Simultaneous determination of clozapine, norclozapine and clozapine-N-oxide in human plasma by high-performance liquid chromatography with ultraviolet detection

• Published in: Biomedical chromatography 2001-06, Vol.15 (4), p.280-286
• Main Authors: Liu, Y. Y., van Troostwijk, L. J. A. E. Doude, Guchelaar, H.-J.
• Format: Article
• Language: English
• Subjects: Chromatography, High Pressure Liquid - methods; Clozapine - analogs & derivatives; Clozapine - blood; Humans; Reproducibility of Results; Sensitivity and Specificity; Spectrophotometry, Ultraviolet - methods
• ISSN: 0269-3879; 1099-0801
• DOI: 10.1002/bmc.73

A reversed‐phase high‐performance liquid chromatographic (HPLC) method for the simultaneous determination of clozapine and its two major metabolites, norclozapine and clozapine‐N‐oxide in human plasma has been developed and validated. The isocratic HPLC assay uses a mobile phase consisting of an acetonitril‐buffered aqueous solution containing 146 µL of triethylamine and 200 µL of 85% phosphoric acid, adjusted to pH 3.3 with 10% potassiumhydroxide solution (400:600, v/v) at a flow‐rate of 0.8 ml/min and a Lichrospher 100 RP‐18 reversed‐phase column and UV detection at 215 nm. Doxepine was used as the internal standard. Mean recoveries for clozapine, norclozapine, clozapine‐N‐oxide and doxepine were 95%, 98%, 96% and 94%, respectively, whereas the respective mean repeatability coefficients of variation were 3.4%, 2.7%, 4.3% and 0.9%. Reproducibility coefficients of variation were 1.3%, 1.8%, 3.6% and 0.5%, respectively. The mean correlation coefficient for the linear calibration curve (n = 2) for clozapine and norclozapine at a concentration range of 100–1600 ng/mL was 0.9998 and 0.9997, respectively; for clozapine‐N‐oxide (20–200 ng/mL) it was found to be 0.9986. The lower limits of quantitation were 12.5 ng/mL, 10 ng/mL and 12.5 ng/mL for clozapine, norclozapine and clozapine‐N‐oxide, respectively. Copyright © 2001 John Wiley & Sons, Ltd. Abbreviations used: LLQ lower limit of quantitation.