Vitamins AACC Method 86-06

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Analysis of Vitamins A and E by High-Performance Liquid

Chromatography
First approval November 8, 2000

Objective
This primary method measures the presence and quantity of vitamin A as retinol
whether it is present as retinol or as retinol esters (retinyl acetate and/or retinyl
palmitate) and vitamin E as a-tocopherol (expressed as a-tocopherol acetate)
whether it is present as a-tocopherol or as a-tocopherol esters (such as a-
tocopherol acetate) in foods. This method is applicable to all foods. The detec-
tion/quantitation limits are 0.15 µg/g for retinol and 0.001 mg/g for a-tocopherol.
This method covers the range of 0.15 µg/g to 100% vitamin A and 0.001 mg/g to
100% vitamin E. Standards and samples are saponified in basic ethanol-water
solution, neutralized, and diluted, converting fats to fatty acids and retinol esters
and tocopherol esters to retinol and tocopherol, respectively. Retinol and a-
tocopherol are quantitated on separate high-performance liquid chromatography
(HPLC) systems, using UV detection at 313 or 328 nm for retinol and fluores-
cence detection (excitation 290 nm, emission 330 nm) for a-tocopherol. Vitamin
concentrations are calculated by comparison of the peak heights or peak areas of
vitamins in samples with those of standards. Caution: see Note 1.

Apparatus
1. HPLC system. See Note 2.
a. Pump, high-pressure, capable of operating continuously at 1.0–2.0
ml/min, with a flow precision of ±1% or better.
b. Injector, manual or autosampling, equipped with a 20-µl fixed loop
having a typical sampling precision of ±0.25% or better.
c. Chromatography columns
For vitamin A: reverse-phase C18, 10-µm (4.6 × 250 mm), capable of
separating cis and trans isomers of retinol with a resolution of 1.0 or
greater. cis-Retinol typically elutes before trans-retinol on columns, pro-
viding effective separation.
For vitamin E: reverse-phase C8, 10-µm (4.6 × 250 mm), capable of
separating a- and b-tocopherol or a- and g-tocopherol with a resolution
of 1.5 or better. The tocopherols will typically elute in the order d-, g-,
and b- (rarely well separated on reverse-phase columns), and then a-
tocopherol. It is important to achieve this resolution; otherwise, low lev-
els of a-tocopherol will be masked by high levels of g- and b-tocopherol
in products with high levels of naturally occurring g- or b-tocopherol
(i.e., oleomargarines produced from soy oil),
d. Detectors. For vitamin A: photometric detector, capable of monitoring absor-
bance at 328 nm. (Alternatively, a wavelength of 313 nm can be used.) For
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Vitamin A and Vitamin E in Foods by HPLC (continued)

vitamin E: fluorescence detector, excitation of 290 nm with emission meas-

ured at 330 nm.
e. Recorder, integrator, or data collection system. Compatible with detec-
tors used.
2. Erlenmeyer flasks, low-actinic, 125 ml, with neck adapted for connecting
reflux condenser.
3. Hot plate, with sufficient heating surface area to handle multiple reflux
apparatus setups preferred. See Fig. 1.
4. Reflux condensers, with adapters (if necessary) to attach to 125-ml low-
actinic Erlenmeyer flasks and nitrogen lines.

Fig. 1. Reflux apparatus.

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Vitamin A and Vitamin E in Foods by HPLC (continued)

5. Volumetric flasks, low-actinic, 100 ml and 10 ml.

6. Nitrogen blanket apparatus, a supply of nitrogen gas with appropriate tubing
and connectors to provide a constant nitrogen atmosphere blanket in the reflux
apparatus during saponification. See Fig. 1.

