Analysis of Clozapine and Norclozapine in Plasma using Automated On-line Sample Preparation and LC–MS/MS

Clozapine is a dibenzodiazepine drug used in the treatment of schizophrenia. It is uniquely effective in patients resistant to therapy with other antipsychotics. In addition to mandatory haematological monitoring to minimize the risk of agranulocytosis, there is a large variation (50-fold) in clozapine dose requirement that complicates dose assessment. Clozapine is metabolized via N-demethylation, N-oxidation, and aromatic hydroxylation, amongst other pathways. N-Desmethylclozapine [norclozapine], accumulates in plasma to concentrations similar to those of clozapine. Clozapine N‑oxide accumulates in plasma to approximately 10% of the plasma clozapine concentration but is not measured routinely. Changes in smoking habit can have a large effect on clozapine dose requirement. On average the clozapine dose for non-smokers is half that required for smokers due to induction of the cytochrome P450 enzyme in CYP1A2 smokers. Accurate measurement of plasma clozapine and norclozapine concentrations may help understand issues related to dose optimization and adherence. Current methodology in our research laboratory for plasma clozapine and norclozapine measurement involves off-line liquid-liquid extraction (LLE) into methyl tert-butyl ether at pH 10.6 with manual transfer of the extract to an HPLC-UV system. Clozapine N-oxide is not extracted.