Journal of Chromatography, 409 (1987) 419-425

Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

CHROM. 19 999

Note

Determination of vitamin A derivatives in fortified foods and commer-

cial vitamin supplements by high-performance liquid chromatography

SAMY H. ASHOOR* and MICHAEL J. KNOX

Division of Agriculture, Arizona State University, Tempe, AZ 85287 (U.S.A.)
(First received June 3rd, 1987; revised manuscript received August 24th, 1987)

Present official AOAC methods for determination of vitamin A in foods and

feed* have several disadvantages related to sample preparation and clean-up, preci-
sion and specificity2-5. A simpler, more precise and specific method is therefore needed
for determination of vitamin A in foods and feed.
Because of the inherent advantages of high-performance liquid chromato-
graphy (HPLC), several HPLC methods were developed recently for accurate deter-
mination of vitamin A in foods and feed 2- l I. However, most of these HPLC methods
require sample saponification and/or sample extract evaporation2-g. These steps may
result in vitamin A losses during analysis lo . The HPLC methods which require no
saponification or evaporation steps were developed for specific foods only such as
margarine and liquid milkslo, and fortified milk powders’ I. They are not versatile
methods. In this study, a simple HPLC method for determination of retinol, retinyl
acetate and retinyl palmitate in foods and commercial vitamin supplements is re-
ported. The method applies to egg yolk and several types of fortified foods and does
not require sample saponification or extract evaporation. It is accurate with recov-
eries higher than 90% and precise with coefficients of variation of less than 5%.

EXPERIMENTAL

Apparatus
HPLC analysis was achieved with a Water Assoc. (Milford, MA, U.S.A.)
liquid chromatograph equipped with a Model 6000 A pump, a Model U6K injector,
and a Model 730 data module. An Autochrom (Milford, MA, U.S.A.) Model 11l-
2 gradient controller was used for gradient elution, and a variable-wavelength Model
222 detector (Gilson, Middleton, WI, U.S.A.) was used for detection at 325 nm and
0.05 a.u.f.s.
A 250 x 4 mm I.D. Bio-Sil ODS-5S reversed-phase column with a 40 x 4.6
mm I.D. ODS-10 cartridge (Bio-Rad Labs, Richmond, CA, U.S.A.) was used for
HPLC analysis.
Food samples were blended with extracting solvent in a Waring blender with
a variable transformer (VWR Scientific, Norwalk, CA, U.S.A.) to regulate speed and
prevent overheating. Stirring and heating were done on a Corning hotplate, stirrer
Model PC-351 (VWR Scientific).

0021-9673/87/$03.50 0 1987 Elsevier Science Publishers B.V.

420 NOTES

Reagents
HPLC mobile phase. HPLC-grade water (double distilled and filtered through
a 0.45~pm membrane filter) was used in preparing HPLC solvents. Solvent A was
0.1% glacial acetic acid in water, and solvent B was acetonitrile-n-butanol-glacial
acetic acid (60:40:0.1). The linear gradient used was 75-lOO-lOO-75% solvent B in
10, 5 and 10 min, respectively. The flow-rate was 1.5 ml/min.
Extracting solvent. Chloroform-acetone (50:50) containing 1 mg/ml antioxi-
dant butylated hydroxytoluene (BHT).
Vitamin A standard solutions. All-trans-retinol, retinyl acetate and retinyl pal-
mitate (with antioxidants) were purchased from Sigma (St. Louis, MO, U.S.A.). Four
standard solutions of each of vitamin A derivatives were prepared in chloroform-
methanol (50:50) containing 1 mg/ml antioxidant BHT with concentrations ranging
from 1 to 10 pg/ml. These standard solutions were used to calibrate the data module
and in constructing standard curves of vitamin A derivatives.

Sample preparation
Preparation of samples and subsequent vitamin A extraction should be done
under subdued light and in the presence of antioxidants (butylated hydroxyanisole
and/or BHT) to prevent degradation of vitamin A derivatives, especially retinol.
Fortified cereals were prepared for extraction by fine grinding using porcelain mortar
and pestle. Fortified cheese and yogurt samples were mixed well with a plastic spoon.
Egg yolk was separated carefully from white then beaten gently with a plastic spoon.
Fortified soft drink mixes were mixed well with a plastic spoon before vitamin A
extraction.

