16.

0 VITAMIN ANALYSIS

16.1 Determination of vitamin A – Using HPLC

Reagents
• Water, HPLC grade
• Potassium hydroxide solution (Dissolve 500 g of KOH in water and dilute to 1 litre)
• Ethanol- 95%
• 2-Propanol
• Light petroleum (40-60ºC)
• Vitamin A standard
• All-trans-retinyl acetate, vitamin A acetate (C22H32O2), 328.5 g/mol, with a purity of at
least 90%.
• All-trans-retinol, vitamin A alcohol (C20H30O), 286.5 g/mol, with a purity of at least
90%
• Methanol, HPLC grade
• Mobile phase for liquid chromatography: Mix together methanol and water (770 +
30 v/v)
• Sodium sulfate (Na2SO4) anhydrous
• Sodium ascorbate solution, p = 100 g/l.
Apparatus
• High-performance liquid chromatograph (Column: length 250 mm, ID 4.6 mm, packed
with a stationary phase consisting of octadea (C18) groups bonded to silica).
• UV-Visible spectrometer
• Boiling water bath
• Rotary vacuum evaporator
• Extraction apparatus
• Membrane filter-0.45 µm pore size

Procedure
Saponification
• Weigh about 0.1 g sample in 1 litre conical flask.
• Add 200 ml of ethanol. Swirl the flask.
• Add 2 ml of sodium ascorbate and 50 ml of potassium hydroxide solution and mix
by swirling.
• Fit a reflux condenser to the flask and immerse the flask in the boiling water bath.
• Allow the contents of the flask to reflux for 60 min.
• Remove and cool the flask to room temperature as rapidly as possible under a stream
of cold water.

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Extraction of vitamin A (retinol)
• Transfer the contents of the flask to extraction cylinder.
• Rinse the flask with two 25 ml portions of ethanol and transfer the rinsing to cylinder.
• Repeat the rinsing of the flask with two 125 ml of petroleum and one 250 ml of
water, each time transferring the rinsing to the cylinder.
• Stopper the cylinder and shake well for 1 min.
• Cool under a stream of cold water while waiting for the two liquid phases to separate,
before removing the stopper.
When the layers have separated, remove the stopper, wash the sides of the stopper
with a few ml of light petroleum and insert the adjustable tube, positioning the lower
open end so that it is just above the level of the interface. By application of a slight
pressure of inert gas to the side-arm tube, transfer the upper, light petroleum layer
to a 1 litre separating funnel.
• Add 125 ml petroleum to cylinder and shake for 1 min.
• Allow the layers to separate and transfer the upper layer to the separating funnel
using the adjustable tube. Repeat above step again.
• Wash the combined light petroleum extracts with four 100 ml portions of water using
at first only gentle inversion then only gentle shaking in order to keep emulsion
formation to a minimum.
• Transfer the washed extract through a medium/fast filter paper containing 60 g of
anhydrous sodium sulfate, into a flask suitable for vacuum evaporation.
• Rinse the separating funnel with two 20 ml of light petroleum and add rinsing, through
the filter to the evaporation flask.
• Wash the filter further with two 25 ml of light petroleum and collect the washings in
the evaporation flask.
• Evaporate the petroleum extract to dryness under vacuum at a temperature not
exceeding 40ºC.
• Restore atmospheric pressure by admitting inert gas.
High performance liquid chromatography
• Dissolve the residue in the minimum volume of 2 propanol and transfer quantitatively
to a 20 ml volumetric flask.
• Rinse the evaporation flask with three small portions of 2-propanol, transferring the
rinsing to the volumetric flask. Dilute to volume with 2 propanol and mix and inject
10 µl of the sample extract into HPLC.
• Calculate the peak area.
Note: 1 IU of vitamin A is equal to 0.300 µg of all-trans-retinol.
1 IU of vitamin A is equal to 0.344 µg of all-trans-retinyl acetate.
Hydrolysis of all-trans-retinyl acetate for calibration
• Prepare a solution of all-trans-retinyl acetate in ethanol, so that 1 ml contains
approximately 15000 IU of vitamin A.
• Transfer 2.5 ml of this solution to a 150 ml flask.
• Add 20 ml of ethanol, 1 ml of potassium hydroxide and 5 ml of sodium ascorbate
solution.
• Fit a condenser to the flask. Immerse the flask in the boiling water bath and allow to
reflux for 60 min.
• Cool the flask to room temperature under a stream of cold water and transfer the
contents to a separating funnel.
• Rinse the flask with 50 ml of water, followed by 25 ml of ethanol, adding the rinsing
to the separating funnel.

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• Extract the aqueous phase with one 80 ml of petroleum and then with two 50 ml
portions of light petroleum.
• Combine petroleum extracts, and then wash with two 50 ml portions of water. Add
2 g of anhydrous sodium sulfate.
• Transfer petroleum extract to 250 ml volumetric flask and dilute to volume. The retinol
concentration of this solution (solution 1) is approximately 150 IU/ml.
Standardization of retinol solution for calibration
• Pipette 5 ml solution 1 into a 50 ml volumetric flask and remove the solvent, at
ambient temperature, with a stream of inert gas.
• Dissolve the residue in 2-propanol, and then dilute to volume with 2-propanol.
• Measure the absorbance (A) of the solution, using 2-propanol as reference,
at wavelengths of 310 nm, 325 nm and 334 nm. The absorbance values will be
approximately 0.7 to 0.8. If necessary, an intermediate dilution may be used.
Using the following equation, calculate the corrected absorbance at 325 nm:
A 325,corr = 6.815 x A 325 – 2.555 x A310 – 4.26 x A334
If A325,corr / A325 is less than 0.97, use the value of A 325,corr for the standardization; otherwise
use A325
The retinol concentration of solution 1 is given by :
Concentration (IU/ml) = A325 x 183 IU/ml, or
Concentration (IU/ml) = A325,corr x 183 IU/ml.
Preparation of a retinol standard for chromatography
• Prepare a retinol solution in 2-propanol. For each 1000 IU of vitamin A/kg of sample,
a retinol concentration of 2.5 IU/ml is expected in the extract.
• Evaporate an aliquot volume of solution 1 to dryness at ambient temperature with a
stream of inert gas. Dissolve the residue in the appropriate volume of 2-propanol to
provide the required retinol concentration and mix it.
• If necessary, filter the standard solution through a membrane filter.
• For calibration purposes, a standard solution of vitamin A (retinol) in 2-propanol
prepared by diluting a stock standard solution of all-trans-retinol made by dissolving
an appropriate quantity of all-trans-retinol standard substance directly in 2-propanol
(5.4) may be used.
• In this case, check the vitamin A standard by measuring the absorbance of the standard
solution in quartz cells at wavelengths of 300 nm, 325 nm, 350 nm and 370 nm against
2-propanol as reference. Determine the A/A325 ratio at each of the wavelengths for
all-trans-retinol. If the ratio does not exceed 0.602, 0.432 and 0.093 at 300 nm, 350
nm and 370 nm respectively, the standard substance is suitable for use.
Calculation
Calculate the numerical value of the vitamin A content of the test sample by the equation.
c
wA = 20,000 x ---
m
where,
wA is the numerical value of the vitamin A content of the test sample, in IU/kg.
c is the numerical value of the retinol concentration of the extract, in IU/ml.
m is the numerical value of the mass of the test sample, in grams.
Reference: Indian Standards, IS 15120 : 2002.

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