5.0 mL 50% aqueous potassium hydroxide
was added. The mixture was mixed again, heated at ca 80°C,
and allowed to reflux for 15 min. The cooled saponification
mixture was extracted with two 100 mL portions of
tert-butyl
methyl ether, and the combined extracts were washed 4 times
with 50 mL
water.
The extract was then either concentrated by
partial vacuum at a temperature at <50°C or adjusted to a de-
fined
volume.
Depending on
the
expected concentration, a por-
tion of the extract was evaporated in a round-bottom flask by
using a rotary evaporator under partial vacuum at temperatures
<50°C. The residue was dissolved in a solvent compatible with
the reversed-phase LC used and made up to a defined volume
(5-50 mL) to obtain a concentration of
up
to 5 (ig/mL.
(b) Direct
extraction
method (sample D).—About
1
g test
sample was weighed accurately in a 250 mL volumetric flask,
10 mL water was added, and the sample was dispersed for
5 min in an ultrasonic bath at room temperature. Then,
100 mL
ethanol was added; the mixture was shaken vigorously, made
up almost to the mark with dichloromethane, and then left in
the dark
to
reach room
temperature.
After
the
volume
was made
up to the mark with dichloromethane, the mixture was shaken
vigorously and then put aside to let solids settle. A
50 mL
por-
tion of the supernatant was concentrated under partial vacuum
at a temperature
<50°C.
The residue was dissolved in a solvent
compatible with the reversed-phase LC used and made up to
a
defined volume (5-50 mL) to obtain a concentration of
up
to
5 ^g/mL.
Chromatographic Determination
LC quantitation of P-carotene
was
required.
A description
of
a working
LC
system was given
to
participants (system of
labo-
ratory 4, Table 1); however, the choice of LC conditions was
left to the analysts, provided there was baseline separation of
lycopene and cc-carotene from P-carotene.
a-Carotene
and ly-
copene standards were sent to participants to allow evaluation
of their LC system. Because different isomers of P-carotene
were not commercially available, their quantitation was based
on the response factor of (all-£)-P-carotene. A compilation of
the LC conditions used by participants is given in Table 1.
Statistical
Methods
Tests for determining outlying laboratories and outlying
in-
dividual replicates were performed according to methods de-
scribed in ISO 5725-2 (29). The Cochran was test used to iden-
tify data showing excess variability among replicate
(within-laboratory) analyses, and the Grubb's test was used to
identify laboratories with extreme averages. Measures of assay
precision were calculated: standard deviations (SD), repeat-
ability standard deviation (s
r
), reproducibility standard devia-
tion (s
R
), repeatability relative standard deviation (RSD
r
), and
reproducibility relative standard deviation (RSD
R
). Maximum
tolerable differences were also calculated for repeatability
(r =
2.83 x s
r
) and reproducibility (R = 2.83 x s
R
).
Results and Discussion
Results are shown in Table
2.
Laboratory 6 quantitated
only
(all-£)-P-carotene. The
LC
system used by laboratory
13
quan-
titated only total P-carotene. Corresponding statistics are sum-
marized in Table 3. RSD
r
values ranged from 2.9 to 5.6% for
total P-carotene and from 3.3 to 5.1% for (all-£)-P-carotene.
RSD
R
values were between 6.5 and 15% for total P-carotene
and between 8.4 and 14% for (all-Zs)-P-carotene.
Total p-carotene concentrations of
the
4 test samples varied
widely. The lowest level was present in margarine (sample
A),
with
a
mean of 0.25 mg/100
g.
Although the matrix of this
sam-
ple was oil-based, the assay was accomplished without
compli-
cations.
