Comprehensive assay for pyrazinamide, rifampicin and isoniazid with its hydrazine metabolites in human plasma by column liquid chromatography

A comprehensive assay for determination of pyrazinamide (PZA), rifampicin (RIF), isoniazid (INH) and hydrazine metabolites is described. The method involves organic solvent extraction of PZA and RIF, followed by derivatization of INH, monoacetylhydrazine (mHYD) and hydrazine (HYD) with salicylaldehyde and extraction with diethyl ether. Acetylisoniazid (acINH) and diacetylhydrazine (dHYD) were hydrolyzed to INH and mHYD, respectively, and processed as above. Using a gradient solvent programmer, PZA and RIF were analyzed on a C₈ (5 μm) column at 248 nm, while INH and metabolites were analyzed on a C₁₈ (5 μm) ODS2 column at 280 nm.