KAMANGAR & FAWZI: J. ASSOC. OFF. ANAL. CHEM. (VOL. 61, NO.
3, 1978) 753
Spectrophotometric Determination of Vitamin A in Oils and Fats
TEYMOUR KAMANGAR and AHMAD B. FAWZF
Tehran University, Department of Food Chemistry, College of Pharmacy, Tehran, Iran
A spectrophotometric method for deter-
mining vitamin A based on interaction with
50% trichloroacetic acid solution in dichloro-
methane was developed. The blue reaction prod-
uct had a maximum absorption at 620 nm and
obeyed Beer's law over the concentration range
of 0.5-5.0 fig retinol/ml solution. The molar
absorptivity of the reaction product was 1.58 X
10 5 . As much as 100 fig vitamin D 2 , and /?-
carotene up to 12 times the vitamin A concen-
tration, did not interfere with the determina-
tion. The results obtained from the determina-
tion of vitamin A in cod liver oil and butter
showed excellent agreement with the Carr-Price
method, 43.013 (d).
The colorimetric Carr-Price reaction is the offi-
cial AOAC method (1) for determining vitamin A
in feeds and foods. Other colorimetric reagents
such as glycerol dichlorohydrin (2-4), 2,3-dichloro-
propanol (5), trichloroacetic acid (6-8), trifluoro-
acetic acid (9, 10), and iodine (11) have been
used for the quantitative determination of vitamin
A.
The reaction of trichloroacetic acid (TCA) with
vitamin A was reported by Rosenheim and Drum-
mond (12), but Carr and Price (13) were unable
to use the reagent for quantitative determination
of the vitamin. Later, Bayfield (6, 7) obtained satis-
factory results for vitamin A in serum and liver by
using a freshly prepared saturated solution of TCA
in alcohol-free and recently distilled chloroform.
Grys (8) improved the method by dissolving the
reagent in anhydrous chloroform and using a fast-
measurement accessory with a dedicated recorder.
Dehydrated magnesium chloride was used for the
preparation of anhydrous chloroform, and the pre-
pared reagent was stable for 5 hr with daylight
excluded. TCA, because of its ready availability
and low toxicity, and the ease of cleaning of cells,
was a suitable reagent for routine analysis for the
vitamin. However, the reported TCA methods (6,
8) had no real advantage over the Carr-Price
method because of instability of the reagent and
special conditions required for its preparation.
Recently, Subramanyam and Parrish tested
antimony trichloride, trifluoroacetic acid, and TCA
in solutions of chloroform, dichloromethane
(DCM), and dichloroethane as colorimetric re-
1
Address all correspondence to this author.
0004-5756/78/6103-0753$00.90
© Association of Official Analytical Chemists, Inc.
agents for determining vitamin A in feeds and
foods (14). They found that, in general, the re-
agents in DCM solutions produced the most stable
blue complex. When we substituted DCM for
chloroform in the TCA reagent solution, the re-
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agent was completely stable in daylight, was more
sensitive, and was easily prepared. The principal
disadvantages of the Carr-Price reagent—rapid
fading of the developed color, interference of
moisture with the determination, difficulty in
cleaning the cells, and possible toxicity of the re-
agent—are avoided. In addition, the color-develop-
ing activity of the reagent is constant, and there
is no need for routine pre-standardization or
checking of the reagent. In the present investiga-
tion, the optimum conditions for the spectrophoto-
metric determination of vitamin A by using TCA
solution in DCM and the application of the
method for the determination of the vitamin in
oils and fats are described.
METHOD
Reagents and Apparatus
Reagents and chemicals used were analytical
grade.
(a) Trichloroacetic acid reagent.—50% (Cau-
tion: TCA is corrosive; handle with care.) Dis-
solve 25 g TCA crystals, with aid of gentle heat,
in 35 ml DCM and dilute to 50 ml with DCM.
(b) Standard vitamin A solutions.—Dissolve ac-
curately weighed amount of USP Reference Stand-
ard Solution of vitamin A in 50 ml DCM. Prepare
solutions containing 0.5-5.0 yjg retinol/ml by dilu-
tion with DCM. Use freshly prepared solutions.
Work in subdued light or use low-actinic glass-
ware.
