Solvent— Use dimethyl sulfoxide.

Internal standard solution— Dissolve testosterone in ethyl acetate to obtain a solution having a concentration of about 0.10mg per mL.

Mobile phase— Prepare a suitable degassed solution of about 6volumes of heptane,6volumes of water-saturated heptane,3volumes of methylene chloride,and 5volumes of ethyl acetate.

Standard preparation— Dissolve an accurately weighed quantity of USP Calcifediol RSin Internal standard solution,and dilute quantitatively and stepwise with Internal standard solutionto obtain a solution having a known concentration of about 20µg per mL.

Assay preparation— Transfer about 10mg of Calcifediol,accurately weighed,to a 50-mLvolumetric flask,dissolve in Internal standard solution,dilute with Internal standard solutionto volume,and mix.Transfer 5.0mLof this solution to a 50-mLvolumetric flask,dilute with Internal standard solutionto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 4-mm ×30-cm column that contains packing L3,a detector that monitors absorption at 254nm,and a pump capable of providing constant flow up to a minimum of 2000psi.

System suitability— The relative standard deviation for peak response ratios for four replicate injections of Standard preparationis not more than 3.0%,and the resolution factor is not less than 3.0.

Procedure— Introduce equal volumes of the Standard preparationand the Assay preparationinto the chromatograph (see Chromatography á621ñ),and measure the peak responses obtained.Calculate the quantity,in mg,of C27H44O2·H2Oin the portion of Calcifediol taken by the formula: in which Cis the concentration,in µg per mL,of USP Calcifediol RSin the Standard preparation;and RUand RSare the ratios of the peak responses of calcifediol to testosterone obtained from the Assay preparationand the Standard preparation,respectively.