Simultaneous Determination of Isoniazid, Pyrazinamide and Rifampin in Human Plasma by High-performance Liquid Chromatography and UV Detection

Therapeutic Drug Monitoring (TDM) of first-line anti-tuberculosis (TB) drugs is a decisive tool, allowing the clinician to successfully treat TB patients. The objective of the study was to develop and optimize a simple, sensitive, and reliable high-performance liquid chromatography (HPLC) method for the simultaneous determination of isoniazid (INH), pyrazinamide (PZA), and rifampin (RIF) levels in human plasma. Nicotinamide was used as the internal standard and the samples were prepared after protein precipitation using acetonitrile and zinc sulfate. The separation was achieved using a C18 reversed-phase applying gradient elution. The mobile phase was a combination of water–methanol solution with a ratio of 95:05 (v/v) at the initial phase. All calibration curves had good linearity (r(2 )> 0.99) and the inter- and intra-day RSDs were lower than 15%. The limit of detection with a signal-to-noise ratio (S/N) of 3:1 was 0.16, 0.5, and 0.33 μg mL(–1) for INH, PZA, and RIF, respectively. The method presented here was selective, sensitive, and reproducible, and could be used for‌ therapeutic drug monitoring in the patients who were under treatment with these drugs.