Mobile phase— Mix 480mLof acetonitrile,3520mLof a 0.005Msolution of sodium 1-heptanesulfonate in water,and 4mLof phosphoric acid.

Standard preparation— Accurately weigh about 25mg of USP Antipyrine RS,about 25mg of USP Benzocaine RS,and about 25mg of USP Phenylephrine Hydrochloride RSinto a 250-mLvolumetric flask.Add 5mLof a 0.5mg per mLsolution of p-aminobenzoic acid in Mobile phase.Add 150mLof Mobile phase,and mix to effect solution,sonicating if necessary.Dilute with Mobile phaseto volume,and mix.

Assay preparation A— Transfer an accurately measured volume of Otic Solution,equivalent to about 100mg of antipyrine,to a 50-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.Pipet 5mLof this solution into a 100-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

Assay preparation B— Transfer an accurately measured volume of Otic Solution,equivalent to about 100mg of benzocaine,to a 50-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.Pipet 5mLof this solution into a 100-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

Assay preparation P— Transfer an accurately measured volume of Otic Solution,equivalent to about 5mg of phenylephrine hydrochloride,to a 50-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

Chromatographic system (see Chromatography á621ñ)—The liquid chromatograph is equipped with a 272-nm detector and a 4.6-mm ×30-cm column that contains packing L11.The flow rate is about 1.5mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative retention times are about 0.19for p-aminobenzoic acid,0.26for phenylephrine,0.64for antipyrine,and 1.0for benzocaine;the resolution,R,between phenylephrine and aminobenzoic acid is not less than 1.5,and the relative standard deviation for replicate injections is not more than 3.0%.

Procedure— Separately inject equal volumes (about 20or 25µL)of the Standard preparationand each of the Assay preparationsinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of antipyrine (C11H12N2O)in each mLof the Otic Solution taken by the formula: in which Cis the concentration,in µg per mLof USP Antipyrine RSin the Standard preparation;Vis the volume,in mL,of Otic Solution taken;and rUand rSare the antipyrine peak responses obtained from Assay preparation Aand the Standard preparation,respectively.Calculate the quantity,in mg,of benzocaine (C9H11NO2)in each mLof the Otic Solution taken by the formula: in which Cis the concentration,in µg per mL,of USP Benzocaine RSin the Standard preparation;Vis the volume,in mL,of Otic Solution taken;and rUand rSare the benzocaine peak responses obtained from Assay preparation Band the Standard preparation,respectively.Calculate the quantity,in µg of phenylephrine hydrochloride (C9H13NO2·HCl)in each mLof the Otic Solution taken by the formula: in which Cis the concentration,in µg per mL,of USP Phenylephrine Hydrochloride RSin the Standard preparation;Vis the volume,in mL,of Otic Solution taken;and rUand rSare the phenylephrine peak responses obtained from Assay preparation Pand the Standard preparation,respectively.