VALIDATION OF HPLC METHODS FOR DETERMINATION OF ISONIAZID, RIFAMPICIN, PYRAZINAMIDE, AND ETHAMBUTOL IN A FIXED-DOSE COMBINATION ANTITUBERCULOSIS

Abstract

Simple HPLC methods using two HPLC systems for the quantification of isoniazid (INH), pyrazinamide (PZA), rifampicin (RIF) and ethambutol (EMB) in a fixed-dose combination (FDC) antituberculosis were developed and validated. The chromatographic separation of  INH, PZA, and RIF  was carried out using Inertsil ODS-3 5µm, C18 (length 150 mm; inner diameter 4.6 mm), UV detection at 254 nm, and a gradient system composed of acetonitrile (A) and 3% acetonitrile in 10 mM monobasic potassium phosphate buffer pH 6.8 (B) at a flow rate of 1.5 mL/min. The gradient profile was (A:B) 0:100 v/v for 5 min, then a linear gradient to 50:50 v/v allowed to maintain at (A:B) 50:50 v/v for 15 min, then return to 0:100 v/v for 1 min, and finally re-equilibrate for another 5 min before the next injection. The retention time of INH, PZA, and RIF was 2.9, 4.0 and 10.4 min, respectively. The limit of quantification of the method for INH, PZA, and RIF was 1.07, 1.74 and 1.13 µg/mL, respectively. The separation of EMB was performed using Hypersil BDS 5 µm, C18 (4.6 mm x 250 cm), UV detection at 254 nm, using an elution isocrartic procedure. The mobile phase mixture composed of 2.0 mM of copper sulfate (CuSO4.5H2O), 3 g/L sodium hexanesulfonate (pH4.5) in distilled water and tetrahydrofuran, 75:25 (v/v) at a flow rate of 0.4 mL/min. The retention time of EMB was 14.92 min. The limit of quantification of the method for EMB was 4.28 µg /mL.