Development and validation of an HPLC method for the simultaneous determination of clozapine and desmethylclozapine in plasma of schizophrenic patients

Abstract

SummaryA high-performance liquid chromatographic method with amperometric detection has been developed for the determination of levels of clozapine (CLZ) and its active metabolite N-desmethylclozapine (DMC) in human plasma. The analysis was performed on a 5 ~m C8 reversed phase column (150 x 4.6 mm i.d.), with acetonitrile-phosphate buffer (pH 3.5), as the mobile phase. The detection voltage was +800 mV and the cell and column temperature were 50 ~ Linear responses were obtained between 2 ng mL -1 and 100 ng mL 1. Absolute recovery for both clozapine and desmethylclozapine exceeded 88 % and the detection limit was 1 ng mL -1. Repeatability, intermediate precision and accuracy were satisfactory. The method, which is rapid, sensitive and selective, has been applied to therapeutic drug monitoring in schizophrenic patients following administration of Leponex | tablets. In 21 patients in steady state at a mean daily clozapine dosage of 358 mg (ranging from 150 to 500 mg day-l), clozapine levels averaged 379 ng mL -~ (ranging from 102 to 818 ng mL -1) and DMC levels averaged 233 ng mL -1 (ranging fi'om 70 to 540 ng mL-1). The method requires only a very small amount of plasma (100 ~L), and thus it is suitable for pharmacokinetic studies, as well as for therapeutic drug monitoring.

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

“…The advantages of the method are numerous: -The high sensitivity, due to the electrochemical detector and the high extraction yield, is highlighted by 1 the values of LOD obtained (0.3 ng mL-for CLZ and DMC, 0.6 ng mL -1 for CLZ-NO). These values are lower than those reported in our previous paper [27] and by other authors [6,18,20,22,25]. -Good selectivity: about 30 drugs active on the central nervous system were examined, and none of them caused any interference; -Satisfactory precision and good accuracy; -A rapid and reliable SPE procedure for the pretreatment of plasma samples.…”

“…The SPE procedures proposed by other authors [21 ] are laborious and do not always give satisfactory yields. Our previously published SPE method [27] for the analysis of CLZ and DMC, based on the use of C8 BondElut cartridges, gave good extraction yields for those two analytes but not for CLZ-NO. Thus, it has been modified to obtain a satisfactory quantification of this analyte as well.…”

“…Few papers simultaneously determine CLZ together with its main metabolites [6,18,22,25]. An HPLC method with electrochemical detection for the determination of CLZ [26], followed by a modified method for the determination of CLZ and DMC [27] have recently been developed by us. Both methods use solid phase extraction (SPE) as the sample pretreatment, and reach detection limits (LOD) of 1.5 ngmL -1 for both CLZ and DMC.…”

Determination of the novel antipsychotic drug olanzapine in human plasma using HPLC with amperometric detection

“…Our previous work on clozapine analysis in the plasma of schizophrenic patients [16] gave us a starting point for the development of this method. The column temperature was kept at 50 ~ and, using the same type of stationary phase (reversed phase C8 column, 150x4.6 mm, 5 Hm) and the same mobile phase (acetonitrile and pH 2.8, 1.35x10 2 mol L 1 aqueous K2HPO4, 40:60 v/v, pH* 3.5), olanzapine was detected as a chromatographic peak at retention time (ta) = 2.5 minutes at an oxidation potential of + 0.550 V (as directed by the previous voltammetric assays).…”

“…In order to apply this method to human plasma samples, a reliable pretreatment of the biological samples was used based on an SPE procedure already developed for the analysis of clozapine in human plasma [16] with minor modifications. A small quantity of plasma sample (250 #L) was loaded onto C8 cartridges and, after suitable washing, olanzapine was eluted by means of 1 mL of mobile phase (i.e.…”