A: Transfer an amount of Topical Aerosol,equivalent to about 5mg of benzocaine,to a beaker,add 20mLof 0.5Nhydrochloric acid,warm gently to disperse the solution,cool,and filter,if necessary,to obtain a clear solution.To 10mLof the clear solution add 5drops of a solution of sodium nitrite (1in 10),and 2drops of methyl red TS,and neutralize with 1Nsodium hydroxide.Add 2mLof a solution of 100mg of 2-naphthol in 5mLof 1Nsodium hydroxide:an orange-red precipitate is formed.

B: The retention time of the major peak for benzocaine in the chromatogram of the Assay preparationcorresponds to that in the chromatogram of the Standard preparation,as obtained in the Assay for benzocaine.

C: The retention time of the major peak for menthol in the chromatogram of the Assay preparationcorresponds to that in the chromatogram of the Standard preparation,as obtained in the Assay for menthol.

Diluent— Prepare a mixture of methanol and water (1:1).

Mobile phase— Prepare a mixture of methanol,water,and glacial acetic acid (56:40:4).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Prepare a solution of USP Benzocaine RSin Diluenthaving a known concentration of about 0.02mg per mL.

Assay preparation— Spray the Topical Aerosol into a flask.Transfer an accurately measured volume of the solution,equivalent to about 200mg of benzocaine,to a 100-mLvolumetric flask,dilute with Diluentto volume,and mix.Transfer 1.0mLof this solution to a second 100-mLvolumetric flask,dilute with Diluentto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 294-nm detector and a 3.9-mm ×30-cm column that contains packing L1.The flow rate is about 1.5mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 50µL)of the Assay preparationand the Standard preparationinto the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantity,in mg,of benzocaine (C9H11NO2)in the portion of Topical Aerosol taken by the formula: in which Cis the concentration,in mg per mL,of USP Benzocaine RSin the Standard preparation,and rUand rSare the benzocaine peak areas obtained from the Assay preparationand the Standard preparation,respectively.

Internal standard solution— Dissolve decanol in n-hexane to obtain a solution having a concentration of about 1mg per mL.

Standard preparation— Dissolve an accurately weighed quantity of USP Menthol RSin n-hexane to obtain a solution having a known concentration of about 1mg per mL.Transfer 5.0mLof this solution and 5.0mLof Internal standard solutionto a 100-mLvolumetric flask,dilute with ether to volume,and mix.

Assay preparation— Spray the Topical Aerosol into a flask.Transfer an accurately measured volume of the solution,equivalent to about 50mg of menthol,to a separator,and extract with three 15-mLportions of ether,collecting the ether extracts in a 50-mLvolumetric flask.Dilute with ether to volume,and mix.Transfer 5.0mLof this solution to a 100-mLvolumetric flask,add 5.0mLof n-hexane and 5.0mLof Internal standard solution,dilute with ether to volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The gas chromatograph is equipped with a flame-ionization detector and contains a 2-mm ×1.8-m column packed with 10%phase G16on support S1AB.The column temperature is maintained isothermally at about 170,the injection port temperature is maintained at about 260,and the detector block temperature is maintained at about 240.Dry helium is used as the carrier gas at a flow rate of about 50mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are about 0.7for menthol and 1.0for decanol;the resolution,R,between the menthol peak and the decanol peak is not less than 2.5;and the relative standard deviation of the ratio of the peak response obtained with menthol to that obtained with decanol is not more than 2%.

Procedure— Separately inject equal volumes (about 2µL)of the Assay preparationand the Standard preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of menthol (C10H20O)in each mLof Topical Aerosol taken by the formula: in which Cis the concentration,in mg per mL,of USP Menthol RSin the Standard preparation;Vis the volume,in mL,of Topical Aerosol taken;and RUand RSare the peak response ratios of menthol to decanol obtained from the Assay preparationand the Standard preparation,respectively.