Diluent— Prepare a mixture of methanol and water (60:40).

Mobile phase— Prepare a mixture of methanol,water,and 0.25Msodium 1-heptanesulfonate (500:500:20).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Transfer about 140mg of USP Benzocaine RS,accurately weighed,to a 100-mLvolumetric flask with the aid of 25mLof methanol,and swirl.Transfer about 140Jmg of USP Butamben RS,accurately weighed,to the same volumetric flask with the aid of 25mLof water,Jbeing the ratio of the labeled amount,in percent,of butamben to the labeled amount,in percent,of benzocaine in the Topical Solution.Transfer about 140J¢mg of USP Tetracaine Hydrochloride RS,accurately weighed,to the same volumetric flask with the aid of 25mLof water,J¢being the ratio of the labeled amount,in percent,of tetracaine hydrochloride to the labeled amount,in percent,of benzocaine in the Topical Solution.Sonicate for about 1minute,dilute with Diluentto volume,and mix.

Assay preparation— Transfer an accurately measured volume of Topical Solution,equivalent to about 1400mg of benzocaine,to a 100-mLvolumetric flask.Add about 75mLof methanol,and mix.Sonicate for about 1minute,dilute with methanol to volume,and mix.Transfer 10.0mLof this solution to a second 100-mLvolumetric flask,add about 75mLof Diluent,shake,dilute with Diluentto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 313-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 0.3for benzocaine,0.8for butamben,and 1.0for tetracaine;the resolution,R,between the benzocaine peak and the butamben peak and between the butamben peak and the tetracaine peak is not less than 2;and the relative standard deviation for replicate injections is not more than 2.0%for each of the three analyte peaks.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the peak areas for the major peaks.Calculate the individual quantities,in mg,of benzocaine (C9H11NO2),butamben (C11H15NO2),and tetracaine hydrochloride (C15H24N2O2·HCl)in the portion of Topical Solution taken by the formula: in which Cis the concentration,in mg per mL,of the appropriate Reference Standard in the Standard preparation,and rUand rSare the peak areas in the corresponding analyte obtained from the Assay preparationand the Standard preparation,respectively.