Mobile phase— Prepare a filtered and degassed mixture of hexane and pentanol (9.97:0.03). Make adjustments if necessary (see System Suitability under Chromatography 621).

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

Standard solution B— Transfer 5.0 mL of Standard solution A to a 50-mL volumetric flask, dilute with Mobile phase to volume, and mix to obtain a solution having a concentration of about 50 µg 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 B with Mobile phase to obtain a solution having a known concentration of about 5 µg per mL. Store this Standard preparation at a temperature not above 0.

Assay preparation— Transfer an accurately measured volume of Solution, equivalent to about 250 µg of cholecalciferol, to a 50-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 2 mL per minute. The relative retention times are about 0.4 for precholecalciferol and 1.0 for 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 which CS is the concentration, in µg per mL, of USP Cholecalciferol RS in the Standard preparation; and rS is the peak response of cholecalciferol.

Precholecalciferol response factor— Transfer 5.0 mL of Standard solution B to a round-bottom flask fitted with a reflux condenser. Displace the air with nitrogen, and reflux for 1 hour 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-mL volumetric flask, dilute with Mobile phase to 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 rS is 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, Fpre for precholecalciferol by the formula: in which rp is the peak response of precholecalciferol.

System suitability— The resolution, R, between the precholecalciferol peak and the choleciferol peak determined for the Precholecalciferol response factor is 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 10 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the cholecalciferol peaks. Calculate the quantity, in µg, of C27H44O in the portion of Solution taken by the formula: in which rc and rpre are the peak responses of cholecalciferol and precholecalciferol, respectively, obtained from the Assay preparation, and the other terms are as previously defined.