Identification—
Transfer a volume of Topical Solution, equivalent to about 10 mg of clotrimazole, to a screw-capped, 50-mL centrifuge tube, and add 5 mL of dilute ammonium hydroxide (1 in 100) and 10 mL of chloroform. Shake vigorously, centrifuge to obtain a clear chloroform phase, and proceed as directed in the
Identification test under
Clotrimazole Cream.
Assay—
Dibasic potassium phosphate solution and Mobile phase—
Prepare as directed in the
Assay under
Clotrimazole.
Internal standard solution—
Transfer about 66 mg of testosterone propionate to a 100-mL volumetric flask, dissolve in 75 mL of methanol, dilute with Dibasic potassium phosphate solution to volume, and mix.
Standard preparation—
Transfer about 50 mg of
USP Clotrimazole RS to a 50-mL volumetric flask, add 5.0 mL of
Internal standard solution, dilute with
Mobile phase to volume, and mix.
Resolution solution—
Prepare a solution of
USP Clotrimazole Related Compound A RS in methanol having a concentration of about 0.2 mg per mL. Transfer 12 mL of this solution to a 25-mL volumetric flask, add 4 mL of
Dibasic potassium phosphate solution and 3 mL of
Standard preparation, dilute with
Mobile phase to volume, and mix.
Assay preparation—
Transfer an accurately measured volume of Topical Solution, equivalent to about 50 mg of clotrimazole, to a 50-mL volumetric flask, add 5.0 mL of Internal standard solution, 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 2.1-mm × 6-cm guard column that contains 10-µm packing L7 and a 3.9-mm × 30-cm analytical column that contains 10-µm packing L1. The flow rate is about 1 mL per minute. Chromatograph the
Resolution solution, and record the peak responses as directed for
Procedure: the relative retention times are about 0.9 for clotrimazole related compound A and 1.0 for clotrimazole; and the resolution,
R, between clotrimazole related compound A and clotrimazole is not less than 1.2. Chromatograph the
Standard preparation, and record the peak responses as directed for
Procedure: the relative retention times are about 1.0 for clotrimazole and 1.5 for testosterone propionate; and the relative standard deviation for replicate injections is not more than 2.0%.
Procedure—
Separately inject equal volumes (about 20 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 mg, of clotrimazole (C
22H
17ClN
2) in each mL of Topical Solution taken by the formula:
50(C / V)(RU / RS),
in which
C is the concentration, in mg per mL, of
USP Clotrimazole RS in the
Standard preparation; V is the volume, in mL, of Topical Solution taken; and
RU and
RS are the peak response ratios of clotrimazole to testosterone propionate obtained from the
Assay preparation and the
Standard preparation, respectively.
and 30