LC–QToF–MS method for quantification of ethambutol, isoniazid, pyrazinamide and rifampicin in human plasma and its application

Abstract In this research, we developed and validated a liquid chromatography coupled to mass spectrometry (LC–QToF–MS) method for simultaneous quantification of the anti‐tuberculosis drugs ethambutol, isoniazid, pyrazinamide and rifampicin in human plasma. Plasma samples spiked with cimetidine (internal standard) were extracted using protein precipitation with acetonitrile containing 1% formic acid. Separation was performed using a C 18 column under flow gradient conditions with water and acetonitrile, both containing 5 m m ammonium formate and 0.1% formic acid. The method was validated according to the ANVISA and US Food and Drug Administration guidelines for bioanalytical method validation. The calibration curve was linear over a concentration range of 0.2–5 μg ml −1 for ethambutol, 0.2–7.5 μg ml −1 for isoniazid, 1–40 μg ml −1 for pyrazinamide and 0.25–2 μg ml −1 for rifampicin, all with adequate precision and accuracy. The method was reproducible, selective and free of carryover and matrix effects. The validated LC–QToF–MS method was successfully applied to real samples and shown to be applicable to future therapeutic and pharmacokinetic monitoring studies.