A sensitive quantitative assay for the determination of propafenone and two metabolites, 5-hydroxypropafenone and N-depropylpropafenone, in human K2EDTA plasma using LC-MS/MS with ESI operated in positive mode

摘要

Propafenone is a potent antiarrhythmic agent; clinically propafenone has been used for a number of cardiac arrhythmias because it possesses multiple modes of action, via beta adrenergic receptor blockade and calcium antagonistic activity. Propafenone (PPF) exhibits extensive saturable presystemic biotransformation (first-pass effect) resulting in two active metabolites: 5-hydroxypropafenone (5-OH PPF) formed by CYP2D6 and N-depropylpropafenone (NDP) formed by both CYP3A4 and CYP1A2 enzymes. A specific and sensitive LC-MS/MS method was developed and validated for quantitation of PPF, 5-OH PPF and NDP using turboion spray in a positive ion mode. A solid-phase extraction was employed for the extraction from human plasma. Chromatographic separation of analytes was achieved using an ACE-5 C-8 (50x4.6mm) column with a gradient mobile phase comprising ammonium acetate containing 0.01% TFA in purified water and acetonitrile. The retention times achieved were 1.36, 1.23, 1.24min and 1.34min for PPF, 5-OH PPF, NDP and IS (carbamazepine), respectively. Quantitation was performed by monitoring multiple reaction monitoring transition pairs of m/z 342.30 to m/z 116.20, m/z 358.30 to m/z 116.20, m/z 300.30 to m/z 74.20 and m/z 237.20 to m/z 194.10, respectively. The developed method was validated for various parameters. The calibration curves of PPF and 5-OH PPF showed linearity from 1 to 500ng/mL, with a lower limit of quantitation of 1.0ng/mL and for NDP linearity from 0.1 to 25ng/mL with a lower limit of quantitation of 0.1ng/mL. The bias and precision for intra- and-inter batch assays were <10 and 5%, respectively. The developed assay was used to evaluate pharmacokinetic properties of propafenone and its major metabolites in healthy human subjects.