A:Infrared Absorption á197Kñ— Do not dry the specimens.

B: The retention time of the major peak in the chromatogram of the Assay preparationcorresponds to that in the chromatogram of the Standard preparation,as obtained in the Assay.

0.025M Ammonium phosphate buffer,Mobile phase,Standard stock solution 1,Standard stock solution 2,Standard stock solution 3,and Resolution solution— Proceed as directed in the Assay.

Standard solution— Transfer 2.0mLof Standard stock solution 1to a 200-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.Transfer 5.0mLof this solution to a 50-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

Test solution— Transfer an accurately weighed quantity of Nevirapine,equivalent to about 24mg of nevirapine anhydrous,to a 100-mLvolumetric flask.Add 4mLof acetonitrile and 80mLof Mobile phase,and sonicate for at least 15minutes.Allow to cool to room temperature,dilute with Mobile phaseto volume,and mix.

Chromatographic system— Proceed as directed in the Assay.Chromatograph the Resolution solution(approximately 25µL),and record the peak responses as directed for Procedure:the relative retention times are about 0.7for nevirapine related compound B,1.0for nevirapine,1.5for nevirapine related compound A,and 2.8for nevirapine impurity C;the resolution,R,between nevirapine related compound Band nevirapine is not less than 5.0;and the resolution between nevirapine and nevirapine related compound Ais not less than 7.4.Chromatograph the Standard solution,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 5.0%.

Procedure— Separately inject equal volumes (about 50µL)of the Standard solutionand the Test solutioninto the chromatograph,record the chromatograms for at least 80minutes,and measure the responses for the major peaks.Calculate the percentage of each impurity in the portion of Nevirapine taken by the formula: in which Fis the relative response factor for each impurity,which is equal to 1.3for nevirapine related compound Band 1.0for all other impurities;Cis the concentration,in mg per mL,of USP Nevirapine Anhydrous RSin the Standard solution;Wis the weight of Nevirapine,in mg,taken to prepare the Test solution;riis the peak response for each impurity obtained from the Test solution;and rSis the nevirapine peak response obtained from the Standard solution:not more than 0.2%each of nevirapine related compound A,nevirapine related compound B,and nevirapine impurity Cis found;not more than 0.1%of any other individual unspecified impurity is found;and not more than 0.6%of total impurities is found.

0.025M Ammonium phosphate buffer— Transfer 2.88g of monobasic ammonium phosphate to a 1000-mLvolumetric flask,dissolve in 800mLof water,adjust with 1Nsodium hydroxide to a pHof about 5.0,dilute with water to volume,and mix.

Mobile phase— Prepare a filtered and degassed mixture of 0.025M Ammonium phosphate bufferand acetonitrile (4:1).

Standard stock solution 1— Transfer an accurately weighed quantity of USP Nevirapine Anhydrous RSto a volumetric flask,add a volume of a mixture of Mobile phaseand acetonitrile (20:1),sonicate for at least 15minutes,allow to cool to room temperature,dilute with Mobile phaseto volume,and mix to obtain a solution having a known concentration of about 0.24mg per mL.[NOTE—Do not use after 78hours.]

Standard stock solution 2— Transfer an accurately weighed quantity of USP Nevirapine Related Compound A RSto a volumetric flask,add a volume of a mixture of Mobile phaseand acetonitrile (3:1),sonicate for at least 15minutes,allow to cool to room temperature,dilute with Mobile phaseto volume,and mix to obtain a solution having a known concentration of about 0.24mg per mL.

Standard stock solution 3— Transfer an accurately weighed quantity of USP Nevirapine Related Compound B RSto a volumetric flask,add a volume of a mixture of Mobile phaseand acetonitrile (2.2:1),sonicate for at least 30minutes,allow to cool to room temperature,dilute with Mobile phaseto volume,and mix to obtain a solution having a known concentration of about 0.06mg per mL.

Resolution solution— Transfer 3.0mLof Standard stock solution 1,3.0mLof Standard stock solution 2,and 6.0mLof Standard stock solution 3to a 25-mLvolumetric flask,dissolve in and dilute with Mobile phaseto volume,and mix.

Standard preparation— Transfer 3.0mLof Standard stock solution 1to a 25-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.[NOTE—Do not use after 78hours.]

Assay preparation— Transfer an accurately weighed quantity of Nevirapine,equivalent to about 24mg of nevirapine anhydrous,to a 100-mLvolumetric flask.Add 4mLof acetonitrile and 80mLof Mobile phase,sonicate for at least 15minutes,allow to cool to room temperature,dilute with Mobile phaseto volume,and mix.Transfer 3.0mLof this solution to a 25-mLvolumetric flask,dilute with Mobile phaseto volume,and mix.

Chromatographic system(see Chromatography á621ñ)— The liquid chromatograph is equipped with a 220-nm detector and a 4.6-mm ×15-cm column that contains 5-µm packing L60(see Chromatography á621ñ).The flow rate is about 1mLper minute.The column temperature is maintained at 35.Chromatograph the Resolution solution(approximately 25µL),and record the peak responses as directed for Procedure:the relative retention times are about 0.7for nevirapine related compound B,1.0for nevirapine,1.5for nevirapine related compound A,and 2.8for nevirapine impurity C;the resolution,R,between nevirapine related compound Band nevirapine is not less than 5.0;and the resolution between nevirapine and nevirapine related compound Ais not less than 7.4.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the relative standard deviation for replicate injections is not more than 2.0%.

Procedure— Separately inject equal volumes (about 25µL)of the Standard preparationand the Assay preparationinto the chromatograph,record the chromatograms,and measure the responses for the major peaks.Calculate the quantity,in mg,of C15H14N4Oin the portion of Nevirapine taken by the formula: in which Cis the concentration,in mg per mL,of USP Nevirapine Anhydrous RSin the Standard preparation;andrUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.USP28