Determination of rifampicin, isoniazid and pyrazinamide by high performance liquid chromatography after their simultaneous extraction from plasma

SETTING:Isoniazid (INH), rifampicin (RIF), and pyrazinamide (PZA) are first-line antituberculosis drugs. Due to difficulties in producing effective combined formulations of these three drugs, the bioavailability of new combination formulations needs to be assessed prior to registration. OBJECTIVE: To develop a rapid, simple and sensitive high performance liquid chromatography (HPLC) assay method suitable for assaying RIF, INH and PZA in large numbers of plasma samples generated in bioavailability studies. METHOD: RIF, desacetyl-rifampicin (DRIF), INH and PZA were extracted simultaneously from plasma using a solid phase extraction column. RIF and DRIF were quantitated by HPLC using an 80% acetonitrile/0.1% trifluoroacetic acid (TFA) mobile phase and a C8 reversed phase column. WH and PZA were also quantitated on a C8 reversed phase column, but a 3% acetonitrile/0.6% TFA mobile phase was used. RESULTS: Mean recovery of RIF, DRIF and PZA from plasma was well over 90%, and over 70% for INH. Calibration graphs were linear for all the drugs in their therapeutic range. Correlation coefficients were all above 0.9995, and between- and within-run coefficients of variation below 10%. CONCLUSION: A simple, accurate, sensitive and cost effective assay for first-line antituberculosis drugs has been developed.