Analytical method development and validation for the determination of Rifampicin, Isoniazid and Pyrazinamide in “Rifampicin, Isoniazid, Pyrazinamide and Ethambutol hydrochloride” tablets

A simple and efficient method for the quantification of rifampicin, isoniazid and pyrazinamide in a fixed dose combination of anti-tuberculosis drug product was developed and validated by using HPLC system. The separation of rifampicin, isoniazid and pyrazinamide was carried out on a Waters 2690, composed of a quaternary pump, autosampler and PDA detector by chromatographic technique. Empower 3 software was used for data acquisition and analysis. Optimization of chromatographic separations was achieved using Phenomenex curosil-pfp column (250 × 4.6 mm, 5 μm particle size), with gradient program using disodium hydrogen phosphate and sodium 1- octane sulfonate buffer solution (pH adjusted to 7.0) as mobile phase A and acetonitrile (100%) as mobile phase B at a flow rate of 0.8 mL/min and UV detection at 238 nm. Approximate relative retention times of isoniazid, pyrazinamide and rifampicin were found to be 0.89, 1.00 and 2.63 respectively.