Mobile phase— Prepare a 0.005Mtetrabutylammonium phosphate solution,and adjust with phosphoric acid to a pHof 2.0.Prepare a filtered and degassed mixture of this solution and methanol (750:250).Make adjustments if necessary (seeSystem Suitability underChromatography á621ñ).

Standard preparation— Dissolve an accurately weighed quantity of USP Ciprofloxacin Hydrochloride RSin 0.1Nhydrochloric acid to obtain a solution having a known concentration of about 0.033mg per mL.

Resolution solution— Dissolve an accurately weighed quantity of USP Ciprofloxacin Ethylenediamine Analog RSin a portion of theStandard preparationto obtain a solution containing about 0.005mg per mL.

Assay preparation— Transfer an accurately weighed quantity of Ophthalmic Ointment,equivalent to about 750µg of ciprofloxacin,to a screw-capped tube.Add 15mLof solvent hexane,and shake vigorously until the Ophthalmic Ointment is dispersed.Loosen the cap,and heat in a water bath at 60for 30minutes,with occasional swirling.Remove from the bath,tighten the cap,and shake for 1.5minutes while still hot.Add 25.0mLof 0.1Nhydrochloric acid,and shake vigorously for 1.5minutes.Allow the layers to separate,and use the lower,aqueous layer.

Chromatographic system(seeChromatography á621ñ)— The liquid chromatograph is equipped with a 280-nm detector and a 4.6-mm ×25-cm column that contains packing L1.The flow rate is about 1.5mLper minute.Chromatograph theResolution solution,and record the responses as directed forProcedure:the relative retention times are about 0.8for the ciprofloxacin ethylenediamine analog and 1.0for ciprofloxacin;and the resolution,R,between ciprofloxacin ethylenediamine analog and ciprofloxacin is not less than 2.0.Chromatograph theStandard preparation,and record the responses as directed forProcedure:the column efficiency is not less than 500theoretical plates;the tailing factor is not less than 0.9and not more than 2.0;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20µL)of theStandard preparationand theAssay preparationinto the chromatograph,record the chromatograms,and measure the areas for the ciprofloxacin peaks.Calculate the quantity,in mg,of ciprofloxacin (C17H18FN3O3)in each g of the Ophthalmic Ointment taken by the formula: in which 331.34and 385.82are the molecular weights of ciprofloxacin and ciprofloxacin hydrochloride monohydrate,respectively;Cis the concentration,in mg per mL,of USP Ciprofloxacin Hydrochloride RSin theStandard preparation;Wis the weight,in g,of Ophthalmic Ointment taken;and rUand rSare the peak responses obtained from theAssay preparationand theStandard preparation,respectively.