A: Infrared Absorption á197Kñ.

B: Ultraviolet Absorption á197Uñ—

Solution: 8µg per mL.

Medium: alcohol.

Test solution: 20mg per mL,in alcohol.

Mobile phase— Prepare a degassed and filtered solution of isooctane and isopropyl alcohol (100:1).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ.

Standard preparation— Dissolve an accurately weighed quantity of USP Dihydrotachysterol RSin Mobile phase,and dilute quantitatively and stepwise,if necessary,with Mobile phaseto obtain a solution having a known concentration of about 10µg per mL.

Assay preparation— Transfer about 50mg of Dihydrotachysterol,accurately weighed,to a 500-mLvolumetric flask,dissolve in and dilute with Mobile phaseto volume,and mix.Pipet 10mLof the resulting solution into a 100-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

System suitability preparation— Prepare a solution of ergocalciferol in Mobile phasehaving a concentration of about 0.7mg per mL.Reflux under nitrogen for 20minutes,and cool to ambient temperature (Solution A).Pipet 3mLof Solution Aand 2mLof a solution in Mobile phasecontaining 0.1mg of dihydrotachysterol per mLinto a 25-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 254-nm detector and a 4.6-mm ×25-cm column that contains packing L3.The flow rate is about 1mLper minute.Chromatograph replicate injections of the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are about 0.6for pre-ergocalciferol,0.7for dihydrotachysterol,and 1.0for ergocalciferol;and the relative standard deviation is not more than 2.5%.Chromatograph the System suitability preparation,and record the peak responses as directed for Procedure:the relative retention times are about 0.6for pre-ergocalciferol,0.7for dihydrotachysterol,and 1.0for ergocalciferol;and the resolution,R,between the pre-ergocalciferol and dihydrotachysterol peaks is not less than 1.5.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of C28H46Oin the portion of Dihydrotachysterol 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 for dihydrotachysterol obtained from the Assay preparationand the Standard preparation,respectively.