Add the following:

A:Infrared Absorption á197Kñ.

B: The retention time of the azithromycin peak in the chromatogram of theAssay preparationcorresponds to that in the chromatogram of theStandard preparation,as obtained in theAssay.

Test solution: 20mg per mL,in dehydrated alcohol.

Change to read:

Add the following:

Mobile phase— Proceed as directed in theAssay.

pH7.5Potassium phosphate buffer— Transfer 2.7g of monobasic potassium phosphate to a 1000-mLvolumetric flask.Dilute with water to volume,and mix.Adjust with 10Npotassium hydroxide to a pHof 7.5±0.1.

Dilution solution— Prepare a mixture ofpH7.5Potassium phosphate buffer and acetonitrile (750:250).

Standard stock solution— Quantitatively dissolve accurately weighed quantities of USP Desosaminylazithromycin RS,USPN-Demethylazithromycin RS,and USP Azithromycin RSwith acetonitrile to obtain a solution having known concentrations of about 45,105,and 160µg per mL,respectively.

Standard solution— Transfer 4.0mLofStandard stock solution to a 200-mLvolumetric flask,dilute withDilution solution to volume,and mix.This solution contains known concentrations of USP Desosaminylazithromycin RS,USPN-Demethylazithromycin RS,and USP Azithromycin RSof about 0.9,2.1,and 3.2µg per mL,respectively.

Test solution— Transfer about 33mg of Azithromycin,accurately weighed,to a 100-mLvolumetric flask,add 5mLof acetonitrile,and sonicate for about 20seconds to dissolve.Dilute withDilution solution to volume,and mix.[NOTE—Use this solution within 6hours.]

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with an amperometric electrochemical detector with dual glassy carbon electrodes operated in the oxidative screen mode with electrode 1set at +0.70±0.05Vand electrode 2set at +0.85±0.05V,and the background current optimized to 95±25nanoamperes,a 4.6-mm ×5-cm guard column that contains 5-µm packing L29,and a 4.6-mm ×15-cm analytical column that contains 5-µm packing L29or 3-µm packing L49without the guard column.[NOTE—In general,maintain electrode 1at 0.12Vless than electrode 2,and maintain the electrodes at a constant temperature of about 26.]The flow rate is about 0.4mLper minute.Chromatograph theStandard solution,and record the peak responses as directed forProcedure:the relative retention times are about 0.38for desosaminylazithromycin,0.54forN-demethylazithromycin,and 1.0for azithromycin;the column efficiency is not less than 1500theoretical plates for the azithromycin peak;the tailing factor for each of these compounds is not more than 1.5;and the relative standard deviation for replicate injections is not more than 5%for each of these compounds.

Procedure— Separately inject equal volumes (about 50µL)of theStandard solution and theTest solution into the chromatograph,record the chromatograms,using an elution period for theTest solution that is 3.3times the elution time of the azithromycin peak in the chromatogram of theStandard solution,and measure the areas for all of the peaks.Calculate the percentages of desosaminylazithromycin andN-demethylazithromycin in the Azithromycin taken by the formula: in whichCis the concentration,in µg per mL,of the appropriate USP Reference Standard in theStandard solution;Pis the designated potency,in percentage,of the relevant USP Reference Standard;Wis the weight,in mg,of Azithromycin taken to prepare theTest solution;andriandrSare the peak area responses for the relevant analyte in the chromatograms obtained from theTest solution and theStandard solution,respectively.Calculate the percentages of other related substances in the Azithromycin taken by the formula: in whichCis the concentration,in µg per mL,of USP Azithromycin RSin theStandard solution;Pis the designated purity,in µg per mg,of USP Azithromycin RS;Wis the weight,in mg,of Azithromycin taken to prepare theTest solution;riis the peak area response for an individual related substance peak in the chromatogram obtained from theTest solution;andrSis the peak area response for the azithromycin peak in the chromatogram obtained from theStandard solution.Not more than 0.3%of desosaminylazithromycin,0.7%ofN-demethylazithromycin,and 1.0%of any other individual related substance is found;and the sum of all related substances is not more than 3.0%.

Mobile phase— Dissolve 5.8g of monobasic potassium phosphate in 2130mLof water,add 870mLof acetonitrile,and mix.Adjust with about 6mLof 10Npotassium hydroxide to a pHof 11.0±0.1,and pass through a filter having a 0.5-µm or finer porosity,and degas.Make adjustments if necessary (seeSystem SuitabilityunderChromatography á621ñ).

Standard stock preparation— Transfer about 16.5mg of USP Azithromycin RS,accurately weighed,to a 100-mLvolumetric flask,add 10mLof acetonitrile,and dissolve by swirling and with the aid of brief sonication.Dilute with acetonitrile to volume,and mix.

Standard preparation— Transfer 2.0mLof theStandard stock preparation to a 100-mLvolumetric flask,dilute withMobile phaseto volume,and mix to obtain aStandard preparationhaving a known concentration of about 0.0033mg of USP Azithromycin RSper mL.

Assay preparation— Transfer about 16.5mg of Azithromycin,accurately weighed,to a 100-mLvolumetric flask,add 10mLof acetonitrile,and dissolve by swirling and with the aid of brief sonication.Dilute with acetonitrile to volume,and mix.Transfer 2.0mLof the solution so obtained to a 100-mLvolumetric flask,dilute withMobile phaseto volume,and mix.

Resolution solution— Transfer about 8mg of USP Azaerythromycin A RSto a 50-mLvolumetric flask,add 5mLof acetonitrile,and dissolve by swirling and with the aid of brief sonication.Dilute withMobile phaseto volume,and mix.Transfer 2.0mLof the solution so obtained and 2.0mLof theStandard stock preparation to a 100-mLvolumetric flask,dilute withMobile phaseto volume,and mix.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with an amperometric electrochemical detector with dual glassy carbon electrodes operated in the oxidative screen mode with electrode 1set at +0.70±0.05Vand electrode 2set at +0.82±0.05V,and the background current optimized to 85±15nanoamperes,a 4.6-mm ×5-cm guard column that contains 5-µm packing L29and a 4.6-mm ×15-cm analytical column that contains 5-µm packing L29or 3-µm packing L49without the guard column.The flow rate is about 1.5mLper minute.Chromatograph theResolution solution,and record the responses as directed forProcedure:the relative retention times are about 0.7for azaerythromycin Aand 1.0for azithromycin with the L29column and about 0.8for azaerythromycin Aand 1.0for azithromycin with the L49column;and the resolution,R,between azaerythromycin Aand azithromycin is not less than 2.5.Chromatograph theStandard preparation,and record the responses as directed forProcedure:the tailing factor for the azithromycin peak is not less than 0.9and not more than 1.5;the column efficiency is not less than 1000theoretical plates;and the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 50µL)of theStandard preparationand theAssay preparationinto the chromatograph,record the chromatograms,and measure the areas for the major peaks.Calculate the quantity,in µg,of azithromycin (C38H72N2O12)in each mg of Azithromycin taken by the formula: in which Wis the quantity,in mg,of USP Azithromycin RStaken to prepare theStandard preparation;Pis the potency,in µg of azithromycin per mg,of USP Azithromycin RS;w is the quantity,in mg,of Azithromycin taken to prepare theAssay preparation;and rUand rSare the azithromycin peak responses obtained from theAssay preparationand theStandard preparation,respectively.