Rapid and simultaneous determination of ten anti-tuberculosis drugs in human plasma by UPLC-MS/MS with applications in therapeutic drug monitoring

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Tuberculosis remains a global challenge, particularly with a growing number of resistant cases, which may become an obstacle to eliminating this disease. Standardized short-course therapy composed of first-line anti-tuberculosis drugs isoniazid (INH), rifampicin (RIF), ethambutol (EMB), and pyrazinamide (PZA) is playing vital roles for curbing the rapid spread of tuberculosis. However, some patients have poor responses to standardized short-course therapy. As the number of drug-resistant tuberculosis increase, some other anti-tuberculous drugs are needed to achieve better treatment outcomes. In this study, we established a UPLC-MS/MS method for simultaneous detection of ten anti-tuberculosis drugs in human plasma including INH, EMB, PZA, RIF, rifampin, rifapentine as well as four second-line antituberculosis drugs, i.e. ethionamide, protionamide, thiosemicarbazone and clofazimine. This study contains almost all the commonly used anti-tuberculosis drugs. The plasma samples were treated with acetonitrile to precipitate proteins, and doped with the isotope internal standard. A Shiseido CAPCELL RAK-ADME (2.1mm×50mm, 3μm) column was used for chromatographic separation, and acetonitrile-water (containing 0.1% formic acid) was the mobile phase. The separation used gradient elution with a flow rate of 0.4mL/min. The column temperature was 40°C, and the sample volume was 1μL. The electrospray ionization source (ESI) and the positive ion multiple reaction monitoring (MRM) mode were used for the detection. The analysis time was as short as 7min. The results show a good linear relationship under optimized conditions in the range of 5.00-7.50×103, 1.00-1.50×103, 5.00-5.00×104, 5.00-7.50×103, 1.00-3.00×103, 1.00×101-1.00×104, 1.00-3.00×103, 1.00-3.00×103, 2.00-4.00×103, and 1.00×10-1-2.00×102 ng/mL for INH, EMB PZA, RIF, rifabutin, rifapentine, ethionamide, protionamide, thiosemicarbazone, and clofazimine, respectively, with a linear correlation coefficient of R>0.99. Finally, 34 patients with pulmonary TB were tested for therapeutic drug monitoring. The results showed that the presented method have significant advances in sensitivity, separation efficiency and simplicity.