A: Infrared Absorption 197K—

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.

Medium: 0.1 M phosphate buffer, pH 2.0, prepared by transferring 3.9 mL of concentrated phosphoric acid and 5.73 g of monobasic sodium phosphate monohydrate to a 1-L volumetric flask, dissolving in and diluting with water to volume, and if necessary, adjusting with phosphoric acid to a pH of 2.0±0.02; 900 mL.

Apparatus 2: 50 rpm. Use stainless steel paddles only. Do not use paddles coated with polytetrafluoroethylene.

Time: 60 minutes.

Mobile phase, Diluent, and Chromatographic system— Proceed as directed in the Assay.

Stock standard solution 1— Transfer 27 mg, accurately weighed, of USP Nevirapine Anhydrous RS into a 500-mL volumetric flask. Add 50 mL of alcohol, followed by 250 mL of Medium. Sonicate for about 20 minutes to dissolve, allow to cool to room temperature, and dilute with Medium to volume.

Stock standard solution 2— Transfer 7 mg, accurately weighed, of USP Nevirapine Related Compound A RS into a 250-mL volumetric flask. Add about 2 mL of Diluent, sonicate until completely dissolved, and dilute with Medium to volume.

Standard solution— Transfer 25.0 mL of Stock standard solution 1 into a 100-mL volumetric flask, dilute with Medium to volume, and mix well.

Resolution solution— Transfer 25.0 mL of Stock standard solution 1 into a 50-mL volumetric flask, and dilute with Medium to volume. Transfer 25.0 mL of this solution into a 50-mL volumetric flask, dilute with Stock standard solution 2 to volume, and mix well.

Test solution— Pass 20 mL of the solution under test through a nylon or glass fiber 0.45-µm filter, and dilute with Medium to obtain a solution having a final concentration of about 0.0135 mg of nevirapine per mL.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the responses for the major peaks. Determine the amount of C15H14N4O dissolved by the formula: in which rU and rS are the peak responses obtained from the Test solution and the Standard solution, respectively; WS is the amount, in mg, of USP Nevirapine Anhydrous RS taken; DS is the dilution factor for the Standard solution; 900 is the volume, in mL, of Medium; 100 is the conversion factor to percentage; DU is the dilution factor for the Test solution; and LC is the Tablet label claim in mg.

Tolerances— Not less than 75% (Q) of the labeled amount of C15H14N4O is dissolved in 60 minutes.

Mobile phase, Diluent, Resolution solution, Stock standard solution 1, and Stock standard solution 2— Proceed as directed in the Assay.

Standard solution— Quantitatively dilute Stock standard solution 1 with Diluent to obtain a solution having a known concentration of about 0.125 µg of nevirapine per mL.

Test solution— Use the Assay preparation, as obtained in the Assay.

Chromatographic system (see Chromatography 621)— Proceed as directed in the Assay except that the relative standard deviation for replicate injections of the Standard solution is not more than 5.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms for at least 13 minutes, and measure the peak responses. Calculate the percentage of each impurity/degradation product in the portion of Tablets taken by the formula: in which C is the concentration, in mg per mL, of USP Nevirapine Anhydrous RS in the Standard solution; W is the weight, in mg, of powdered Tablets taken to prepare the Test solution; A is the average weight of each Tablet, in mg; L is the labeled amount, in mg, of nevirapine in each Tablet; ri is the peak response obtained for each impurity/degradation product in the Test solution; and rS is the peak response for nevirapine in the Standard solution. Disregard all peaks due to the solvent or excipients and impurity peaks less than 0.1%. Not more than 0.1% of any individual unknown impurity/degradation product is found; and not more than 0.2% of total unknown impurities/degradation products is found.

Mobile phase— Prepare a filtered and degassed mixture of water and acetonitrile (77:23). Make adjustments if necessary (see System Suitability under Chromatography 621).

Diluent— Prepare a mixture of dehydrated alcohol and water (1:1).

Stock standard solution 1— Transfer about 25 mg of USP Nevirapine Anhydrous RS, accurately weighed, into a 250-mL volumetric flask, and dissolve in and dilute with Diluent to volume.

Stock standard solution 2— Transfer about 5 mg of USP Nevirapine Related Compound A RS, accurately weighed, into a 50-mL volumetric flask, and dissolve in and dilute with Diluent to volume.

Standard preparation— Transfer 25.0 mL of Stock standard solution 1 into a 100-mL volumetric flask, and dilute with Diluent to volume. The final concentration is about 0.025 mg of nevirapine per mL.

Resolution solution— Transfer 25.0 mL of Stock standard solution 1 and 25.0 mL of Stock standard solution 2 into a 100-mL volumetric flask, dilute with Diluent to volume, and mix well.

Assay preparation— Weigh and finely powder not fewer than 20 Tablets. Transfer an accurately weighed portion of the powder, equivalent to 200 mg of nevirapine into a 200-mL volumetric flask, and add about 150 mL of Diluent. Sonicate the solution for about 20 minutes, and then shake for about 20 minutes. Cool to room temperature, dilute with Diluent to volume, and mix. Centrifuge a portion of the resulting solution at about 1500 rpm for about 5 minutes. Transfer 5.0 mL of the supernatant into a 200-mL volumetric flask, and dilute with Diluent to volume. Filter, and discard the first 2 mL of the filtrate.

Chromatographic system (see Chromatography 621)— The liquid chromatograph is equipped with a 214-nm detector and a 3.9-mm × 15-cm column containing L1 packing. The column is maintained at ambient temperature. The flow rate is about 1.0 mL per minute. Chromatograph the Resolution solution (about 20 µL), and record the peak responses as directed for Procedure: the resolution, R, between nevirapine and nevirapine related compound A is not less than 3.0. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the relative standard deviation for replicate injections of the Standard preparation is not more than 2.0%.

Procedure— Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the responses for the nevirapine peak. Calculate the quantity, in mg, of nevirapine (C15H14N4O) in the portion of Tablets 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.