A: To a quantity of powdered Tablets,equivalent to about 1g of pyrazinamide,add about 75mLof isopropyl alcohol,heat on a steam bath,and filter while hot.Allow to cool,filter the crystals that form,and dry at 105for 1hour:the IRabsorption 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 Identificationtest Ameet the requirements for Identificationtest Bunder Pyrazinamide.

C: To 20mg of the dried crystals obtained in Identificationtest Aadd 5mLof 5Nsodium hydroxide,and heat gently over an open flame:the odor of ammonia is perceptible.

Medium: water;900mL.

Apparatus 2: 50rpm.

Time: 45minutes.

Procedure— Determine the amount of C5H5N3Odissolved by employing UVabsorption at the wavelength of maximum absorbance at about 268nm 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 RSin the same Medium.

Tolerances— Not less than 75%(Q)of the labeled amount of C5H5N3Ois dissolved in 45minutes.

Mobile phase— Prepare pH8.0phosphate buffer (see Buffer Solutionsin the section Reagents,Indicators,and Solutions),and adjust with phosphoric acid to a pHof 3.0.Mix 10mLof acetonitrile with 1liter of this solution,filter,and degas.Make adjustments if necessary (see System Suitabilityunder Chromatography á621ñ).

Standard preparation— Transfer an accurately weighed quantity of USP Pyrazinamide RSto 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 0.1mg per mL.Transfer 20.0mLof the solution to a 50-mLvolumetric flask,dilute with water to volume,and mix.

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

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

Chromatographic system (see Chromatography á621ñ)—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 1mLper minute.Chromatograph the Standard preparation,and record the peak responses as directed for Procedure:the column efficiency is not less than 2500theoretical 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 0.45for pyrazinoic acid and 1.0for pyrazinamide;and the resolution,R,between pyrazinamide and pyrazinoic acid is not less than 6.0.

Procedure— Separately inject equal volumes (about 20µL)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 pyrazinamide (C5H5N3O)in the portion of Tablets taken by the formula: in which Cis the concentration,in µg,of USP Pyrazinamide RSin the Standard preparation;and rUand rSare the peak responses obtained from the Assay preparationand the Standard preparation,respectively.