Development and validation of RP-HPLC method for simultaneous estimation of rifampicin, isoniazid and pyrazinamide in human plasma

A rapid, simple, sensitive and cost effective stability indicating high performance liquid chromatographic method for the simultaneous determination of rifampicin, isoniazid and pyrazinamide in human plasma was developed and validated in accordance to Food and Drug Administration (FDA) guidelines. The three drugs were eluted under isocratic mode using a 250 × 4.0 mm i.d., 5 μm Phenomenex ODS 2 C18 column. The mobile phase was composed of a mixture of acetonitrile, methanol and water in the ratio of 30: 5: 65 (v/v, pH adjusted to 5.2) at a flow rate of 1.0 mL/min. The limits of detection and quantification for rifampicin were 0.13 and 0.4 μg/mL, for isoniazid—0.6 and 1.8 μg/mL; and for pyrazinamide—0.5 and 1.6 μg/mL, respectively. The method can be successfully applied for pharmacokinetic, bioavailability or bioequivalence studies of rifampicin, isoniazid and pyrazinamide combination in human subjects.