Determination Of Isoniazid, Ethambutol And Fudosteine In Plasma By Liquid Chromatographic-tandem Mass Spectrometry

Objective: To develop and validate rapid, sensitive and specific methods for quantitative analyses of isoniazid, ethambutol and fudosteine in plasma by liquid chromatographic-tandem mass spectrometry and be applied to pharmacokinetic studies.Method: After a simple protein precipitation using methanol, isoniazid, ethambutol and the internal standard metformin were chromatographed on an Atlantis? dC₁₈ column and detected by MS/MS with an atmospheric pressure chemical ionization interface in a Thermo Finnigan TSQ Ultra tandem mass spectrometer. Selected reaction monitoring (SRM) using the precursor → product ion combination of m/z 138→ m/z 79, m/z 206 → m/z 116 and m/z 130→m/z 60 was used to quantify isoniazid, ethambutol and the internal standard, respectively. Fudosteine was prepared from plasma samples by protein precipitation, separated on a Zorbax SB C₁₈ column. The mobile phase consisted of acetonitrile-2 mM ammonium acetate (15: 85, v/v), at a flow rate of 0.50 ml/min. An MDS SCIEX API 4000 triple quadrupole mass spectrometer, equipped with a Turbo IonSpray interface, was operated in the negative ion mode. Multiple reaction monitoring (MRM) using the precursor → product ion combination of m/z 178 → m/z 91, and m/z 364 → m/z 223 was used to quantify fudosteine and the internal standard, respectively.Results: The linear calibration curves were obtained in the concentration range of 10.0-5000 ng/mL for both isoniazid and ethambutol. The intra- and inter-run precisions, expressed as the relative standard deviation (RSD), were less than 5.1% and 6.4%, determined from QC samples for isoniazid and ethambutol, and accuracy was within ±2.1% and ± 4.5% in terms of relative error, respectively. The standard calibration curve for fudosteine was linear in the concentration range of 0.020-20.0 μg/mL in human plasma. The intra- and inter-run precisions were less than 5.6% and 5.4%,...