Simultaneous Determination of Pyrazinamide, Rifampicin, Ethambutol, Isoniazid and Acetyl Isoniazid in Human Plasma by LC-MS/MS Method

Monitoring of anti-tuberculosis drug concentrations in human plasma is very important in the treatment of TB disease. In this study, we described a method that can rapidly and simultaneously measure the plasma concentrations of four anti-tuberculosis drugs (isoniazid, rifampicin, pyrazinamide, and ethambutol) and one major metabolite (acetylisoniazid) using high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). To adjust for degradation and losses during sample preparation, Diltiazem hydrochloride was used as an internal standard. Samples were prepared by using protein precipitation with methanol. Four drugs and one metabolite were simultaneously separated by using the high-performance liquid chromatography system with Gemini C18 column and a mobile phase consisting of MeOH and Ammonium acetate 5mM, pH 3.5. The method was found to have high selectivity. Precision estimated by the coefficient of variation was <15% for all drugs. The linear range of the calibration curve for PZA 1.0-100 g/mL; RIF 0.2-20 g/mL; INH 0.1-10 g/mL; AcINH 0.1-10 g/mL; and EMB 20-5000 ng/mL. The lower limit of quantification was 1 g/mL; 0.2 g/mL; 0.1 g/mL; 0.1 g/mL and 20 ng/mL, respectively. The results of validation for specificity/selectivity, precision, accuracy, linearity, limit of quantification, recovery, and stability show that the method meets the requirements of a bioanalytical method. Our method may serve well for routine therapeutic monitoring of the first-line anti-TB drugs in patient plasma.