Assay
Internal standard solution
Dissolve testosterone in ethyl acetate to obtain a solution having a concentration of about 0.10 mg per mL.
Mobile phase
Prepare a suitable degassed solution of about 6 volumes of heptane, 6 volumes of water-saturated heptane, 3 volumes of methylene chloride, and 5 volumes of ethyl acetate.
Standard preparation
Dissolve an accurately weighed quantity of
USP Calcifediol RS in
Internal standard solution, and dilute quantitatively and stepwise with
Internal standard solution to obtain a solution having a known concentration of about 20 µg per mL.
Assay preparation
Transfer about 10 mg of Calcifediol, accurately weighed, to a 50-mL volumetric flask, dissolve in Internal standard solution, dilute with Internal standard solution to volume, and mix. Transfer 5.0 mL of this solution to a 50-mL volumetric flask, dilute with Internal standard solution to volume, and mix.
Chromatographic system
(see
Chromatography 621)The liquid chromatograph is equipped with a 4-mm × 30-cm column that contains packing L3, a detector that monitors absorption at 254 nm, and a pump capable of providing constant flow up to a minimum of 2000 psi.
System suitability
The relative standard deviation for peak response ratios for four replicate injections of Standard preparation is not more than 3.0%, and the resolution factor is not less than 3.0.
Procedure
Introduce equal volumes of the
Standard preparation and the
Assay preparation into the chromatograph (see
Chromatography 621), and measure the peak responses obtained. Calculate the quantity, in mg, of C
27H
44O
2·H
2O in the portion of Calcifediol taken by the formula:
0.5C(RU / RS),
in which
C is the concentration, in µg per mL, of
USP Calcifediol RS in the
Standard preparation; and
RU and
RS are the ratios of the peak responses of calcifediol to testosterone obtained from the
Assay preparation and the
Standard preparation, respectively.