APPLICATION OF HPLC FOR SIMULTANEOUS DETERMINATION OF ISONIAZID, RIFAMPICIN AND PYRAZINAMIDE IN A FIXED DOSE FORMULATION

Abstract

A precise, accurate, sensitive and robust RP-HPLC method was developed and validated for Isoniazid (INH), Rifampicin (RIF) and Pyrazinamide (PYZ) in fixed-dose combination (FDC) antitubercular pharmaceutical dosage form. Chromatographic analysis was performed on a 250 × 4.6 mm I.D. C18column packed with 5 mm-in-size particles applying gradient elution with a mobile phase composed of 20 mM monobasic sodium phosphate buffer (pH 7) and acetonitrile (B). A:B ratio was 48:52 v/v for the initial 5 min, and then it was maintained at 96:4 v/v; the flow rate was 1 ml/min. UV detection was performed at 264 nm. The total run time was 20 min. The retention time was found to be 5.43 min, 7.31 min and 17.52 min for INH, PYZ and RIF respectively. The method was validated with respect to linearity, accuracy, precision, specificity and sensitivity in accordance with ICH guidelines. Limits of detection were of 0.063, 0.036 and 0.059 μg/ml and limits of quantification were of 0.19, 0.11 and 0.18 μg/ml for isoniazid, pyrazinamide and rifampicin respectively. High recovery and low coefficients of variance confirmed the suitability of the method for the simultaneous analysis of the three considered drugs.