Abstract

VALIDATION OF AN LC-MS/MS METHOD FOR THE RIFAMPICIN (SPIKED WITH ETHAMBUTOL, ISONIAZID AND PYRAZINAMIDE) QUANTIFICATION IN HUMAN PLASMA

Abdullah Al Zou’bi, Dipal Patel, Mohammad Farhan, Mahmoud Mustafa, Meet Shah, Mosab Rawashdeh, Dr. Mohammed Abufara and Dr. Rabab Tayyem*

ABSTRACT

A sensitive, rapid and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) analytical method based on liquid- liquid extraction method was developed and validated for analysis of Rifampicin (RIF) spiked with Ethambutol (EMB), Isoniazid (INH) and Pyrazinamide (PZA) in human plasma. Monitoring of anti-tuberculosis drug concentrations in human plasma is very important in the treatment of TB disease. Here we described a method that can rapidly measure the plasma concentrations of Rifampicin (spiked with Ethambutol, Isoniazid and Pyrazinamide) using LC-MS/MS. To adjust for degradation and losses during sample preparation IS mix solution of Rifampicin-d4, Isoniazid-d4, Ethambutol-d4, Pyrazinamide-d3 were used as an internal standard. Rifampicin drug was separated by using the high-performance liquid chromatography system with Agilent Eclipse XDB CN (150*4.6 mm, 5.0 μm) and a mobile phase consisting of acetonitrile with ammonium acetate buffer (20 mM) in a ratio of 70:30 (V/V), 300 μl diluted ammonia solution (25%) was added per one litre of mobile phase. The method used simple isocratic chromatographic condition and mass spectrometric detection in the positive ionization mode. The method was found to be highly selective. Precision estimated by the coefficient of variation was <15%. The linear range of the calibration curve for RIF 0.100–10.000 μg/mL spiked with EMB 0.100–10.000 μg/mL; INH 0.100–10.000 μg/mL and PZA 1.0–100 μg/mL. The lower limit of quantification was 0.100 μg/mL. The results of specificity/selectivity, precision, accuracy, linearity, limit of quantification, recovery, and stability show that the method meets the regulatory requirements of a bioanalytical method validation.

Keywords: Anti TB, Rifampicin, Isoniazid, ethambutol, pyrazinamide, LC-MS/MS, Human Plasma, Liquid chromatography; Tandem mass spectroscopy; Pharmacokinetic study; Bioequivalence study.