A: Infrared Absorption 197K— Do not dry the specimens.

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

0.025 M 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 2.0 mL of Standard stock solution 1 to a 200-mL volumetric flask, dilute with Mobile phase to volume, and mix. Transfer 5.0 mL of this solution to a 50-mL volumetric flask, dilute with Mobile phase to volume, and mix.

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

Chromatographic system— Proceed as directed in the Assay. Chromatograph the Resolution solution (approximately 25 µL), and record the peak responses as directed for Procedure: the relative retention times are about 0.7 for nevirapine related compound B, 1.0 for nevirapine, 1.5 for nevirapine related compound A, and 2.8 for nevirapine impurity C; the resolution, R, between nevirapine related compound B and nevirapine is not less than 5.0; and the resolution between nevirapine and nevirapine related compound A is 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 50 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms for at least 80 minutes, 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 F is the relative response factor for each impurity, which is equal to 1.3 for nevirapine related compound B and 1.0 for all other impurities; C is the concentration, in mg per mL, of USP Nevirapine Anhydrous RS in the Standard solution; W is the weight of Nevirapine, in mg, taken to prepare the Test solution; ri is the peak response for each impurity obtained from the Test solution; and rS is 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 C is 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.

(Official January 1, 2007)

0.025 M Ammonium phosphate buffer— Transfer 2.88 g of monobasic ammonium phosphate to a 1000-mL volumetric flask, dissolve in 800 mL of water, adjust with 1 N sodium hydroxide to a pH of about 5.0, dilute with water to volume, and mix.

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

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

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

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

Resolution solution— Transfer 3.0 mL of Standard stock solution 1, 3.0 mL of Standard stock solution 2, and 6.0 mL of Standard stock solution 3 to a 25-mL volumetric flask, dissolve in and dilute with Mobile phase to volume, and mix.

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

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

Chromatographic system (see Chromatography 621)— 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 621). The flow rate is about 1 mL per minute. The column temperature is maintained at 35. Chromatograph the Resolution solution (approximately 25 µL), and record the peak responses as directed for Procedure: the relative retention times are about 0.7 for nevirapine related compound B, 1.0 for nevirapine, 1.5 for nevirapine related compound A, and 2.8 for nevirapine impurity C; the resolution, R, between nevirapine related compound B and nevirapine is not less than 5.0; and the resolution between nevirapine and nevirapine related compound A is 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 25 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Calculate the quantity, in mg, of C15H14N4O in the portion of Nevirapine taken by the formula: in which C is the concentration, in mg per mL, of USP Nevirapine Anhydrous RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.