Vitamin A extraction
An appropriate weight of prepared samples (5-10 g cereal; 10 g cheese, yogurt
or soft drink mix; 20 g egg yolk) was transferred to a glass blender jar and blended
for 5 min with 80 ml extracting solvent. Blend of each solid sample (cereals, soft
drink mix, and cottage cheese) was filtered directly through anhydrous sodium sulfate
with rinsing the blender jar twice with 10 ml extracting solvent and adding rinses to
blend. The filtrate was received in a lOO-ml volumetric flask and the volume was
made to mark with extracting solvent. Each of the liquid or semi-solid samples blends
(yogurt and egg yolk) was transferred to a 250-ml separatory funnel. The blender jar
was rinsed twice with 10 ml extracting solvent and rinses were added to blend in the
separatory funnel. The blend was shaken vigorously then left still for a few seconds
for phase separation. The lower phase was passed through anhydrous sodium sulfate
into a lOO-ml volumetric flask and volume was adjusted with extracting solvent.
Sample extracts were all refrigerated until HPLC analysis.
For vitamin supplements, two tablets or capsules were stirred with 25 ml warm
water in a 150-ml beaker at 50-55°C for 5 min. A volume of 80 ml extracting solvent
was then added, and stirring at 50-55°C was continued for additional 3 min. Tablets
or capsules should be dissolved completely at this time. The blend was then trans-
ferred to a 250-ml separatory funnel and treated as in liquid samples. An additional
dilution of each vitamin supplement extract was required. This was done by diluting
l-2 ml of original extract to 50 ml with extracting solvent.
NOTES 421

HPLC analysis
Refrigerated sample extracts and one standard solution of each vitamin A
derivative were first warmed up to room temperature. A volume of 25 ~1 of each of
the standard solutions was injected into chromatograph and the data module was
calibrated according to retention time and amount of derivative injected. Calibration
was checked regularly using standard solutions. A volume of 25-50 ~1 of each sample
extract was injected into chromatograph and the amount of vitamin A derivative was
obtained directly from calibrated data module. Each HPLC run was at least 25 min.

Standard curves
A volume of 25 ~1 of each of the four standard solutions for each vitamin A
derivative was injected into liquid chromatograph three times, and the correspond-
ing data module area units were recorded. A standard curve for each vitamin A
derivative was constructed by plotting average area units against equivalent amounts
of vitamin A derivative injected.

Vitamin A recovery
Each sample analyzed in this study was spiked with known amount of a vit-
amin A derivative (known volume of a particular standard solution) so that vitamin
A content would approximately double in the spiked sample. Spiked samples were
analyzed for vitamin A content as mentioned above for unspiked samples. Recovery
percent of vitamin A was then calculated for each spiked sample.

RESULTS AND DISCUSSION

Constructed standard curves of retinol, retinyl acetate and retinyl palmitate

indicated a linear detector response for each compound over a range of 20-300 ng.
They also indicated that the method is sensitive and can detect as low as 20 ng of
any of vitamin A derivatives used. It should be noted that on weight basis, retinol
has the highest detector response followed by retinyl acetate then retinyl palmitate
(lOO%, 86% and 53%, respectively).
The HPLC conditions used to separate standard vitamin A derivatives resulted
in good separation with sharp peaks (Fig. 1). The gradient elution system used was
essential for the separation of peaks of minor isomers from major ones. This resulted
in more accurate data. A run time of about 22 min was required to separate the three
vitamin A derivatives. However, if the analysis involves retinol or its acetate ester
only, the run time can be shortened to about 11 min. If palmitate is the only vitamin
A derivative present, solvent B can be used isocratically to shorten the retention time
of palmitate ester to about 6 min. Preliminary runs with complete run time would be
very helpful in choosing optimum conditions for the analysis of one or more of
vitamin A derivatives.
In the preliminary phase of this study, different conditions for vitamin A ex-
traction were tried. Food samples were extracted with the solvent systems:
ethanol-hexane, ethanolchloroform, acetone-hexane, and acetone-chloroform at
different proportions. The optimum extraction time was also studied. Blending food
samples in acetone-chloroform (50:50) for 5 min was most convenient and yielded
high recoveries of vitamin A derivatives. Blending food samples in acetone alone for
 NOTES

A B

I 1 I I I

0 IO 20 30 0 IO 20 xl

REI-ENTION TIME, MIN RETENTION TIME. MIN

C D

G
I

0
RETENTION
I

IO
I

20 so

TIME, MIN
I I_
0 IO

RmENTION
20

TIME,
30

MIN

Fig. 1. Chromatograms of standard retinol (A), standard retinyl acetate (B), standard retinyl pahnitate
(C), and a mixture of the three standards (D).

