Pyrazinamide

This article is compiled based on the United States Pharmacopeia (USP) – 2025 Edition

Issued and maintained by the United States Pharmacopeial Convention (USP)

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C₅H₅N₃O 123.12 

Pyrazinecarboxamide CAS RN®: 98-96-4; UNII: 2KNI5N06TI. 

1 DEFINITION

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Pyrazinamide contains NLT 98.0% and NMT 102.0% of pyrazinamide (C₅H₅N₃O), calculated on the anhydrous basis. 

2 IDENTIFICATION 

A. Spectroscopic Identification Tests 〈197〉, Infrared Spectroscopy: 197M 

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B. The retention time of the major peak of the Sample solution corresponds to that of the Standard solution, as obtained in the Assay. ) 

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C. Procedure 

Sample: 20 mg 

Analysis: Boil the Sample with 5 mL of 5 N sodium hydroxide. 

Acceptance criteria: The odor of ammonia is perceptible. ) 

3 ASSAY 

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Procedure 

Mobile phase: Transfer 2.0 g of tetrabutyl ammonium hydrogen sulphate to a 1000-mL volumetric flask. Add 800 mL of water and sonicate to dissolve. Add 2.0 mL of ammonia water, 25 percent and mix. Adjust with 10% phosphoric acid to a pH of 6.8, and dilute with water to volume. Pass through a suitable lter of 0.45-µm pore size. 

Diluent: Water 

System suitability solution: 0.01 mg/mL of USP Pyrazinamide RS and 0.001 mg/mL of USP Pyrazinamide Related Compound A RS in Diluent Standard stock solution: 1.0 mg/mL of USP Pyrazinamide RS in Diluent 

Standard solution: 0.02 mg/mL of USP Pyrazinamide RS in Diluent, from Standard stock solution 

Sample stock solution: 1.0 mg/mL of Pyrazinamide in Diluent 

Sample solution: 0.02 mg/mL of Pyrazinamide in Diluent 

Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: UV 269 nm 

Column: 4.6-mm × 15-cm; 5-µm packing L1 

Column temperature: 25° 

Flow rate: 1.0 mL/min 

Injection volume: 20 µL 

Run time: NLT 4.0 times the retention time of pyrazinamide 

System suitability 

Samples: System suitability solution and Standard solution 

Suitability requirements 

Resolution: NLT 3.0 between pyrazinamide and pyrazinamide related compound A, System suitability solution Tailing factor: NMT 3.0, Standard solution 

Relative standard deviation: NMT 1.0%, Standard solution 

Analysis 

Samples: Standard solution and Sample solution 

Calculate the percentage of pyrazinamide (C₅H₅N₃O) in the portion of Pyrazinamide taken:

Result = (r_U/r ) × (C /C_U) × 100 

r_U = peak response of pyrazinamide from the Sample solution 

r = peak response of pyrazinamide from the Standard solution 

C = concentration of USP Pyrazinamide RS in the Standard solution (mg/mL) 

C_U = concentration of Pyrazinamide in the Sample solution (mg/mL) 

Acceptance criteria: 98.0%–102.0% on the anhydrous basis ) 

4 IMPURITIES 

Residue on Ignition 〈281〉: NMT 0.1% 

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Organic Impurities 

Mobile phase: Transfer 6.8 g of potassium dihydrogen phosphate to a 1000-mL volumetric flask. Add 800 mL of water and 1.84 g of sodium hydroxide and dissolve. Adjust with dilute phosphoric acid to a pH of 3.0, and dilute with water to volume. Add 10 mL of acetonitrile and 1 mL of tetrahydrofuran and mix. 

Diluent: Water 

System suitability solution: 0.08 mg/mL of USP Pyrazinamide RS and 0.004 mg/mL of USP Pyrazinamide Related Compound B RS in Diluent Standard solution: 0.0002 mg/mL each of USP Pyrazinamide RS and USP Pyrazinamide Related Compound A RS and 0.0004 mg/mL of USP Pyrazinamide Related Compound B RS in Diluent 

Sample solution: 0.4 mg/mL of Pyrazinamide in Diluent 

Chromatographic system 

(See Chromatography 〈621〉, System Suitability.) 

Mode: LC 

Detector: UV 270 nm 

Column: 4.6-mm × 25-cm; 5-µm packing L1 

Column temperature: 30° 

Flow rate: 2.0 mL/min 

Injection volume: 40 µL 

Run time: NLT 5.2 times the retention time of pyrazinamide 

System suitability 

Samples: System suitability solution and Standard solution 

[Note—See Table 1 for relative retention times.] 

Suitability requirements 

Resolution: NLT 4.0 between pyrazinamide and pyrazinamide related compound B, System suitability solution Relative standard deviation: NMT 10.0%, Standard solution 

Analysis 

Samples: Standard solution and Sample solution 

Calculate separately the percentage of pyrazinamide related compound A, pyrazinamide related compound B, and any unspecified impurity in the portion of Pyrazinamide taken: 

Result = (r_U/r ) × (C /C_U) × 100 

r_U = peak response of pyrazinamide related compound A, pyrazinamide related compound B, or any unspecified impurity from the Sample solution 

r = peak response of pyrazinamide, pyrazinamide related compound A, or pyrazinamide related compound B from the Standard solution 

C = concentration of USP Pyrazinamide RS in the Standard solution (mg/mL) 

C_U = concentration of Pyrazinamide in the Sample solution (mg/mL) 

Acceptance criteria: See Table 1. The reporting threshold is 0.003%. 

Table 1 

^a Pyrazinecarboxylic acid. b Pyrazinecarbonitrile. 

5 SPECIFIC TESTS 

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Melting Range or Temperature 〈741〉: 188°–191° ) 

Water Determination 〈921〉, Method I: NMT 0.5% 

6 ADDITIONAL REQUIREMENTS 

Packaging and Storage: Preserve in well-closed containers. 

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USP Reference Standards 〈11〉 

USP Pyrazinamide RS 

 USP Pyrazinamide Related Compound A RS 

Pyrazinecarboxylic acid. 

C₅H₄N₂O₂ 124.10 

USP Pyrazinamide Related Compound B RS 

Pyrazinecarbonitrile. 

C₅H₃N₃ 105.10