On-line fully automated determination of clozapine and desmethylclozapine in human serum by solid-phase extraction on exchangeable cartridges and liquid chromatography using a methanol buffer mobile phase on unmodified silica.

A fully automated method for determination of clozapine and desmethylclozapine in human serum using high-performance liquid chromatography was developed. On-line solid-phase extraction was performed on an exchangeable cyanopropyl cartridge. The analytes were eluted with a methanol-ammonium acetate buffer mobile phase, separated on a silica column, and measured by ultraviolet detection at 261 nm. The total time for one analysis was 13 min. Inter-day variation was < 6% and < 8% for clozapine and desmethylclozapine, respectively. Detector response was linear in the range of 30-300 ng/ml. Comparison with liquid-liquid extraction showed good agreement. The patients had clozapine serum concentrations in the range 40-1500 ng/ml. Desmethylclozapine concentrations were 25% lower and closely related.