Rapid and Sensitive LC-MS/MS Method for Simultaneous Determination of Three First-Line Oral Antituberculosis Drug in...

A bioanalytical method for simultaneous quantification of isoniazid (INH), pyrazinamide (PZA) and ethambutol (EMB) in plasma was developed and validated using high-performance liquid chromatography with tandem mass spectrometry. After extracted by protein precipitation with acetonitrile, the analytes were separated on a Waters XBridge Amide column by isocratic elution with acetonitrile and 5 mM ammonium acetate solution containing 0.3% formic acid (77:23, v/v) at a flow rate of 0.5 mL/min. The detection was carried out on a triple quadrupole tandem mass spectrometer equipped with an electrospray ionization source in positive mode by monitoring the selected ion transitions at m/z 205.2 → 116.1, m/z 137.9 → 121.2, m/z 124.3 → 78.9 and m/z 213.1 → 122.4 for EMB, INH, PZA and EMB-d8 Internal standard (IS), respectively. The calibration curves were linear over the range of 0.0125-2.00 μg/mL for EMB, 0.0625-10.0 μg/mL for INH and 0.250-40.0 μg/mL for PZA. Neither cross-analytes inter-conversion nor matrix effects were observed. The intra- and inter-assay precision (%RSD) values were within 8.80%, and accuracy (%RE) ranged from -11.13 to 13.49%, indicating that the precision and accuracy were well within the acceptable limits of variation. The method would be helpful for analysis of EMB, INH and PZA in plasma samples from clinical pharmacokinetics and therapeutic drug monitoring.