An improved HPLC-ED method for monitoring plasma levels of clozapine and its active metabolites in schizophrenic patients

An HPLC method with electrochemical detection has been developed for the de termination of clozapine and its main metabolites, desmethylclozapine and c lozapine N-oxide, in human plasma. An accurate pretreatment of the biologic al samples was implemented by means of solid phase extraction (SPE) on HLB cartridges. This improved pretreatment, together with a new mobile phase, a llows for the accurate determination of clozapine N-oxide, which could not be quantitated by a previous method. The method uses only 100 mu L of plasm a for one complete analysis and shows good recovery values for all three an alytes. The eluates from the SPE procedure were chromatographed in a revers ed phase C18 column using a mobile phase composed of phosphate buffer, acet onitrile and methanol. Clozapine, desmethylclozapine and clozapine N-oxide were eluted in less than 10 minutes, without any interference from the biol ogical matrix. Linearity was observed over the 2.50 - 150 ng mL(-1) (clozap ine and desmethylclozapine) or 1.25 - 75 ng mL(-1) (clozapine N-oxide) rang e for the three analytes, with satisfactory repeatability values. The limit of detection was 0.3 ng mL(-1) for clozapine and desmethylclozapine, and 0 .6 for clozapine N-oxide. The application to plasma samples of patients tre ated with Leponex gave good results, No interference from other common cent ral nervous system drugs was found. This method seems to be a useful tool f or pharmacokinetic studies and for clinical monitoring, because of its need for small plasma samples and its high sensitivity and selectivity.