A: To a quantity of powdered Tablets, equivalent to about 1 g of pyrazinamide, add about 75 mL of isopropyl alcohol, heat on a steam bath, and filter while hot. Allow to cool, filter the crystals that form, and dry at 105 for 1 hour: the IR absorption spectrum of a mineral oil dispersion of the dried crystals so obtained exhibits maxima only at the same wavelengths as that of a similar preparation of USP Pyrazinamide RS. If a difference appears, dissolve portions of both the dried crystals and the Reference Standard in acetone, evaporate the solutions to dryness, and repeat the test on the residues.

B: The dried crystals obtained in Identification test A meet the requirements for Identification test B under Pyrazinamide.

C: To 20 mg of the dried crystals obtained in Identification test A add 5 mL of 5 N sodium hydroxide, and heat gently over an open flame: the odor of ammonia is perceptible.

Medium: water; 900 mL.

Apparatus 2: 50 rpm.

Time: 45 minutes.

Procedure— Determine the amount of C5H5N3O dissolved by employing UV absorption at the wavelength of maximum absorbance at about 268 nm on filtered portions of the solution under test, suitably diluted with Dissolution Medium, if necessary, in comparison with a Standard solution having a known concentration of USP Pyrazinamide RS in the same Medium.

Tolerances— Not less than 75% (Q) of the labeled amount of C5H5N3O is dissolved in 45 minutes.

(Official January 1, 2007)

Mobile phase— Prepare pH 8.0 phosphate buffer (see Buffer Solutions in the section Reagents, Indicators, and Solutions), and adjust with phosphoric acid to a pH of 3.0. Mix 10 mL of acetonitrile with 1 L of this solution, filter, and degas. Make adjustments if necessary (see System Suitability under Chromatography 621).

Standard preparation— Transfer an accurately weighed quantity of USP Pyrazinamide RS to a suitable volumetric flask, dissolve in water, sonicating to dissolve, dilute with water to volume, and mix to obtain a solution having a known concentration of about 0.1 mg per mL. Transfer 20.0 mL of the solution to a 50-mL volumetric flask, dilute with water to volume, and mix.

System suitability solution— Transfer 1 mL of hydrochloric acid to a 5-mL volumetric flask, dilute with Standard preparation to volume, and mix. Keep this solution on a boiling water bath for 5 minutes, and cool.

Assay preparation— Accurately weigh not fewer than 20 Tablets, and grind to a fine powder. Transfer an accurately weighed quantity of the powder, equivalent to about 100 mg of pyrazinamide, to a 500-mL volumetric flask, add 300 mL of water, and sonicate for 10 minutes. Dilute with water to volume, and mix. Filter a portion of this solution, discarding the first few mL of the filtrate. Transfer 20.0 mL of this filtrate to a 100-mL volumetric flask, dilute with water to volume, and mix.

Chromatographic system (see Chromatography 621)—The liquid chromatograph is equipped with a 270-nm detector and a 3.9-mm × 15-cm column that contains packing L1. The flow rate is about 1 mL per minute. Chromatograph the Standard preparation, and record the peak responses as directed for Procedure: the column efficiency is not less than 2500 theoretical plates; and the tailing factor for the pyrazinamide peak is not more than 1.3. Chromatograph the System suitability solution, and record the peak responses as directed for Procedure: the relative retention times are about 0.45 for pyrazinoic acid and 1.0 for pyrazinamide; and the resolution, R, between pyrazinamide and pyrazinoic acid is not less than 6.0.

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 major peaks. Calculate the quantity, in mg, of pyrazinamide (C5H5N3O) in the portion of Tablets taken by the formula: in which C is the concentration, in µg, of USP Pyrazinamide RS in the Standard preparation; and rU and rS are the peak responses obtained from the Assay preparation and the Standard preparation, respectively.