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

Abstract

SummaryAn HPLC method with electrochemical detection has been developed for the determination ofclozapine and its main metabolites, desmethylclozapine and clozapine Noxide, in human plasma. An accurate pretreatment of the biological samples was implemented by means of solid phase extraction (SPE) on HLB cartridges. This improved pretreatment, together with a new mobile phase, allows for the accurate determination of clozapine Noxide, which could not be quantitated by a previous method. The method uses only 100 #L of plasma for one complete analysis and shows good recovery values for all three analytes. The eluates from the SPE procedure were chromatographed in a reversed phase C 18 column using a mobile phase composed of phosphate buffer, acetonitrile and methanol. Clozapine, desmethylclozapine and clozapine N-oxide were eluted in less than 10 minutes, without any interference from the biological matrix. Linearity was observed over the 2.50 -150ng mL 1 (clozapine and desmethylclozapine) or 1.25 -75 ng mL 1 (clozapine N-oxide) range for the three analytes, with satisfactory repeatability values. The limit of detection 1 was 0.3 ng mL-for clozapine and desmethylclozapine, and 0.6 for clozapine N-oxide. The application to plasma samples of patients treated with Leponex gave good results. No interference from other common central nervous system drugs was found. This method seems to be a useful tool for pharmacokinetic studies and for clinical monitoring, because of its need for small plasma samples and its high sensitivity and selectivity.

High throughput therapeutic drug monitoring of clozapine and metabolites in serum by on-line coupling of solid phase extraction with liquid chromatography–mass spectrometry

“…This result is in agreement with the limited accuracy and precision obtained for this compound at this concentration level. The high recovery percentages obtained are in accordance with literature data for most other methods where sample preparation is performed over a reversed phase extraction material [14,16,17,23,26,29].…”

“…Though amperometric detection is more sensitive and selective than UV detection, the performance of MS detection, which is intrinsically highly selective, can be even better in this respect. Though, detection limits published by Aravagiri and Marder for a method using tandem MS [25] and Kollroser and Schober for a method using ion trap MS 2 [29] are not as good as those presented by Raggi et al for a method using amperometric detection [23].…”

“…In accordance with the majority of literature dealing with monitoring of CLZ and metabolites, a reversed phase (RP) chromatographic system, utilising a C18 stationary phase [14,[16][17][18]20,23,[24][25][26][27][28][29], has been chosen to separate CLZ, DMC and NOX. Other RP stationary phases reported in literature include C6 [21], C8 [17,20] and cyanopropyl (operated under RP conditions) [19,30].…”

“…Amperometric detection of CLZ and metabolites has been suggested by Raggi et al [23] and, more recently, mass spectrometric detection bas been applied [25,29]. Though amperometric detection is more sensitive and selective than UV detection, the performance of MS detection, which is intrinsically highly selective, can be even better in this respect.…”

“…Over the last decade, several liquid chromatographic methods for monitoring CLZ (and metabolites) in serum and/or plasma have been published. Sample clean-up is performed either by LLE [18,[20][21][22]25,28,30], or by SPE, off-line [14,16,17,23,26,27], or on-line [15,19,24,29] with LC separation. In the case of on-line SPE, a cyanopropyl [15,24], C18 [19], or divinylbenzene co-polymer resin SPE stationary phase [29] is used.…”