Mobile phase— Prepare a filtered and degassed mixture of methanol and water (74:26). Make adjustments if necessary (see System Suitability under Chromatography 621) so that the retention time for calcitriol is not less than 20 minutes.

Standard preparation— Transfer 3.0 mL of USP Calcitriol Solution RS, equilibrated to room temperature, to a container; add 3.0 mL of water; and mix.

Assay preparation— Transfer an accurately measured volume of Injection, equivalent to about 3 µg of calcitriol, to a container; add a sufficient amount of water to dilute to a total volume of 3.0 mL; add 3.0 mL of methanol; and mix.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 264-nm detector, a 4.6-mm × 4.5-cm guard column that contains 5-µm packing L1, and a 4.6-mm × 7.5-cm analytical column that contains 3-µm packing L1. The flow rate is about 1 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 100 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 µg, of calcitriol (C27H44O3) in each mL of the Injection taken by the formula: in which C is the concentration, in µg per mL, of calcitriol in the USP Calcitriol Solution RS; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.