Reagents
1. USP-vitamin A acetate concentrate, equivalent to approximately 30 mg of
retinol per g of oil (content certified by USP).
or
Retinyl palmitate, all-trans. Request certificate of lot analysis when ordering.
If manufacturer’s certification is unavailable or purity of standard needs to be
verified, test vitamin A palmitate purity with method given in Note 3. Store at 0–
4° to allow for easier handling while weighing.
2. Vitamin E acetate.
3. Mixed tocopherol standard. Prepare a 50:50 chromatographic resolution test
mixture of a- and b- or a- and g-tocopherol in the mobile phase, at the concen-
tration level of the highest a-tocopherol standard.
4. Acetic acid, glacial, AR grade.
5. Methanol, HPLC grade.
6. Ethanol, 95%, AR grade.
7. Tetrahydrofuran (THF), AR grade.
8. Hexane, AR grade.
9. Pyrogallic acid, crystals, AR grade.
10. Vitamin A mobile phase. Combine 860 ml methanol (HPLC grade) and
140 ml deionized water. Mix well. Let stir overnight to degas or mechanically
degas before use.
11. Vitamin E mobile phase. Combine 940 ml methanol (HPLC grade) and 60
ml deionized water. Mix well. Let stir overnight to degas or mechanically degas
before use.
12. THF:ethanol, 50:50. Combine 500 ml THF and 500 ml 95% ethanol. Mix
well.
13. 50% potassium hydroxide (KOH) solution. Slowly add 500 g of KOH pellets
to 500 ml distilled water contained in a 2-liter thick-walled Erlenmeyer flask.
Caution: The solution gives off substantial heat while KOH is dissolving, so the
KOH should be added in 100-g portions while the flask is being cooled with cold
water. The flask should be swirled gently to aid in dissolution of KOH. Store in
glass container with cork stopper.
14. Standard solutions (use low-actinic glassware).
a. Vitamin A working standard (approximately 15 µg/ml).
Using USP standard: Weigh 50 mg vitamin A acetate concentrate into
a 100-ml volumetric flask. Record weight to nearest 0.1 mg. Record con-
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Vitamin A and Vitamin E in Foods by HPLC (continued)

centration in mg/g per USP certification. Add small amount of acetone

(less than 3 ml) to aid dissolution. Dilute to volume with 95% ethanol.
Store at 4° in dark. Solution is stable for two weeks.
Using retinyl palmitate: Weigh 55 mg retinyl palmitate into 100-ml
volumetric flask. Record weight to nearest 0.1 mg. Record purity per
supplier certification or purity test. Add pea-sized piece of pyrogallic
acid. Dissolve and dilute to volume with hexane. Pipet 5 ml solution to
second 100-ml flask and dilute to volume with 95% ethanol. Store at 4°
in dark. Solution is stable for two weeks.
b. Vitamin E stock solution (approximately 500 µg/ml). Weigh 50 mg vitamin
E acetate into 100-ml volumetric flask. Record weight to nearest 0.l mg.
Add small amount of acetone (less than 3 ml) to aid dissolution. Dilute to
volume with 95% ethanol. Store at 4° in dark. Solution is stable for one
month.
c. Vitamin E working standard (approximately 50 µg/ml). Pipet 10 ml
vitamin E stock solution into a 100-ml volumetric flask. Dilute to volume
with 95% ethanol. Store at 4° in dark. Solution is stable for one month.

Procedure
Preparation of sample
Solid samples should be ground to pass a 40-mesh sieve. Liquid or wet sam-
ples should be blended to homogeneity and stored at or below 4°. All samples
should be stored in the dark.

Saponification and extraction of sample

1. Turn on hot plate to preheat. Start and adjust cooling water flow to precool
reflux condensers.
2. Standards
a. High standard. Pipet 5 ml vitamin A working standard and 10 ml vitamin
E working standard into 125-ml Erlenmeyer flask. Add 25 ml 95% etha-
nol. Proceed to step 4.
b. Intermediate standard. Pipet 2 ml vitamin A working standard and 5 ml
vitamin E working standard into a second 125-ml Erlenmeyer flask. Add
33 ml 95% ethanol. Proceed to step 4.
c. Low standard. Pipet 0.5 ml vitamin A working standard and 2 ml vitamin
E working standard into a third 125-ml Erlenmeyer flask. Add 37.5 ml
95% ethanol. Proceed to step 4.
3. Samples
a. Low-fat (less than 40% fat). Weigh sample (not more than 5 g) to give
approximately 50 µg vitamin A and/or 1.0 mg vitamin E into 125-ml
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Vitamin A and Vitamin E in Foods by HPLC (continued)

