title: Therapeutic drug monitoring of clozapine and norclozapine in human serum using ultra-performance liquid chromatography- tandem mass spectrometry.
abstract: A rapid, sensitive, and specific method was developed and validated using ultra-performance liquid chromatography- tandem mass spectrometry (UPLC-MS-MS) for simultaneous determination of clozapine and its major metabolite norclozapine in human serum. The compounds were extracted from serum by a single step protein precipitation and analyzed using a UPLC-triple-quadrupole detection (TQD) system. Separation of compounds was achieved on a BEH C18 (50 mm x 2.1 mm, 1.7 microm) analytical column using methanol and water (both containing 0.2% ammonium hydroxide) as the mobile phase at a flow rate of 0.40 mL/min. The compounds were ionized in the electrospray ionization ion source of the TQD and were detected in the multiple reaction monitoring (MRM) mode. The MRM transitions m/z 327 --> 270 and m/z 313 --> 192 for clozapine and norclozapine, respectively, were used for the quantification ions. Clozapine transition 327 --> 192 and norclozapine transition 313 --> 270 were used as confirmation ions. Linear calibration curves in human serum were generated over the range of 10-2000 ng/mL for both clozapine and norclozapine with a correlation coefficient (r(2)) > 0.9970. Calibration curves exhibited consistent linearity and reproducibility. Interassay coefficients of variation (CV) (n = 20) were 3.04-4.94% for clozapine and 2.84-6.07% for norclozapine. Intra-assay CVs (n = 6, 20 days) were 0.61-1.26% and 1.62-2.21% for clozapine and norclozapine, respectively. The extraction recoveries were larger than 95% for both clozapine and norclozapine. The method was applied to the quantification of clozapine and norclozapine in the sera of schizophrenic patients, and the data revealed that the concentrations of two compounds varied significantly in the patients treated with clozapine.
authors.affiliation: Department of Chemistry, BC Biomedical Laboratories, Inc., Surrey, British Columbia, Canada, V3W 1H8. DMing@bcbio.com
authors.forename: Dong Sheng
authors.initials: DS
authors.lastname: Ming
authors.forename: John
authors.initials: J
authors.lastname: Heathcote
mesh_terms: D014150:Antipsychotic Agents / Q000097:blood* / Q000627:therapeutic use; D001711:Biotransformation; D002138:Calibration; D002853:Chromatography, Liquid / Q000592:standards; D003024:Clozapine / Q000031:analogs & derivatives* / Q000097:blood / Q000627:therapeutic use; D016903:Drug Monitoring / Q000379:methods* / Q000592:standards; D004355:Drug Stability; D006801:Humans; D015203:Reproducibility of Results; D012559:Schizophrenia / Q000097:blood* / Q000188:drug therapy; D021241:Spectrometry, Mass, Electrospray Ionization / Q000592:standards; D053719:Tandem Mass Spectrometry / Q000592:standards; D013696:Temperature; D013997:Time Factors
chemical_list: D014150:Antipsychotic Agents; C058272:norclozapine; D003024:Clozapine