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Table 1. LC conditions used by participants zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
Retention times, min
Detection zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
RT
B
22.5
30
450
450
445
10.5
16.0
18.6
17.9
20.4
25.7
18.8
22.8
28.0
19.8
24.2
29-32.1
Flow, Column wavelength, (all-£)-(5- (Z)-p-
Laboratory Precolumn Stationary phase Mobile phase mL/min temp., °C nm Lycopene a-Carotene Carotene Carotene
1 None Spherisorb ODS-2; 5 nm; 250 x CH
3
CN-O.05M AcONH
4
a
in 1.0
4.0 mm CH
3
OH-CH
2
CI
2
(75 + 20 + 5)
2 Spherisorb ODS-2; 10 x 4.6 mm + Vydac 201 TD; 5 urn; 250 x 4.6 mm CH
3
CN-Ch30H-CH2CI
2
(75 + 20 + 5) 1.5
100x4.6 mm
3 None Nucleosil 100-10Cia; 10um;250x Ch
3
OH-CH
3
CN-2-propanol-0.2% 1.0
4.0 mm AcONrU in H;>0-N-ethyldiiso-
propylamine (500 + 455 + 20 + 25
+ 0.2) + 50 ppm BHT
C
4 SuplexpKb-100;5um;30x4.6mm SuplexpKb-100; 5um; 250 x CH;3OH-CH
3
CN-2-propanol-0.2% 1.0 30 445 16.0 21.3 22.9 24.4-27.5
4.6 mm AcONH
4
in H20-W-ethyl-
diisopropylamine (500 + 455 + 20
+ 25 + 0.2) + 50 ppm BHT
5 RP18; 10x4.6 mm Brownlee Spheri-5 ODS; 5 nm CH3CN-CH3OH-THF (55 + 35 + 15) 0.8 RT 12.3 17.9 19.9 20.7
6 SuplexpKb-100 5um;30x4.6mm SuplexpKb-100; 5um; 250x CH
3
OH-CH
3
CN-2-propanol-0.2% 1-0 21 445 17.8 24.1 26.2
4.6 mm AcONH
4
in H20-/V-Ethyldiiso-
propylamine (500 + 455 + 20 + 25
+ 0.2) + 50 ppm BHT
7 Lichrospher 100 RP18; 5 um; 4 x Lichrospher 100 RP18 ; 5 um; 250 x CH
3
OH-CH
3
CN-2-propanol-0.2% 2.2 25 453 31.7 45.5 50.2 53.2
4.0 mm 4.0 mm AcONhU in H2O (500 + 455 + 20 +
25) + 50 ppm BHT
8 None Nucleosil-100Ci8AB;5um; 250x CH
3
OH-CH
3
CN-2-propanol-0.2% 1.5 25 455 14 15 17 18-19
4.0 mm AcONH
4
in H2O (500 + 455 + 20 +
25) + 50 ppm BHT
9 None Lichrospher 100 RP18; 5 um; 250 x CH
3
CN-CH
3
OH-THF (40 + 55 + 5)
4.0 mm
10 Lichrospher 100 RP18; 5 um; 4 x YMC, C
3
o polymeric non-endcapped; CH
3
OH-MTBE
d
gradient (30-80%
4.0 mm 5 um; 250 x 4.6 mm MTBE) +
0.1%
BHT
11 None Vydac 218 TP 54 Protein & Peptide CH
3
CN-0.05 M AcONH
4
in
Cia 5 um; 250 x 4.0 mm CH3OH-CH2CI2 (75 + 20 + 5)
12 None Vydac 218 TP 54 Protein & Peptide CH
3
OH-THF
e
(99 + 1)
Cia 5
um;
250 x 4.6 mm
13 None Ultrasphere ODS; 5 um; 250 x CH
3
OH-THF (95 + 5)
4.6 mm
14 Vydac 201 TP 54; 10x4.6 mm Vydac 201 TP54; 10 um; 250 x CH
3
OH-THF(95 + 5) +
0.1%
BHT
4.6 mm
a
AcONH
4
, ammonium acetate. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
b
RT, room temperature.
c
BHT, butylated hydroxytoluene.
d
MTBE, methyl tert-butyl ether.
e
THF, tetrahydrofuran.
2.0
1.0
1.0
0.6
3.0
1.0
30
22
15
RT
RT
20
450
452
445
450
450
445
9.2
18.4
20.3
22.8
12
24.8
13.1
11.3
19.6
17.2
13.6
13.5
14.1
12.4
22.3
19.6
14.6
15.6
14.6-15.2
10.8;
13.4
24-28
21.5-23.5
17.6
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Downloaded from https://academic.oup.com/jaoac/article/80/5/1057/5684295 by guest on 21 August 2022

SCHUEP
&
SCHIERLE:
JOURNALOF
AOAC
INTERNATIONAL
VOL.