Erlenmeyer flask. For samples high in sugar, add 3 ml water and disperse
sample as a slurry. Add 40 ml 95% ethanol.
b. High-fat. Weigh sample (not more than 2 g) to give approximately 50 µg
vitamin A and/or 1.0 mg vitamin E into 125-ml Erlenmeyer flask. Add 40
ml 95% ethanol.
4. Add a pea-sized piece (approximately 50 mg) of pyrogallic acid
(antioxidant) to each standard and sample flask. Add a glass bead to promote
even boiling.
5. Swirl all flasks to ensure that all samples are thoroughly dispersed in the
solution.
6. Turn on nitrogen flow and ensure a nitrogen atmosphere for all flasks while
refluxing.
7. Pipet 10 ml 50% KOH solution into each flask and immediately place flask
on hot plate under reflux condenser. Swirl.
8. Reflux 45 min. Swirl flasks every 10 min.
9. Remove reflux flasks from hot plate, stopper with corks, and quickly cool
flasks to room temperature, using cold water or ice water.
10. Pipet 10 ml glacial acetic acid solution into each flask to neutralize the
KOH. Mix well and let flasks cool again to room temperature.
11. Quantitatively transfer solution in each flask to 100-ml volumetric flask,
using 50:50 THF:ethanol. Dilute to volume with same.
12. Stopper and invert volumetric flask 10 times.
13. Allow samples to set for at least 1 hr at room temperature and preferably
overnight in refrigerator to allow fatty acid salts formed during saponification to
precipitate. In some cases, centrifugation may be helpful to reduce settling time.

Determination
1. Start HPLC system(s) and allow to warm up and equilibrate for minimum of
30 min with mobile phase flowing. Flow rate should be 1.0 ml/min for either
vitamin.
2. Inject vitamin A standard onto vitamin A HPLC system. Adjust mobile
phase to achieve a resolution of 1.5 or better for cis and trans forms. All trans
retinol should elute in approximately 6 min or longer. Inject chromatographic
resolution test mixture onto vitamin E HPLC system. Adjust mobile phase to
achieve a resolution of 1.5 or better for the two forms of tocopherol present in
the resolution test mix. a-Tocopherol should elute in approximately 6 min or
longer. See Figs. 2 and 3.
3. Inject high, medium, and low standards. Adjust detector sensitivity to give
peak heights of 50–90% of full scale for the vitamin of interest in the high stan-
dard. Repeat injection of standards until peak height(s) are reproducible.
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Vitamin A and Vitamin E in Foods by HPLC (continued)

4. Inject sample solutions. Intersperse with standard solution injections after

every nine samples to assure accurate quantitation. (If either retinol or a-
tocopherol exceeds the peak height of its high standard by more than 25%, dilute
sample solutions using a solution of 10 ml 50% KOH solution, 40 ml 95%
ethanol, 10 ml glacial acetic acid, and 40 ml 50:50 THF:ethanol solution.)

Calculations
For vitamin A
Calculate µg/g vitamin A (as retinol) as follows.
1. Measure peak heights or areas of standards.
a. Using USP standard
Response factor for vitamin A (RFA):
mgstd ™ mlstd ™ Cstd
RFA =
PHstd ™ 10,000

where mgstd = mg of USP standard weighed in reagents step 14a; mlstd =

ml of standard used in procedure step 2a, 2b, or 2c; Cstd = concentration
of USP vitamin A (as retinol) per USP certification (mg/g); PHstd = peak
height or area of standard from chromatogram; and 10,000 = combined
dilution factors for vitamin A standard.

Fig. 2. Chromatogram of vitamin A. histd = high standard.

 Component mg/g Peak PRT RRF Area Height
13-cis 1.99 001 5.78 1.8819 1.062 0.093
All-trans 60.970 001 6.29 1.8410 33.118 2.333
Total 62.969 34.180
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Vitamin A and Vitamin E in Foods by HPLC (continued)

b. Using retinyl palmitate

mgstd ™ mlstd ™ Pstd ™ 0.5458
RFA =
PHstd ™ 200
where: mgstd = mg retinyl palmitate weighed in reagents step 14a; Pstd =
percent purity certified by supplier (or determined by method in Note 3),
divided by 100; 0.5458 = ratio of retinol to retinyl palmitate molecular
weights; and 200 = combined dilution factors and conversion from mg to µg.
RFA values of low, medium, and high standards should agree with each other
within 3% relative since detector response should be linear across the
concentration range used here. Average of RFA values calculated from high,
medium, and low standards should be used for sample quantitation.
2. Measure peak heights or areas corresponding to retinol (vitamin A) in sample
extracts. The 13-cis isomer of retinol (eluting immediately before the all-trans
isomer) might be present in some samples. See Fig. 2. Measure 13-cis peak also.
3. Multiply height or area of 13-cis retinol peak by 1.08 (to compensate for
difference in absorbance compared to that of the trans isomer).
4. Add corrected peak height or area for 13-cis isomer to that of the all-trans
isomer to give total sample peak height or area.
RFA ™ PHsam ™ 100
Vitamin A, mg/g (as retinol) =
W

Fig. 3. Chromatogram of vitamin E. histd = high standard

Component IU/100 g Peak PRT RRF Area Height
0001 0.000 BCV 5.17 0.0000 4.406 0.492
Vitamin E 75.500 VCB 5.64 0.1036 728.559 48.455
Total 75.500 732.966
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Vitamin A and Vitamin E in Foods by HPLC (continued)

where PHsam = total sample peak height or area of all-trans and 13-cis retinol;
100 = dilution volume of sample; and W = weight of sample in g.

