Simultaneous determination of clozapine and its N-desmethyl and N-oxide metabolites in plasma by liquid chromatography/electrospray tandem mass spectrometry and its application to plasma level monitoring in schizophrenic patients.
A liquid chromatography tandem mass spectrometry (LC-MS-MS) assay method for the simultaneous determination of clozapine and its N-desmethyl (norclozapine) and N-oxide metabolites in human plasma is described. The compounds were extracted from plasma by a single step liquid-liquid extraction procedure and analyzed using a high performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a C-18 column, ionized using positive ion atmospheric pressure electrospray ionization method by a TurboIonspray source and analyzed using multiple reaction monitoring mode. The ion transitions monitored were m/z 327 --> m/z 270 for clozapine, m/z 313 --> m/z 192 for norclozapine, m/z 343 --> m/z 256 for clozapine-N-oxide and m/z 421--> m/z 201 for internal standard. The standard curves of clozapine, norclozapine and clozapine-N-oxide were linear over the range of 1 ng/ml to 1000 ng/ml when 0.5 ml of plasma was used for the analysis (r(2) >0.998). Three pooled plasma samples collected from patients who were treated with clozapine were used as long-term quality control samples to check the validity of spiked standard curve samples made at various times. The intra- and inter-assay variations for the spiked standard curve and quality control samples were less than 14%. These variations for the long-term patient quality control samples were less than 11%. The LC-MS-MS assay for simultaneous determination of clozapine, norclozapine and clozapine-N-oxide reported here is highly specific, sensitive, accurate and rapid. This method is currently being used for the plasma level monitoring of clozapine and its N-desmethyl and N-oxide metabolites in patients treated with clozapine. The plasma levels of clozapine, norclozapine and clozapine-N-oxide varied widely within and among patients. The data revealed that the norclozapine and clozapine N-oxide metabolites were present at about 58%+/-14% and 17%+/-6% of clozapine concentrations in plasma, respectively.
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Psychopharmacology Unit, Room 4 (B151H) Building 210, VA Greater Los Angeles Healthcare System, 11301 Wilshire Blvd, Los Angeles, CA 90073, USA. kannan@ucla.edu
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D014150:Antipsychotic Agents / Q000097:blood* / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods; D003024:Clozapine / Q000031:analogs & derivatives / Q000097:blood* / Q000737:chemistry; D016903:Drug Monitoring / Q000379:methods; D006801:Humans; D015203:Reproducibility of Results; D012559:Schizophrenia / Q000097:blood*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods
D014150:Antipsychotic Agents; C058272:norclozapine; D003024:Clozapine; C079149:clozapine N-oxide