Development of validation of RP-HPLC method for simultaneous determination of pyridoxine hydrochloride, isoniazid, pyrazinamide and rifampicin in pharmaceutical formulation

Abstract

A simple, rapid and precise liquid chromatographic method for simultaneous determination of pyridoxine hydrochloride. isoniazid, pyrazinamide and rifampicin in a tablet dosage form has been developed. Chromatographic analysis was performed on a 250 x 4.6 mm I.D. C 18 column packed with 5 μm-in-size particles applying gradient elution with a mobile phase composed of acetonitrile (A) and 15 mmol L -1 potassium dihydrogen phosphate buffer of pH adjusted to 4.0 ± 0.1 with o-phosphoric acid (B). A:B ratio was 11:89 v/v for the initial 4.5 min, and then it was maintained at 50:50 v/v; the flow rate was 1 mL min -1 . UV detection was performed at 235 nm. The total run time was 20 min. Retention times for pyridoxine hydrochloride, isoniazid. pyrazinamide and rifampicin were 3.687, 4.113, 5.041 and 12.829 min, respectively. The method was validated with respect to linearity, accuracy, precision, specificity and sensitivity in accordance with ICH guidelines. Limits of detection were 0.043. 0.063, 0.036 and 0.059 μg mL -1 and limits of quantification were 0.13, 0.19, 0.11 and 0.18 μg mL -1 for pyridoxine hydrochloride, isoniazid, pyrazinamide and rifampicin, respectively. High recovery and low coefficients of variance confirmed the suitability of the method for the simultaneous analysis of the four considered drugs.