A: Transfer a portion of powdered Tablets,equivalent to about 2mg of dihydrotachysterol,to a glass-stoppered flask,add about 25mLof methylene chloride,shake for 15minutes,and filter.Evaporate the filtrate to dryness,and dissolve the residue in 0.4mLof methylene chloride.Apply 10µLof this solution and 10µLof a Standard solution of USP Dihydrotachysterol RSin methylene chloride containing 5mg per mLat 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 1in 5solution of p-toluenesulfonic acid in a mixture of alcohol and propylene glycol (9:1),and heat at 80until reddish brown spots appear (about 10minutes):the RFvalue 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 preparationcorresponds 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 Assayunder Dihydrotachysterol.

Assay preparation— Weigh and finely powder not less than 20Tablets.Transfer an accurately weighed portion of the powder,equivalent to about 2.5mg of dihydrotachysterol,to a suitable container,add 100.0mLof dehydrated alcohol,shake by mechanical means for 10minutes,sonicate,and centrifuge.Transfer 20.0mLof the supernatant to a 50-mLvolumetric 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 phaseto volume,and mix.

Procedure— Proceed as directed for Procedurein the Assayunder Dihydrotachysterol.Calculate the quantity,in µg,of C28H46Oin the portion of Tablets taken by the formula: in which Cis the concentration,in µg per mL,of USP Dihydrotachysterol RSin the Standard preparation,and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.