A: Infrared Absorption 197M.

B: Visualizing solution—Dissolve 0.85 g of bismuth subnitrate in 10 mL of glacial acetic acid, dilute with water to 50 mL, and mix. Mix 10 mL of this solution, 50 mL of potassium iodide solution (2 in 25), and 20 mL of glacial acetic acid, dilute with water to 100 mL, and mix.

Procedure— Prepare a test solution by dissolving 50 mg of Tioconazole in 1 mL of methanol. Separately apply 10 µL of the test solution and 10 µL of a Standard solution of USP Tioconazole RS, similarly prepared, to a thin-layer chromatographic plate (see Chromatography 621), coated with a 0.25-mm layer of chromatographic silica gel mixture. Allow the spots to dry, and develop the chromatogram using a solvent system consisting of a mixture of chloroform, methanol, and glacial acetic acid (40:5:1) until the solvent front has moved about three-fourths of the length of the plate. Remove the plate from the developing chamber, mark the solvent front, and locate the spots on the plate by viewing under short- and long-wavelength UV light after drying the plate at 80 for 5 minutes. Spray the plate with Visualizing solution, air-dry for 2 minutes, and overspray with sodium nitrite solution (1 in 20). Air-dry the plate for 5 minutes, and examine it for brown spots on a pale yellow background: the RF value of the principal spot from the test solution corresponds to that obtained from the Standard solution.

C: The chromatogram of the Assay preparation obtained as directed in the Assay exhibits a major peak for tioconazole, the retention time of which corresponds to that exhibited in the chromatogram of the Standard preparation obtained as directed in the Assay.

Mobile phase and Chromatographic system—Prepare as directed in the Assay.

Standard preparation— Transfer about 1 mg each, accurately weighed, of USP Tioconazole Related Compound A RS, USP Tioconazole Related Compound B RS, and USP Tioconazole Related Compound C RS to a 25-mL flask. Add 15.0 mL of methanol, and shake until the contents are completely dissolved.

Test preparation— Transfer about 100 mg of Tioconazole, accurately weighed, to a 25-mL flask, add 15.0 mL of methanol, and shake until the substance is completely dissolved.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Test preparation into the chromatograph, record the chromatograms, and measure the responses for the peaks. Calculate, in turn, the percentages of 1-[2,4-dichloro--[(3-thenyl)-oxy]phenethyl]imidazole hydrochloride (tioconazole related compound A), 1-[2,4-dichloro--[(2,5-dichloro-3-thenyl)-oxy]phenethyl]imidazole hydrochloride (tioconazole related compound B), and 1-[2,4-dichloro--[(5-bromo-2-chloro-3-thenyl)-oxy]phenethyl]imidazole hydrochloride (tioconazole related compound C) in the portion of Tioconazole taken by the same formula: in which WI is the weight, in mg, of the respective USP Reference Standard taken to prepare the Standard preparation, WU is the weight, in mg, of Tioconazole taken to prepare the Test preparation, and rU and rS are the peak responses at corresponding retention times, obtained from the Test preparation and the Standard preparation, respectively. The limit of each related compound is 1.0%.

Mobile phase— [Note—Prepare the Mobile phase fresh daily.]

Standard preparation— Dissolve an accurately weighed quantity of USP Tioconazole RS in methanol, and dilute quantitatively and stepwise, if necessary, with methanol to obtain a solution having a known concentration of about 200 µg per mL.

Assay preparation— Transfer about 100 mg of Tioconazole, accurately weighed, to a 100-mL volumetric flask, dissolve in methanol, dilute with methanol to volume, and mix. Transfer 10.0 mL of the resulting solution to a 50-mL volumetric flask, dilute with methanol to volume, and mix.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 219-nm detector, a 4-mm × 10-cm precolumn that contains packing L4, installed between the pump and the injector, and a 5-mm × 25-cm analytical column that contains packing L1. [note—Replace the precolumn daily.] The flow rate is adjusted to obtain a retention time of between 12 and 17 minutes for tioconazole. Chromatograph the Standard preparation, and record the peak responses as directed under Procedure. The column efficiency determined from the analyte peak is not less than 1000 theoretical plates, the tailing factor for the analyte peak is not more than 2.0, 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 C16H13Cl3N2OS in the Tioconazole taken by the formula: in which C is the concentration, in µg per mL, of USP Tioconazole RS, calculated on the anhydrous basis, in the Standard preparation, and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.