80,
No.
5,1997
1061 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
Table 2.zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA (continued) zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
Laboratory
11
12
13
14
Assay No.
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
Sample A (margarine)
Total (All-E)-
p-carotene p-carotene
0.257
0.261
0.265
0.268
0.276
0.233
0.235
0.235
0.247
0.235
0.255
0.273
0.227
0.229
0.234
0.267
0.270
0.266
0.262
0.216
0.217
0.218
0.222
0.23
0.203
0.213
0.208
0.222
0.212
0.224
0.224
0.221
0.219
Sample
Total
p-carotene
2.38
2.38
2.37
2.26
2.36
2.02
2.06
2.14
2.24
2.05
2.24
2.18
2.07
2.14
2.20
2.18
2.13
2.24
1.99
2.20
p-carotene,
B (drink)
(All-E)-
P-carotene
1.93
1.94
1.99
1.84
1.93
1.98
2.01
2.09
2.20
2.01
1.73
1.69
1.77
1.58
1.74
mg/100g
Sample C
(pudding powder)
Total
p-carotene
1.67
1.50
1.47
1.74
1.56
1.63
1.70
1.53
1.60
1.56
1.60
1.39
1.44
1.43
1.24
1.73
1.63
1.68
1.56
1.63
(All-E)-
P-carotene
0.94
0.85
0.83
0.99
0.88
1.58
c
1.65
c
1.49
c
1.55
c
1.54
c
1.00
0.95
0.99
0.90
0.96
Sample D
(vegetable mix)
Total
P-carotene
p
21.0
21.2
21.3
21.5
20.8
8.8°
7.6
C
6.4°
9.0°
6.5
C
15.45
14.83
22.8
23.6
22.0
23.5
24.8
(All-E)-
-carotene
18.0
18.0
18.1
18.2
17.8
8.7
C
7.5
C
6.2
C
8.9
C
6.3
C
18.8
19.1
18.4
19.8
20.5
a
, No result. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
" Cochran test outlier.
' Grubb's test outlier.
The mean
total [3-carotene concentration of sample
B,
a sup-
plemented drink, was 2.25 mg/100 g, 10-fold higher than that
of sample
A.
This sample showed the best repeatability and re-
producibility values. Because this sample was a
liquid,
it might
be concluded that the (3-carotene in it was better distributed
compared with other samples.
Sample C, a pudding powder, had a mean level of supple-
mented total (3-carotene of 1.53 mg/100 g. The RSD
r
values
Table 3. Statistical results and precision data for determination of (all-£)- and total p-carotene
Parameter
Total number of laboratories
Number of outliers
Number of laboratories after
elimination of outliers
Number of accepted results
Mean
content,
mg/1 OOg
Repeatability s
r
,
mg/1 OOg
Relative repeatability RSD
r
, %
Repeatability value r,
mg/1 OOg
Reproducibility s
R
,
mg/1 OOg
Relative reproducibility RSD
R
, %
Reproducibility value R,
mg/1 OOg
Sample A
Total
13
1
12
55
0.253
0.011
4.5
0.032
0.024
9.7
0.069
(margarine)
All-E
13
1
12
53
0.223
0.009
3.9
0.025
0.026
12
0.073
Sample
Total
13
1
12
59
2.248
0.065
2.9
0.19
0.15
6.5
0.41
Value for indicated sample
B(drink)
All-E
13
1
12
57
1.933
0.064
3.3
0.18
0.16
8.4
0.46
Sample C (pudding
powder)
Total
13
2
11
51
1.531
0.085
5.6
0.24
0.14
9.3
0.40
All-E
13
3
10
44
0.974
0.050
5.1
0.14
0.14
14
0.38
Sample
Total
13
1
12
56
18.05
0.71
3.9
2.0
2.7
15
7.6
D (vegetable
mix)
All-E
11
1
10
49
16.01
0.75
4.7
2.1
2.1
13
5.8
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