Mobile phase— Prepare a filtered and degassed mixture of hexane and pentanol (9.97:0.03).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard solution A— Dissolve an accurately weighed quantity of USP Cholecalciferol RSin toluene,and dilute quantitatively with Mobile phaseto obtain a solution having a known concentration of about 500µg per mL.Prepare this solution fresh daily.

Standard solution B— Transfer 5.0mLof Standard solution Ato a 50-mLvolumetric flask,dilute with Mobile phaseto volume,and mix to obtain a solution having a concentration of about 50µg per mL.Reserve a portion of this solution for the determination of the Precholecalciferol response factor.

Standard preparation— Quantitatively dilute an accurately measured volume of Standard solution Bwith Mobile phaseto obtain a solution having a known concentration of about 5µg per mL.Store this Standard preparationat a temperature not above 0.

Assay preparation— Transfer an accurately measured volume of Solution,equivalent to about 250µg of cholecalciferol,to a 50-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 2mLper minute.The relative retention times are about 0.4for precholecalciferol and 1.0for cholecalciferol.

Calibration— Chromatograph the Standard preparation,and record the peak responses as directed under Procedure.Calculate the cholecalciferol response factor,FC,by the formula: in whichCSis the concentration,in µg per mL,of USP Cholecalciferol RSin the Standard preparation,and rSis the peak response of cholecalciferol.

Precholecalciferol response factor— Transfer 5.0mLof Standard solution Bto a round-bottom flask fitted with a reflux condenser.Displace the air with nitrogen,and reflux for 1hour in a water bath under a nitrogen atmosphere to obtain a solution containing cholecalciferol and precholecalciferol.Cool,transfer the solution,with the aid of several portions of Mobile phase,to a 50-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.Chromatograph this solution,and record the peak responses as directed under Procedure.Calculate the concentration,C¢S,in µg per mL,of cholecalciferol by the formula: in which rSis the peak response for cholecalciferol.Calculate the concentration,C¢pre,in µg per mL,of precholecalciferol by the formula: in which the terms are defined as above.Calculate the response factor,Fprefor precholecalciferol by the formula: in which rpis the peak response of precholecalciferol.

System suitability— The resolution,R,between the precholecalciferol peak and the choleciferol peak determined for the Precholecalciferol response factoris not less than 1.0,and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the cholecalciferol peaks.Calculate the quantity,in µg,of C27H44Oin the portion of Solution taken by the formula: in which rcand rpreare the peak responses of cholecalciferol and precholecalciferol,respectively,obtained from the Assay preparation,and the other terms are as previously defined.