up to 5 min then adding chloroform and blending for additional 5 min did not
improve recoveries significantly. As for capsules or tablets of vitamin supplements,
heated water (50-55°C) had to be used in order to completely dissolve them in a short
time. Mild heating did not affect the stability of vitamin A derivatives as judged by
high recoveries.
Results obtained from HPLC analysis of foods and vitamin supplements by
the HPLC method are shown in Tables I and II. Chromatograms of selected samples
are shown in Fig. 2. These results indicate that the method is precise and accurate
with coefficients of variation of 0.949% and recoveries of 93.2 f 1.8 to 100.3 f
1.6%. They also indicate that the method is versatile and can be applied to a wide
variety of foods. It should be mentioned that fortification with retinyl acetate was
 NOTES 423

TABLE I
HPLC ANALYSIS OF VITAMIN A IN FOODS

Sample Vitamin A Vitamin A Coeficien t of Recovery

derivative content variation (%) (%)*
(mgllo0 g)*

Raw egg yolk Retinol 0.82 f 0.04 4.9 100.3 f 1.6

Fortified unsweetened soft drink mix** Retinyl acetate 10.1 f 0.22 2.2 95.7 f 3.5
Fortified lowfat cottage cheese** Retinyl acetate 1.52 f 0.06 4.0 95.4 f 3.0
Fortified plain yogurt** Retinyl pahnitate 2.35 f 0.03 1.3 97.1 f 2.7
Fortified raspberry flavored yogurt*** Retinyl palmitate 2.42 f 0.03 1.2 99.2 f 2.1
Fortified unsweetened soft drink mix** Retinyl palmitate 15.8 f 0.7 4.4 97.3 f 3.2
Fortified breakfast cereal brand I** Retinyl palmitate 3.52 f 0.16 4.5 96.0 f 2.3
Fortified breakfast cereal brand 2*** Retinyl pahnitate 12.5 f 0.5 4.0 94.8 f 1.7
Fortified breakfast cereal brand 3*** Retinyl palmitate 21.5 f 0.9 4.3 95.3 f 2.0

* Mean of 6 determinations f standard deviation.

l* Level of fortification was at least 25% of U.S. RDA per serving (5.1 g soft drink; 28.4 g cereal brand 1).
*** Level of fortification was at least 100% of U.S. RDA per serving(113 g cheese; 227 g yogurt; 28.4 g cereal
brands 2 and 3).

done in our laboratory since foods fortified with the acetate ester are not commer-
cially available. Fortification of yogurt with retinyl palmitate was done for a com-
mercial product under actual production conditions.
In all chromatograms obtained by the HPLC method from food and vitamin
supplements analysis, peaks of vitamin A derivatives were well separated with no
interference from other compounds. This indicates that the method is specific to

TABLE II
HPLC ANALYSIS OF VITAMIN A IN COMMERCIAL VITAMIN SUPPLEMENTS

Sample Vitamin A Vitamin A Coejicien t of Vitamin A activity

derivative content variation (%) (W/tablet or
(mg/tablet)* capsule)

Found Declared

Multiple Retinyl acetate 3.17 f 0.07 2.2 9193 5000

vitamin
supplement
Vitamin A Retinyl pahnitate 7.86 f 0.21 2.7 14 305 10 t-JO0
supplement
brand 1
Vitamin A Retinyl palmitate 21.5 f 0.2 0.9 39 130 25 000
supplement
brand 2

* Mean of 6 determinations f standard deviation. Recoveries of vitamin A derivatives from spiked

samples were 93.2 f 1.8%, 96.1 f 2.2% and 94.5 f 2.5% for multiple vitamin supplement and vitamin
A supplement brands 1 and 2, respectively.
424 NOTES

B
A

RElENTION Tl ME, MIN RETENTION TIME. MIN

c
L 1 1 I

0 IO 20 30 0 IO 20 30

RETENTION TIME, MIN RETENTION TIME, MIN

Fig. 2. Chromatograms of egg yolk (A), multiple vitamin supplement (B), fortified breakfast cereal brand
2 (C), and vitamin A supplement brand 1 (D). Arrows indicate peaks of retinol, retinyl acetate, retinyl
palmitate, and retinyl palmitate in A, B, C and D, respectively.

vitamin A derivatives used in this study. The identity of the peak of each vitamin A
derivative in sample chromatograms was verified by co-injection and spiking with
the corresponding standard. No attempts were made to identify other minor isomers
of retinol.
It is hoped that because of the advantages mentioned above plus simplicity
and convenience, this HPLC method receives wide acceptability.
NOTES 425

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