Determination of clozapine, and its metabolites, N-desmethylclozapine and clozapine N-oxide in dog plasma using high-performance liquid chromatography

J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Jan 15;783(2):377-82. doi: 10.1016/s1570-0232(02)00655-4.


Clozapine and its two major metabolites, N-desmethylclozapine and clozapine N-oxide were quantified using a high-performance liquid chromatographic method with UV detection in dog plasma following a single dose of clozapine. The analysis was performed on a 5-micrometer Hypersil CN (CPS-1; 250x4.6 mm) column. The mobile phase consisted of acetonitrile-water-1 M ammonium acetate (50:49:1, v/v/v), which was adjusted to pH 5.0 with acetic acid. The detection wavelength was 254 nm. A liquid-liquid extraction technique was used to extract clozapine and its metabolites from dog plasma. The recovery rates for clozapine, N-desmethylclozapine, and the internal standard (I.S.) were close to 100% using this method. The recovery rate for clozapine N-oxide (62-66%) was lower as expected because it is more polar. The quantitation limits for clozapine, clozapine N-oxide, and N-desmethylclozapine were 0.11, 0.05 and 0.05 microM, respectively. Intra-day reproducibility for concentrations of 0.1, 1.0 and 5.0 microM were 10.0, 4.4 and 4.2%, respectively, for N-oxide; 11.2, 4.3 and 4.9%, respectively, for N-desmethylclozapine; and 10.8, 2.2 and 4.9%, respectively, for clozapine. Inter-day reproducibility was <15% for clozapine N-oxide, <8% for N-desmethylclozapine and <19% for clozapine. This simple method was applied to determine the plasma concentration profiles of clozapine, N-desmethylclozapine and clozapine N-oxide in dog following administration of a 10 mg/kg oral dose of clozapine.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Chromatography, High Pressure Liquid / methods*
  • Clozapine / analogs & derivatives*
  • Clozapine / blood
  • Clozapine / pharmacokinetics*
  • Dogs
  • Reference Standards
  • Reproducibility of Results
  • Sensitivity and Specificity


  • norclozapine
  • Clozapine
  • clozapine N-oxide