Simultaneous determination of isoniazid, rifampicin, ethambutol, pyrazinamide and levofloxacin in human plasma by HPLC-MS/MS

ZHAO Guan-ren; PENG Ming-li; SHEN Jian; LUAN Xiao-xiao; XI Na

Abstract：Objective:To establish a method for the simultaneous determination of isoniazid, rifampicin, ethambutol, pyrazinamide and levofloxacin in human plasma by HPLC-MS/MS, and provide the convenience about blood drug monitoring of pulmonary tuberculosis patients.Methods:Acetaminophen was used as internal standard. The proteins of plasma samples were precipitated with methanol. The supernatant fluid was separated on the Agilent Poroshell 120SB C18 column (4.6 mm × 50 mm, 2.7μm) and guarded by the ZORBAX SB C18 column (2.1 mm × 12.5 mm, 5 μm). The mobile phase consisted of 0.2% of methanol and 0.2% of acetic acid in gradient elution. Electrospray ionization (ESI) source was applied and operated in the positive multiple reaction monitoring (MRM) mode. The transition ofm/z was 138.0→121.0 for isoniazid, 823.2→791.2 for rifampicin, 205.2→116.1 for ethambutol, 124.1→79.0 for pyrazinamide, 362.2→261.1 for levofloxacin and 152.0→110.0 for acetaminophen. Each analysis was completed within 12 minutes.Results:Chromatograms showed no endogenous interfering peaks with blank samples. The linear ranges were 0.20– 9.81 μg·mL-1 for isoniazid, 0.60– 30.12 μg·mL-1 for rifampicin, 0.10– 4.89 μg·mL-1 for ethambutol, 1.01– 50.60μg·mL-1 for pyrazinamide, 0.10– 5.00 μg·mL-1 for levofloxacin respectively. The intra-day and inter-day precision (RSDs) were less than 10%. The accuracy (RE) was less than 10%.Conclusion:The method is specific, sensitive and accurate, and proved to be suitable for the simultaneous determination of isoniazid, rifampicin, ethambutol, pyrazinamide and levofloxacin in human plasma from clinical plasma samples.