A: Transfer a quantity of Ointment,equivalent to about 5mg of erythromycin,to a separator containing 50mLof solvent hexane.Shake until dissolved.Extract with three separate 20-mLportions of methanol.Combine the methanol extracts in a beaker,and evaporate to dryness.Dissolve the residue in 2mLof methanol (test solution).Proceed as directed in theIdentification test underErythromycin Delayed-Release Capsules,beginning with “Prepare a Standard solution of USP Erythromycin RS.”

B: The retention times of the peaks for erythromycin A,erythromycin B,and erythromycin Cin the chromatogram of theAssay preparation correspond to those in the chromatograms of theStandard preparation andErythromycins Band Cstandard preparation,as obtained in theAssay.

Solution A— Prepare a degassed mixture of acetonitrile and water (90:10).Store in a reservoir protected from air by sparging with helium.

Solution B— To 1000mLof degassed water add 0.5mLof sodium hydroxide solution (1in 2),using a suitable syringe and needle to minimize exposure to air.Degas,and store in a reservoir protected from air by sparging with helium.

Solution C— Use degassed water,and store in a reservoir protected from air by sparging with helium.

Mobile phase— Using a suitable pumping system,pumpSolution A,Solution C,andSolution Bfrom the respective reservoirs in the ratio of 56:37:7.Make any necessary adjustments (seeSystem Suitability underChromatography á621ñ).

Diluent— Prepare a mixture of methanol and water (50:50).

Standard preparation— Quantitatively prepare a solution of USP Erythromycin RSinDiluent having a known concentration of about 0.66mg per mL.

Erythromycins Band Cstandard preparation— Quantitatively prepare a solution inDiluent having known concentrations of about 34µg each of USP Erythromycin B RSand USP Erythromycin C RSper mL.

System suitability solution— Transfer about 2mg of USP Erythromycin Related Compound N RSto a 10-mLvolumetric flask,add 0.4mLof theStandard preparation and 6mLofErythromycins Band Cstandard preparation,and mix.Dilute withErythromycins Band Cstandard preparation to volume,and mix.

Assay preparation— Transfer an accurately weighed portion of Ointment,equivalent to about 60mg of erythromycin,to a 125-mLseparator.Add 50mLof solvent hexane,and shake until dissolved.Extract with four separate 20-mLportions ofDiluent,collecting the extracts in a 100-mLvolumetric flask.Dilute the combined extracts withDiluent to volume,mix,and pass a portion of the solution through a filter having a 0.45-µm porosity.Use the clear filtrate as theAssay preparation.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with an electrochemical detector,a glassy carbon electrode fitted with three gaskets,a 4-mm ×5-cm guard column that contains 8-µm packing L52,and a 4-mm ×25-cm analytical column that contains 8-µm packing L52.The electrochemical detector is used in the pulsed integrated amperometric mode with a range of 10nC,an output of 1Vfull scale,a rise time of 0.6second,positive polarity,E=0.9V;t1=400ms;E2=0.9V;t2=100ms;E3=–0.9V;t3=100ms.The flow rate is about 1mLper minute.Chromatograph theSystem suitability solution,and record the peak responses as directed forProcedure:the relative retention times are about 0.4for erythromycin related compound N,0.5for erythromycin C,1.0for erythromycin A,and 1.6for erythromycin B;the resolution,R,between erythromycin related compound Nand erythromycin Cis not less than 0.6,between erythromycin Cand erythromycin Anot less than 2.5,and between erythromycin Aand erythromycin Bnot less than 2.5.Chromatograph theStandard preparation,and record the peak responses as directed forProcedure:the tailing factor is not more than 2;and the relative standard deviation for replicate injections is not more than 3%.[NOTE—Turn off the electrochemical detector before stopping the flow of theMobile phase.]

Procedure— Separately inject equal volumes (about 10µL)of theStandard preparation,Erythromycins Band Cstandard preparation,and theAssay preparation into the chromatograph,record the chromatograms,and measure the peak areas.Calculate the percentage of erythromycin Ain the portion of Ointment taken by the formula: in whichCis the concentration,in mg per mL,of USP Erythromycin RSin theStandard preparation;Pis the designated percentage of erythromycin Ain USP Erythromycin RS;Wis the quantity,in g,of Ointment taken to prepare theAssay preparation;andrUandrSare the erythromycin Apeak areas obtained from theAssay preparation and theStandard preparation,respectively.Calculate the percentages of erythromycin Band erythromycin Cin the portion of Ointment taken by the formula: in whichCis the concentration,in µg per mL,of the relevant USP Reference Standard in theErythromycins Band Cstandard preparation;Pis the designated percentage of erythromycin Bor erythromycin Cin the relevant USP Reference Standard;Wis the quantity,in g,of Ointment taken to prepare theAssay preparation;andrUandrSare the peak areas for the relevant analyte obtained from theAssay preparation and theErythromycins Band Cstandard preparation,respectively.Calculate the percentage content of erythromycin in the Ointment by adding the percentages of erythromycin A,erythromycin B,and erythromycin Cfound.