A: The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that of the Standard preparation,both relative to the internal standard,as obtained in the Assay.

B: Place a quantity of finely powdered Lozenges,equivalent to about 50mg of clotrimazole,into a screw-capped 50-mLtest tube,add 20.0mLof dichloromethane,and mix.Shake by mechanical means for 10minutes,and allow the suspension to settle.Use the supernatant as the test solution.Separately apply 20µLof this solution and 20µLof a Standard solution of USP Clotrimazole RSin dichloromethane containing 2.5mg per mLto a thin-layer chromatographic plate (see Chromatography á621ñ)coated with a 0.25-mm layer of chromatographic silica gel mixture.Position the plate in a chromatographic chamber,and develop the chromatograms in a solvent system consisting of a mixture of diethyl ether and ammonium hydroxide (8:1)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 allow the solvent to evaporate.Examine the plate under short-wavelength UVlight:the RFvalue of the principal spot obtained from the test solution corresponds to that obtained from the Standard solution.Dissolve 3g of bismuth subnitrate and 30g of potassium iodide in 10mLof dilute hydrochloric acid (1in 4),dilute with water to 100mL,mix,and prepare a spray reagent by diluting 10mLof this solution and 10mLof dilute hydrochloric acid (1in 4)with water to 100mL.Spray the plate with the spray reagent,and visually locate the clotrimazole spots on the plate:the spots are orange.

Buffer solution— Dissolve 1g of ammonium carbonate in 1000mLof water.Adjust with 10%sulfuric acid solution to a pHof 6.0.

Mobile phase— Prepare a filtered and degassed mixture of methanol and Buffer solution(75:25).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Internal standard stock solution— Dissolve an accurately weighed quantity of triphenylmethane in methanol,and dilute quantitatively and stepwise with methanol to obtain a solution having a concentration of 5mg per mL.

Internal standard solution— Pipet 10.0mLof Internal standard stock solutioninto a 250-mLvolumetric flask,and dilute with Mobile phaseto volume.

Standard preparation— Transfer about 20mg of USP Clotrimazole RS,accurately weighed,to a 100-mLvolumetric flask.Add 4.0mLof Internal standard stock solution,dissolve in and dilute with Mobile phaseto volume,and mix.

Assay preparation— Weigh and pulverize not fewer than 10Lozenges.Transfer an accurately weighed portion of the powder,equivalent to about 5mg of clotrimazole,to a 50-mLscrew-capped centrifuge tube.Pipet 25mLof Internal standard solutioninto the tube.Sonicate for 10minutes,and then shake for 10minutes.Centrifuge at 2500rpm for 30minutes.Use the clear supernatant layer as the Assay preparation.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 215-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 2mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are about 1.0for clotrimazole and 1.4for triphenylmethane;the resolution,R,between clotrimazole and triphenylmethane is not less than 1.5;the column efficiency is not less than 1500theoretical plates;the tailing factor is not more than 2,and the relative standard deviation for replicate injections is not more than 2.0%.

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 C22H17ClN2in the portion of Lozenges taken by the formula: in which Cis the concentration,in mg per mL,of USP Clotrimazole RSin the Standard preparation;and RUand RSare the ratios of the peak responses for clotrimazole and triphenylmethane obtained from the Assay preparationand the Standard preparation,respectively.