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 fo r the simultaneous determination of clozapine and its N-desmethyl (norcloza pine) and N-oxide metabolites in human plasma is described, The compounds w ere extracted from plasma by a single step liquid-liquid extraction procedu re and analyzed using a high performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically o n a C-18 column. ionized using positive ion atmospheric pressure electrospr ay 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. Th e standard curves of clozapine, norclozapine and clozapine-N-oxide were lin ear 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 co ntrol samples to check the validity of spiked standard curve samples made a t various times. The intra- and inter-assay variations for the spiked stand ard curve and quality control samples were less than 14%. These variations for the long-term patient quality control samples were less than 11%. The L C-MS-MS assay for simultaneous determination of clozapine, norclozapine and clozapine-N-oxide reported here is highly specific. sensitive, accurate an d rapid. This method is currently being used for the plasma level monitorin g of clozapine and its N-desmethyl and N-oxide metabolites in patients trea ted with clozapine. The plasma levels of clozapine. norclozapine and clozap ine-N-oxide varied widely within and among patients. The data revealed that the norclozapine and clozapine N-oxide metabolites were present at about 5 8% +/- 14% and 17% +/- 6% of clozapine concentrations in plasma. respective ly. (C) 2001 Elsevier Science B.V. All rights reserved.