Field-amplified sample stacking in capillary electrophoresis for the determination of clozapine, clozapine N-oxide, and desmethylclozapine in schizophrenics’ plasma

A method of field-amplified sample stacking in capillary electrophoresis is described for the simultaneous determination of clozapine (CZP) and its metabolites, clozapine N-oxide (CNO), and desmethylclozapine (DMC), in human plasma. Plasma (0.2 mL) was extracted with organic solvents (ethyl acetate/n-hexane/isopropyl alcohol, 8/1/1 by volume) and centrifuged. An aliquot of supernatant was evaporated and suitably reconstituted with water for CE analysis. An untreated fused-silica capillary was used (31.2 cm; effective length, 20 cm; 50 μm i.d.) for the analysis. The background buffer was phosphate buffer (400 mM, pH 3.0) containing 50% ethylene glycol. The separation voltage was 25 kV with a detection wavelength of 214 nm. In the method validation, the calibration curves were linear (r ≧ 0.98) over a range of 50–800 ng/mL for CZP, 30–180 ng/mL for CNO, and 25–600 ng/mL for DMC. The relative standard deviation (R.S.D.) and relative error (R.E.) were all less than 11% for the intra- and inter-day assays. The limits of detection (S/N = 3, electric-driven injection, 99.9 s) of CZP, DMC, and CNO were 5, 5, and 10 ng/mL, respectively. After continuing treatment with the CZP tablets, a blood sample from one male schizophrenic patient (41-year-old, 62 kg) who had been receiving ongoing treatment with the CZP tablets was prepared and analyzed. The levels of CZP, DMC, and CNO were determined and the feasibility of the method’s application in clinical treatment was proven.