(c) Potassium hydroxide solution.—See 43.009
(g) (1).
(d) Spectrophotometer.—Beckman DB-GT with
1.00 cm cells and Beckman 10 in. recorder.
Preparation of Sample Assay Solution
Carry out the following experiments as rapidly
as consistent, in subdued light, and at low labo-
ratory temperature.
(a) Saponification.—Weigh accurate amount of
oil or fat containing 12.5-125 /tg retinol into 250
or 500 ml boiling flask. Digest sample as in
Received May 23, 1977. Accepted October 5, 1977.
754 KAMANGAR & FAWZI: J. ASSOC. OFF. ANAL. CHEM. (VOL. 61, NO. 3, 1978)
43.013 (a) (1), ending with "Cool rapidly to room
temp.".
(b) Extraction.—Transfer solution to 125 or
250 ml separatory funnel. Wash flask 4 and 2 times
with 5 ml portions of water and alcohol, respec-
tively, and add washings to the separatory funnel.
Extract vitamin A by vigorous shaking with 20,
15, and five 10 ml portions of hexane. Combine
extracts in 125 ml separatory funnel. Wash extract
3 times with 30 ml portions of tap water, followed
by successive 30 ml portions of distilled water
until free of alkali. Let extract stand 15 min and
draw off any separated water.
(c) Solvent removal.—Evaporate extract to dry-
ness under vacuum or nitrogen in water bath at
60-65°C. Dissolve residue immediately in DCM
and adjust volume of solution to 25 ml.
Procedure
Adjust spectrophotometer at 620 nm, using mix-
ture of 1 ml DCM and 2 ml TCA reagent solution.
Pipet exactly 1 ml DCM sample solution con-
taining 0.5-5.0 fig retinol into spectrophotometer
cell and place cell in instrument. Cover cell com-
partment with piece of carton with small hole on
cell side. Set chart speed to 0.1 in./sec, switch in-
strument on, and start recorder. Pipet exactly 2
ml TCA reagent solution from rapid delivery
pipet, within 1-2 sec, directly into cell and record
absorbance for 30 sec.
Calculation
Calculate amount of vitamin A by comparison
with standard curve or factor. Determine recovery
factor, R, for vitamin A by adding known amount
of vitamin A to samples, and proceeding with
entire analysis.
Vitamin A, fig/g = C X 25/(R X W)
where C = fig vitamin A/ml DCM solution calcu-
lated from curve or factor; and W = g sample.
Results and Discussion
The resulting bright blue complex from the re-
action between vitamin A and TCA reagent solu-
tion, following the recommended procedure,
showed maximum absorption at 620 nm. Color
intensity at 620 nm was maximum 5 sec after re-
agent addition, remained constant 5 sec, and then
decreased slowly.
Concentrations of 25, 50, 75, and 100% TCA
reagent in DCM and chloroform were prepared
separately and treated, as described in the recom-
mended procedure, with a known concentration of
vitamin A solution prepared in the same solvent.
Reagent solution in DCM showed maximum color
intensity with 50% solution and was constant with
higher reagent concentrations. The color intensity
increased as the reagent concentration in chloro-
form increased. The color obtained by using 50%
reagent solution in DCM was more intense than
that produced by the saturated reagent solution in
chloroform prepared by Grys (8) and also by the
antimony trichloride reagent 43.009(h) (1).
The 50% reagent, solution in DCM was prepared
and stored in tightly stoppered containers in the
daylight. The colo • intensity of the reaction prod-
uct between the reagent and a known amount of
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vitamin A was measured at selected intervals of
time for as long JLS 4 months, and was the same
as that produced by using freshly prepared re-
agent. This finding is in close agreement with the
observations of Subramanyam and Parrish (14).
Nine standard series of 10 samples, ranging in
concentration from 0.4 to 9.2 /ig retinol/ml, were
prepared and treated by the proposed procedure.
The optimum cone entration range for the measure-
ments, 0.5-5.0 fug retinol/ml solution, conformed
to Beer's law, at 620 nm and 1.00 cm optical path-
length. The molar absorptivity of the colored re-
action product at 620 nm was 1.58 X 105. The ab-
sorbance of a solution containing 3.70 fig retinol/
ml was 0.680±0.004. The relative standard deviation
of the calculated absorptivities in the optimum
concentration range was 0.8%. The percentage ac-
curacy and the confidence limits of a solution con-
taining 2.67 fig retinol/ml were calculated to be
0.4% and 2.68±0.02 fig, respectively.
