Determination of Assay of Vitamin D : Pharmaceutical Guidelines

Determination of Assay of Vitamin D

Learn how to determine the Vitamin D by Chromatography.

The assay should be completed promptly and care should be taken throughout the procedure to keep to a minimum the exposure to air and to actinic light by the use of inert gas and low actinic glassware. All the operations should be carried out in subdued light.

Special Reagents

Adsorbent: A chromatographic grade of kaolin such as Florex XXS or of fuller's earth having a water content corresponding to not less than 8.5 per cent and not more than 9.0 per cent of loss on drying at 105° for 6 hours.
NOTE - Adjust the water content, if necessary, by drying in vacuum oven at room temperature, restoring the water required and equilibrating by shaking for 2 hours.

Standard preparation of vitamin D: 

Dissolve 0.01 g of ergocalciferol RS or cholecalciferol RS (for assaying substances labelled to contain Vitamin D as ergocalciferol or as cholecalciferol respectively) in sufficient purified 1,2-dichloroethane to produce 100.0 m!. Dilute 10.0 ml of this solution to 100.0 ml with purified 1,2-dichloroethane to give a solution containing 10 J..l.g(400 Units) of vitamin D per m!.


Chromatographic Tubes

Column No.1. A chromatographic tube (25 em x 2.5 mm) fitted at the lower end with a sintered glass disc or a small plug of glass wool and a tap.
Column No.2. A chromatographic tube (20 em x 5 mm) fitted at the lower end with a sintered glass disc and a tap.

Chromatographic columns

Column No.1. Shake 200 ml of 2, 2,4-trimethylpentane with sufficient polyethylene glycol 600 so that, on separation, two layers are obtained. To 100 ml of the upper layer add 25 g of chromatographic siliceous earth, shake vigorously to form a thin slurry, add, in small portions and with vigorous stirring, 10 ml of polyethylene glycol 600 and continue to stir for a further 2 minutes to produce a uniform suspension. Transfer the suspension in small portions to the chromatographic tube, apply gentle suction and pack each portion carefully with the aid of a glass plunger. Add sufficient of the suspension to produce a column IS em in length and discard any eluate.

Standardisation of column - 

Determine the volume in which ergocalciferol is recovered from the column by the following method.
Transfer 2 ml of a 0.03 per cent w/v solution of ergocalciferol in 2,2,4-trimethylpentane to the top of the column and rinse it into the column with not more than 5 ml of trimethylpentane. Elute the column with 2,2,4-trimethylpentane, adjusting the rate of flow from the bottom of the column to 2 to 3 ml per minute and collect successive 5 ml fractions of the eluate.
Measure the absorbance of each fraction at the maximum at about 263 nm and there from determine the position, relative to volume of eluate, at which the elution of ergocalciferol begins and finishes.
Column No.2. Mix 5 g of adsorbent with sufficient 2,2, 4-trimethylpentane to form a slurry and transfer the slurry to the chromatograph tube. Pack the tube carefully with the aid of glass plunger and discard the eluate.


Weigh accurately a quantity of the substance under examination equivalent to about 400 Units of vitamin D. For capsules, the mixed contents of 20 capsules may be used as the sample. Add 10 ml of a freshly prepared 0.01 per cent w/v solution of butylated hydroxy toluene in ethanol (95 per cent), 15 ml of a 50 per cent w/v solution of potassium hydroxide and 5 ml of ethanol (95 per cent). Reflux on a water-bath for 30 minutes, cool, transfer the solution to a separator with the aid of 50 ml of water, add 75 ml ether and shake vigorously.
Allow to separate, transfer the aqueous layer to a second separator and extract with three successive quantities, each of 30 ml, of ether, adding each ethereal extract to the liquid in the first separator and finally discarding the aqueous solution. Pour two successive quantities, each of 100 ml, of water through the ethereal solution without shaking and discard the aqueous layers. Add successive quantities, each of 10 ml, of water to the ethereal solution, agitate gently each time and discard the aqueous extracts. Continue the process until the aqueous extracts are neutral to phenolphthalein solution.
Dry the ethereal solution by stilTing with anhydrous sodium sulphate, decant the ethereal solution, wash the residue with successive small portions of ether and evaporate the combined solution and washings on a water-bath to a volume of about 5 ml. Cool and evaporate to dryness in a cUlTent of nitrogen. Dissolve the residue in 5.0 ml of trimethylpentane to obtain the sample preparation.
Transfer the sample preparation to the top of chromatographic column No.1 with the aid of 5 ml of 2,2,4-trimethylpentane, elute the column with 2,2,4-trimethylpentane, adjusting the rate of flow of eluate from the bottom of the column to 2 to 3 ml per minute, and collect the fraction of eluate estimated to contain the calciferol, as indicated by the standardisation of the column.
Transfer the eluate collected from chromatographic column No.1 to the top of chromatographic column No.2, allow the liquid to flow, add to the top of the column 10 ml of trimethylpentane and discard the eluate. Elute the column with 50 ml of benzene, evaporate the eluate on a water-bath to a volume of about 5 ml, cool, evaporate to dryness in a current of nitrogen and dissolve the residue in 4.0 ml of purified 1,2-dichloroethane to obtain the sample solution (solution A).
Add 1.0 ml of solution A to each of three tubes. To the fIrst tube add 1.0 ml of a mixture of equal volumes of purified 1,2-dichloroethane and acetic anhydride and 10.0 ml of antimony trichloride solution in purified 1,2-dichloroethane. To the second tube add 1.0 ml of purified 1,2-dichloroethane and 10.0 ml of antimony trichloride solution in purified 1,2-dichloroethane. To the third tube add 1.0 ml of the standard preparation of vitamin D and 10.0 ml of antimony trichloride solution in purified 1,2 -dichloroethane.
Measure the absorbance of each solution exactly 1 minute after the addition of the antimony trichloride solution at the maximum at about 500 nm using purified 1,2 -dichloroethane as the blank (2.4.7). The amount of vitamin D, in mg, in the weight of the sample taken is given by the expression:
A1 = the absorbance due to the solution in the fIrst tube;
A2 = the absorbance due to the solution in the second tube;
A3 = the absorbance due to the solution in the third tube.
1mg of ergocalciferol or cholecalciferol is equivalent to 40,000 Units of vitamin D.

Ankur Choudhary is India's first professional pharmaceutical blogger, author and founder of Pharmaceutical Guidelines, a widely-read pharmaceutical blog since 2008. Sign-up for the free email updates for your daily dose of pharmaceutical tips.
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