A: Dissolve a quantity of powdered Lozenges,equivalent to about 20mg of benzocaine,in 10mLof water with the aid of a few drops of 3Nhydrochloric acid,and filter,if necessary,to obtain a clear solution.Add 5drops of a solution of sodium nitrite (1in 10),followed by 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 chromatograms of the Assay preparationscorresponds to that in the chromatograms of the respective Standard preparations,as obtained in the Assay.

Mobile phase— Prepare a filtered and degassed mixture of water,acetonitrile,and 1.0Mmonobasic potassium phosphate solution previously adjusted with phosphoric acid to a pHof 3.0(700:250:50).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation 1— Prepare a solution of USP Benzocaine RSin 0.1Nhydrochloric acid having a known concentration of about 0.01mg per mL.

Standard preparation 2— Prepare a solution of USP Benzocaine RSin a mixture of acetonitrile and water (1:1)having a known concentration of about 0.01mg per mL.

Assay preparations— Weigh and finely powder not fewer than 20Lozenges.Transfer accurately weighed portions of the powder,each equivalent to about 40mg of benzocaine,to two separate 200-mLvolumetric flasks.To one flask add about 150mLof 0.1Nhydrochloric acid,and stir for not less than 2hours.Dilute with 0.1Nhydrochloric acid to volume,and mix.Transfer 5.0mLof this solution to a 100-mLvolumetric flask,dilute with 0.1Nhydrochloric acid to volume,and mix (Assay preparation 1).To the second flask add about 150mLof a mixture of acetonitrile and water (1:1),and stir for not less than 30minutes.Dilute with the mixture of acetonitrile and water (1:1)to volume,and mix.Transfer 5.0mLof this solution to a 100-mLvolumetric flask,dilute with the mixture of acetonitrile and water (1:1)to volume,and mix (Assay preparation 2).

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm ×25-cm column that contains packing L7.The flow rate is about 1.5mLper minute.Chromatograph the Standard preparations,and record the peak responses as directed for Procedure:the tailing factor is not more than 1.5,and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20µL)of Assay preparation 1,Assay preparation 2,Standard preparation 1,and Standard preparation 2into the chromatograph,record the chromatograms,and measure the area responses for the major peaks.Calculate the quantity,in mg,of total C9H11NO2in the portion of Lozenges taken to prepare Assay preparation 1by the formula: in which Cis the concentration,in mg per mL,of USP Benzocaine RSin Standard preparation 1,and rUand rSare the benzocaine peak areas obtained from Assay preparation 1and Standard preparation 1,respectively.Calculate the quantity,in mg,of free C9H11NO2in the portion of Lozenges taken to prepare Assay preparation 2by the formula: in which Cis the concentration,in mg per mL,of USP Benzocaine RSin Standard preparation 2,and rUand rSare the benzocaine peak responses obtained from Assay preparation 2and Standard preparation 2,respectively.