For Vitamin E
Calculate mg/g of vitamin E (as a-tocopherol acetate) as follows.
1. Measure the peak height or area of a-tocopherol in standards.
Response factor for vitamin E (RFE):
mgstd ™ mlstd
RFE =
PHstd ™ 100,000

where mgstd = mg a-tocopherol acetate standard weighed in reagents step 14b;

mlstd = ml standard used in procedure step 2a, 2b, or 2c; PHstd = peak height or
area of standard from chromatogram; and 100,000 = combined dilution factors in
vitamin E standard.
RFE values of low, medium, and high standards should agree with each other
within 3% relative since detector response should be linear across the
concentration range used here. Average of low, medium, and high RFE values
should be used for sample quantitation.
2. Measure peak height or area of peak corresponding to a-tocopherol on
chromatogram of sample extract.
RFE ™ PHsam ™ 100
Vitamin E, mg/g (as a - tocopherol acetate) =
W

where PHsam = sample peak height or area of a-tocopherol; 100 = dilution vol-
ume; W = weight of sample in g.
Alternatively, a three-level calibration using a zero polynomial fit (linear) can
be used to calculate vitamins A and E. The three standards (high, intermediate,
and low) should have a correlation coefficient between 0.995 and 1.000.

Notes
1. Caution. KOH is extremely caustic and can cause severe bums. Protect skin
and eyes. When using flammable liquids, perform operations behind a safety
barrier when using, steam, or electric mantle heating. Use an effective fume
removal device to remove flammable vapors as produced. Leave ample head-
room in flasks, and add boiling chips before heating is begun. All controls,
unless vapor sealed, should be located outside of vapor area. For toxic liquids,
use an effective fume removal device to remove vapors as produced. Avoid
contact with skin.
2. Two separate simple isocratic HPLC systems can be used to carry out the
determination steps of this method for vitamins A and E simultaneously. Alter-
natively, the retinol and a-tocopherol can be analyzed sequentially since the a-
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Vitamin A and Vitamin E in Foods by HPLC (continued)

tocopherol is sufficiently stable in the diluted saponification solution to be stored

while vitamin A is being run.
3. Purity of vitamin A palmitate may be tested as follows. Dissolve 50 mg
(record to nearest 0.1 mg) of retinol palmitate standard in 2-propanol (UV-
spectroscopy grade) in a 500-ml flask and dilute to volume. Dilute 10 ml of this
solution to 100 ml with 2-propanol (final concentration is approximately 10 mg
per liter). Measure maximum absorbance obtained at 325–328 nm using a 1-cm
pathlength cell and 2-propanol as blank. Calculate purity of retinol palmitate (4)
as
A ™ (5 ™ 106 )
Purity,% = max
960 ™ W
where Amax = absorbance maximum; (5 × 10–6) = combined dilution factors, con-
version to 1% equivalent solution, and conversion to percent; 960 = absorbance
of pure retinol palmitate (1% solution in 1-cm cell), and W = weight of sample in
mg.

References
1. Egberg, D. C., Heroff, J. C., and Potter, R. H. 1977. Determination of all-trans and 13-cis vita-
min A in food products by high-pressure liquid chromatography. J. Agric. Food Chem. 25:1127.
2. DeVries, J. W., Egberg, D. C., and Heroff, J. C. 1979. Concurrent analysis of vitamin A and
vitamin E by reversed-phase high-performance liquid chromatography. Page 477 in: Liquid
Chromatographic Analysis of Food and Beverages, Vol. 2. G. Charalambous, ed. Academic
Press, New York.
3. DeVries, J. W. 1985. Chromatographic assay of vitamins. Page 65 in: Methods of Vitamin
Assay, 4th ed. J. Augustin, B. Klein, D. Becker, and P. Venugopat, eds. Wiley Interscience, New
York.
4. Olson, J. A. 1990. Vitamin A. Page 8 in: Handbook of Vitamins. L. J. Machlin, ed. Marcel
Dekker, New York.