Mobile phase— Use chloroform containing alcohol as preservative.

Standard solution A— Dissolve an accurately weighed quantity of USP Ergocalciferol RSin chloroform,and dilute quantitatively with chloroform to obtain a solution having a known concentration of about 500µg per mL.[NOTE—Prepare this Standard solution Afresh daily.]

Standard solution B— Transfer 5.0mLof Standard solution Ato a 50-mLvolumetric flask,dilute with chloroform to volume,and mix to obtain a solution having a known concentration of 50µg per mL.[NOTE—Reserve a portion of this solution for the test for Pre-ergocalciferol response factor.]

Standard preparation— By quantitative dilution,with chloroform,of 5mLof Standard solution B,prepare a solution having a known concentration of about 5µg per mL.[NOTE—Store this Standard preparation at a temperature not above 0.]

Assay preparation— Transfer an accurately measured volume of Oral Solution,equivalent to about 250µg of ergocalciferol,to a 50-mLvolumetric flask,dilute with chloroform to 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 5-µm packing L3.The flow rate is about 1mLper minute.The relative retention times of pre-ergocalciferol and ergocalciferol are about 0.8and 1.0,respectively.

Calibration— Inject a suitable volume (10µLto 20µL)of the Standard preparationinto the chromatograph,record the chromatogram,and measure the response for the major peak.Calculate the Ergocalciferol response factor,FD,by the formula: in which CSis the concentration,in µg per mL,of USP Ergocalciferol RSin the Standard preparation,and RSis the peak response of ergocalciferol.

Pre-ergocalciferol response factor— Transfer 5.0mLof Standard solution Binto a round-bottomed 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 ergocalciferol and pre-ergocalciferol.Cool,transfer,with the aid of several portions of chloroform,to a 50-mLvolumetric flask,dilute with chloroform to volume,and mix to obtain the Working mixture.Inject a suitable volume (about 10µLto 20µL)of the Working mixtureinto the chromatograph,record the chromatogram,and measure the peak responses for ergocalciferol and pre-ergocalciferol.Calculate the concentration,C1S,in µg per mL,of ergocalciferol in the (heated)Working mixturetaken by the formula: in which REis the peak response for ergocalciferol.Calculate the concentration,C1Pre,in µg per mL,of pre-ergocalciferol in the (heated)Working mixturetaken by the formula: Calculate the response factor,FPre,for pre-ergocalciferol taken by the formula: in which RPis the peak response of pre-ergocalciferol.

System suitability— The resolution,R,between the pre-ergocalciferol peak and the ergocalciferol peak determined during the test for Pre-ergocalciferol response factoris not less than 1.0,and the relative standard deviation for the peak response of replicate injections of the Standard preparationis not more than 2.0%.

Procedure— Separately inject equal volumes (10µLto 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the ergocalciferol peaks.Calculate the quantity,in µg,of C28H44Oin the Assay preparationtaken by the formula: in which SEand SPreare the peak responses of ergocalciferol and pre-ergocalciferol,respectively.