A: Transfer a portion of powdered Tablets, equivalent to about 2 mg of dihydrotachysterol, to a glass-stoppered flask, add about 25 mL of methylene chloride, shake for 15 minutes, and filter. Evaporate the filtrate to dryness, and dissolve the residue in 0.4 mL of methylene chloride. Apply 10 µL of this solution and 10 µL of a Standard solution of USP Dihydrotachysterol RS in methylene chloride containing 5 mg per mL at separate points on a thin-layer chromatographic plate coated with a 0.25-mm layer of chromatographic silica gel. Develop the chromatogram using a solvent system consisting of a mixture of ether and cyclohexane (1:1) until the solvent front has moved about three-fourths of the length of the plate. Air-dry, spray lightly with a 1 in 5 solution of p-toluenesulfonic acid in a mixture of alcohol and propylene glycol (9:1), and heat at 80 until reddish brown spots appear (about 10 minutes): the RF value of the principal spot obtained from the solution under test corresponds to that obtained from the Standard solution.

B: The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, obtained as directed in the Assay.

Mobile phase , Standard preparation, System suitability preparation, and Chromatographic system—Prepare as directed in the Assay under Dihydrotachysterol.

Assay preparation— Weigh and finely powder not less than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to about 2.5 mg of dihydrotachysterol, to a suitable container, add 100.0 mL of dehydrated alcohol, shake by mechanical means for 10 minutes, sonicate, and centrifuge. Transfer 20.0 mL of the supernatant to a 50-mL volumetric flask, and evaporate with the aid of a stream of nitrogen to dryness, observing precautions to avoid exposure to moisture and light. Dissolve the residue in Mobile phase, dilute with Mobile phase to volume, and mix.

Procedure— Proceed as directed for Procedure in the Assay under Dihydrotachysterol. Calculate the quantity, in µg, of C28H46O in the portion of Tablets taken by the formula: in which C is the concentration, in µg per mL, of USP Dihydrotachysterol RS in the Standard preparation, and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.