A: Infrared Absorption 197K.

B: Ultraviolet Absorption 197U—

Solution: 8 µg per mL.

Medium: alcohol.

Test solution: 20 mg 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 Suitability under Chromatography 621.

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

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

System suitability preparation— Prepare a solution of ergocalciferol in Mobile phase having a concentration of about 0.7 mg per mL. Reflux under nitrogen for 20 minutes, and cool to ambient temperature (Solution A). Pipet 3 mL of Solution A and 2 mL of a solution in Mobile phase containing 0.1 mg of dihydrotachysterol per mL into a 25-mL volumetric flask, dilute with Mobile phase to volume, and mix.

Chromatographic system (see Chromatography 621)—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 1 mL per minute. Chromatograph replicate injections of the Standard preparation, and record the peak responses as directed for Procedure: 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 0.6 for pre-ergocalciferol, 0.7 for dihydrotachysterol, and 1.0 for ergocalciferol; and the resolution, R, between the pre-ergocalciferol and dihydrotachysterol peaks is not less than 1.5.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C28H46O in the portion of Dihydrotachysterol 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 for dihydrotachysterol obtained from the Assay preparation and the Standard preparation, respectively.