LC-MS-MS Determination and Pharmacokinetic Study of Clozapine in Human Plasma

Abstract

A sensitive and selective liquid chromatographic method coupled with tandem mass spectrometry was established and validated for the determination and pharmacokinetic study of clozapine in human plasma. Ethyl acetate extraction was used for plasma sample preparation with mirtazapine as internal standard. Chromatographic separation was achieved on a Hanbon Kromasil C18 (250 mm × 4.6 mm, 5 μm) column by isocratic elution with a mixture of 70 volumes of methanol and 30 volumes of water containing 0.2% ammonium acetate and 0.1% formic acid as mobile phase delivered at 1.0 mL min−1. The MS-MS detection was carried out on a tandem mass spectrometer using positive electrospray ionization and multiple reaction monitoring with argon for collision-induced dissociation. The ion transitions were monitored as follows: m/z 327 to m/z 270 for clozapine and m/z 266 to m/z 195 for the internal standard (mirtazapine), respectively. Calibration curves were generated over the concentration range from 0.10 to 200 ng mL−1 with the lower limit of quantification of 0.10 ng mL−1, and two segments of linear calibration curves were established by regressing in the way of least-square in the range from 0.10 to 5.0 and 5.0 to 200 ng mL−1, respectively. The intra- and inter-day precision and accuracy were determined at three different concentration levels, 0.20, 10.0 and 100 ng mL−1, and were all better than 15% (n = 5). This specific and sensitive liquid chromatography coupled with tandem mass spectrometry has been successfully applied to a pharmacokinetic study of clozapine after a single oral dose of 25 mg in healthy Chinese volunteers.