Acetate buffer— Dissolve 1.4g of potassium acetate in 980mLof water,adjust with about 2mLof glacial acetic acid to a pHof 4.3±0.1,dilute with water to 1000mL,and mix.Adjust the buffer molarity (0.018-0.072M)as necessary to obtain suitable chromatographic performance.Increased retention time may be achieved by a decrease in the buffer molarity.

Mobile phase— Prepare a filtered and degassed mixture of methanol and Acetate buffer(65:35).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Diluent— Prepare a mixture of methanol and Acetate buffer(6:4).

Internal standard solution— Dissolve 1-benzylimidazole in methanol to obtain a solution containing about 1.6mg per mL.

Standard preparation— Dissolve an accurately weighed quantity of USP Butoconazole Nitrate RSin methanol,and dilute quantitatively,and stepwise if necessary,with methanol to obtain a stock solution having a known concentration of about 400µg per mL.Transfer 2.0mLof this stock solution and 3.0mLof Internal standard solutionto a 50-mLflask,add 35.0mLof Diluent,and mix.

Assay preparation— Place about 200mLof methanol in a 250-mLvolumetric flask.Transfer to the flask an accurately weighed quantity of Vaginal Cream,equivalent to about 100mg of butoconazole nitrate,and sonicate until the Vaginal Cream is dissolved completely.Cool to room temperature,dilute with methanol to volume,and mix.Transfer 2.0mLof this solution to a 50-mLflask,add 3.0mLof Internal standard solutionand 35.0mLof Diluent,and mix.Allow the precipitated excipients which form to rise to the top of the solution,remove them by aspiration,and discard.Centrifuge or filter the remaining solution.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 225-nm detector and a 4.6-mm ×25-cm column that contains packing L9which has been converted to the potassium form by the use of 0.555Mpotassium acetate solution.The flow rate is about 1mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are about 0.6for butoconazole nitrate and 1.0for 1-benzylimidazole;the resolution,R,between the analyte and internal standard peaks is not less than 4.0;the column efficiency determined from the analyte peak is not less than 1100theoretical plates;the tailing factor for the analyte peak is not more than 2.1;and the relative standard deviation for replicate injections is not more than 1.5%.

Procedure— Separately inject equal volumes (about 20µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of butoconazole nitrate (C19H17Cl3N2S·HNO3)in the portion of Vaginal Cream taken by the formula: in which Cis the concentration,in µg per mL,of USP Butoconazole Nitrate RSin the stock solution used to prepare the Standard preparation;and RUand RSare the peak response ratios of butoconazole nitrate to the internal standard obtained from the Assay preparationand the Standard preparation,respectively.