Bioanalytical Method Development for Simultaneous Estimation of Standard First-Line Anti-Tuberculosis Drugs in Human Plasma Using Liquid Chromatography-Tandem Mass Spectrometry

Standardized tuberculosis treatment involves four major drugs: rifampicin, isoniazid, pyrazinamide, and ethambutol. Suboptimal concentrations of these drugs are one of the reasons for treatment failure, relapse, or resistance to the disease, though most patients respond to treatment. It is necessary to monitor the concentrations of these drugs and make the required dose adjustments to achieve the desired therapeutic effect. We developed and validated a simple and practical LC–MS/MS method for the simultaneous estimation of these four main drugs. A single-step protein precipitation technique was employed, using methanol as the precipitating agent to extract the drugs from the plasma sample. Chromatographic separation was achieved on a Phenomenex Luna® C18 column (150 × 4.6 mm, 3 µm) using a mobile phase composed of 0.1% formic acid in water and methanol, following isocratic elution. The flow rate was fixed at 0.7 mLmin −1 , and the run time was 5 min. Quantification was performed in ESI (+ ve) mode. The method demonstrated good linearity, with R 2 between 0.9912 and 0.9969. The mean extraction recoveries for all analytes ranged from 85.84% to 97.49%, and they were reproducible and consistent; no significant matrix effects were observed. Stability studies: bench-top (5 h), autosampler (26 h), freeze–thaw (3 cycles), and long-term stability in plasma (10 days) met the % CV acceptance criteria of ± 15%. The developed LC–MS/MS method was successfully applied to a healthy volunteer study administered with these drugs.