RP-HPLC method has been developed and validated for assay to three of four constituents in anti-tuberculosis 4- FDC tablet simultaneously: rifampicin, isoniazid and pyrazinamid. To increase UV absorbance of the three components, phenyl isothiocyanate (PITC) is used in pre-column derivatization. Derivatization parameters were optimized and the results are molar ratio of the analytes to PITC, 1:40 and time for vortex processing is 60 seconds. Derivatization result was separated using stationary phase Phenomenex Luna C18, 5 μm (250 x 4.6 mm). Phosphate buffer 8 mM pH 4.8 and acetonitrile were used as mobile phase with gradient system, flow rate 1.0 mL/minute and detector UV 254 nm. This method was validated with some parameters: selectivity, linearity, precision, accuracy, LOD, LOQ and stability. This method cannot be performed on a compound of ethambutol because its reaction produced two derivatives. So this method can only be applied to assay to three constituent in anti-tuberculosis 4- FDC tablet simultaneously.