The bluish violet of the reaction product of /?-
carotene and the reagent developed 5 sec after
the reagent addition, and reached maximum in-
tensity at 620 nm within 15-20 sec. Solutions con-
taining a known amount of vitamin A and dif-
ferent concentrations of /3-carotene were prepared
and analyzed by the recommended procedure. At-
tempts at absorbimce addition failed. Since the
color developed only after 5 sec, the speed of the
chart recorder wa« set at 1.0 in./sec, and the ab-
sorbance was recorded at 5 sec exactly. This al-
lowed accurate determination of the vitamin in
the presence of 12 times its concentration of /3-
carotene.
Vitamin D 2 reacts with the reagent, forming a
yellow color which changes rapidly to yellowish
brown. At low concentrations, the yellow color has
no absorbance at 620 nm and does not interfere
in the determination of vitamin A. Above 100 fig
vitamin D 2 /ml, the blue vitamin A reaction prod-
uct fades quickly and the absorbance recording
is not accurate.
The vitamin A contents of cod liver oil and
butter were determined by the recommended pro-
cedure. To evaluate the accuracy of the method,
the results were compared with the Carr-Price
 KAMANGAR & FAWZI: J. ASSOC. OFF. ANAL. CHEM. (VOL. 61, NO. 3, 1978) 755

Table 1. Comparison of proposed and Carr-Price (2) Sobel, A. E., & Werbin, H. (1945) J. Biol.
methods for determining vitamin A Chem. 159, 681-691
(fig retinol/g sample)
(3) Sobel, A. E., & Werbin, H. (1946) Ind. Eng.
Rel. std Found, Chem. Anal. Ed. 18, 570-573
Sample Carr-Price° TCA° dev. .% %b (4) Sobel, A. E., & Werbin, H. (1947) Anal.
Chem. 19, 107-112
Cod liver oil 240. 0 247.0 1. 0 102.9
Butter 3.,12 3.19 1. 4 102.2
(5) Blake, J. A., & Moran, J. J. (1976) Can. J.
Chem. 54, 1757-1764
° Mean of 5 determinations. (6) Bayfield, R. F. (1971) Anal. Biochem. 39,
6
Calculated on the basis of Carr-Price measure- 282-287
ments. (7) Bayfield, R. F. (1975) Anal. Biochem. 64,

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method, 43.013(d) (1) (Table 1). Excellent agree-
ment of the 2 methods validates the proposed pro-
cedure for determining vitamin A in oils and fats.
Acknowledgment
The authors wish to express their sincere thanks
to Ali Shafiee for the facilities to carry out this
work.
REFERENCES
(1) Official Methods oj Analysis (1975) 12th Ed.,
AOAC, Washington, DC, sees 43.008-43.013
403-413
(8) Grys, S. (1975) Analyst 100, 637-639
(9) Dugan, R. E., Frigerio, N. A., & Siebert,
J. M. (1964) Anal. Chem. 36, 114-117
(10) Gharbo, S. A., & Gosser, L. A. (1975) Analyst
100, 703-707
(11) Al-Sulimany, F., & Townshend, A. (1973)
Anal. Lett. 6, 1029-1037
(12) Rosenheim, 0., & Drummond, J. C. (1925)
Biochem. J. 19, 753-756
(13) Carr, F. H., & Price, E. A. (1926) Biochem.
J. 20, 497-501
(14) Subramanyam, G. B., & Parrish, D. B. (1976)
J. Assoc. Off. Anal. Chem. 59, 1125-1130

CORRECTION
/ . Assoc. Off. Anal. Chem. 60, 1345-1349 (1977), Change average from 500 to 505.
"Comparison of Methods for Determining Pro-
benecid in Tablets and Flavored Oral Suspen-
sions Containing Ampicillin," by P. J. Vollmer,
R. Chastonay, C. Haneke, N. Kantor, and R.
Blyth, Table 2, Sample M