A: Transfer 5mLto a separator containing 25mLof water,and extract the solution with two 25-mLportions of a mixture of equal volumes of ether and solvent hexane.Combine the extracts,and retain the water solution for Identificationtest B.Extract the ether-hexane solution with 50mLof water,and discard the water layer.Evaporate the ether-hexane solution to dryness,dry the residue in vacuum at 40to 50for 1hour,and dissolve the residue in 1mLof chloroform:the IRabsorption spectrum of this solution exhibits maxima at the same wavelengths as that of a similar solution of USP Benzocaine RS,concomitantly measured.

B: Add 5mLof 1Nsodium hydroxide solution to the water solution retained from Identificationtest A,and extract with two 25-mLportions of chloroform.Evaporate the combined extracts to dryness,dry the residue in vacuum at 40to 50for 1hour,and dissolve the residue in 3mLof chloroform:the IRabsorption spectrum of this solution exhibits maxima at the same wavelengths as that of a similar solution of USP Antipyrine RS,concomitantly measured.

C: The retention times of the major peaks in the chromatogram of the Assay preparationcorrespond to those in the chromatogram of the Standard preparation,as obtained in the Assay.

Ammonium acetate solution— Dissolve 7.7g of ammonium acetate in water,dilute with water to 1000mL,and mix.

Mobile phase— Prepare a filtered and degassed mixture of Ammonium acetate solutionand acetonitrile (3:1).Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Transfer about 15mg of USP Benzocaine RS,accurately weighed,to a 100-mLvolumetric flask,add 15Jmg of USP Antipyrine RS,accurately weighed,Jbeing the ratio of the labeled amount,in mg,of antipyrine to the labeled amount,in mg,of benzocaine per mLof Otic Solution.Add 50mLof methanol,dissolve in methanol,dilute with methanol to volume,and mix.Transfer 10.0mLof this solution to a 100-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

Assay preparation— Transfer an accurately measured volume of Otic Solution,equivalent to about 15mg of benzocaine,to a 100-mLvolumetric flask.Dissolve in 50mLof methanol,dilute with methanol to volume,and mix.Transfer 10.0mLof this solution to a 100-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 280-nm detector and a 4-mm ×15-cm column that contains 5-µm packing L15.The flow rate is about 1.0mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the tailing factor for the benzocaine peak is not more than 2.5,the column efficiency for the benzocaine peaks is not less than 1500theoretical plates,and the relative standard deviation for replicate injections is not more than 2%.

Procedure— Separately inject equal volumes (about 10µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.The relative retention times are about 0.35for antipyrine,and 1.0for benzocaine.Calculate the quantity,in mg,of benzocaine (C9H11NO2)in each mLof the Otic Solution taken by the formula: in which Cis the concentration,in mg per mL,of USP Benzocaine RSin the Standard preparation;Vis the volume,in mL,of Otic Solution taken;and rUand rSare the peak responses due to benzocaine in the Assay preparationand the Standard preparation,respectively.Calculate the quantity,in mg,of antipyrine (C11H12N2O)in each mLof the Otic Solution taken by the same formula,changing the terms to refer to antipyrine instead of benzocaine.