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Clozapine and its metabolites require close therapeutic monitoring (TDM) in patients due to poor correlation between the administrated doses and resulting plasma concentrations, the narrow therapeutic interval, high inter-individual variability, and the risk of serious side effects once toxic levels are exceeded. The aim of the study was to develop a simple (relatively cheap) LC-UV method for the quantification of clozapine and its metabolites in plasma and urine samples. For sample preparation, liquid-liquid extraction (LLE) in n -octanol was more efficient and less limiting in injection volumes compared to the in-situ formation of SUPRAS. When analyzing urine, an alkalinization step before extraction was required. The proposed method produced linear concentration responses with/without internal standard (IS) for the target analytes, with LLOQs within the targeted range of 50 ppb and %RSD within the acceptable 15% range. Furthermore, sample stability studies proved that pre-extracted samples were stable for the short term at room temperature and long-term when frozen.

Antipsychotics are the drugs most often involved in drug poisoning cases, and therefore, therapeutic drug monitoring (TDM) is necessary for safe and effective medication administration of these drugs. In this study, a coffee ring effect-based surface-enhanced Raman spectroscopy (CRE-SERS) method was developed and successfully used to monitor antipsychotic poisoning by using urine samples for the first time. The established method exhibited excellent SERS performance since more hot spots were obtained in the "coffee ring". Using the optimized CRE-SERS method, the sensitivity was improved one order more than that of the conventional method with reasonable reproducibility. The antipsychotic drug clozapine (CLO) spiked into urine samples at 0.5-50 μg mL -1 was quantitatively detected, at concentrations above the thresholds for toxicity. The CRE-SERS method allowed CLO and its metabolites to be ultimately distinguished from real poisoning urine samples. The coffee-ring effect would provide more opportunities for practical applications of the SERS-based method. The frequent occurrence of drug poisoning may have created a new area for the application of the CRE-SERS method. It is anticipated that the developed method will also have great potential for other drug poisoning monitoring., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In this study, conventional thin-film microextraction (TFME) was endowed with magnetic by introducing superparamagnetic SiO2@Fe3O4 nanoparticles in thin-films. Novel magnetic octadecylsilane (ODS)-polyacrylonitrile (PAN) thin-films were prepared by spraying, and used for the microextraction of quetiapine and clozapine in plasma and urine samples, followed by the detection of HPLC-UV. The influencing factors on the extraction efficiency of magnetic ODS-PAN TFME, including pH, extraction time, desorption solvent, desorption time, and ion strength were investigated systematically. Under the optimal conditions, both analytes showed good linearity over ranges of 0.070-9.000μgmL(-1) and 0.012-9.000μgmL(-1) in plasma and urine samples, respectively, with correlation coefficients (R(2)) above 0.9990. Limits of detection (LODs) for quetiapine in plasma and urine samples were 0.013 and 0.003μgmL(-1), respectively. LODs for clozapine in plasma and urine samples were 0.015 and 0.003μgmL(-1), respectively. The relative standard deviations (RSDs) for quetiapine and clozapine were less than 9.23%. After the validation, the protocol was successfully applied for the determination of quetiapine and clozapine in patients' plasma and urine samples with satisfactory recoveries between 99-110%. The proposed magnetic ODS-PAN TFME was very simple, fast and easy to handle. It showed high potential as a powerful pretreatment technology for routine therapeutic drug monitoring (TDM) in plasma and urine samples., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Sequential injection chromatography (SIC) is a recent, simple, and inexpensive green miniaturized separation technique. In the current study, SIC was exploited for the first time for biochemical analysis. A new SIC method for the separation and quantification of chlorpromazine in human urine, as well as pharmaceutical formulations, was developed and validated. Clozapine was used as an internal standard. Chlorpromazine was successfully separated on a C18 monolithic column (25x4.6 mm id). The UV detection was carried out at 250 nm using miniaturized fiber optic spectrometric devices. The optimum mobile phase composition was 30 mmol/L phosphate-acetonitrile-methanol (55.0 + 31.5 + 13.5, v/v/v) at pH 3.0. The sample volume was 40 microL and flow rate was 40 microL/s. Acceptable chromatographic results were obtained. The resolution was 2.7, peak symmetry was 1.1, and number of theoretical plates was more than 1 x 10(6). Good linearity (r = 0.9997) in the range of 25-100 microg/mL was also obtained. The method offered acceptable recovery for both human urine (89.6-93.1%) and pharmaceutical formulations (96.9-98.5%), which was sensitive enough to detect chlorpromazine. The LOD and LOQ in human urine were 61 and 204 ng/mL, respectively. The method was rapid and reagent-saving, and hence safe for the environment. The sample throughput was 26.3 samples/h and the total volume of consumed reagents was 4.0 mL.

The method for the determination of clozapine during chemical studies of cadaveric blood, urine, and liver (kidneys) has been developed based on the use of high performance liquid chromatography. Clozapine losses were estimated at 40-60% with the fractional uncertainty below 10-11% and the detection limit of 0.001 mg%. An algorithm for the estimation of the quality of clozapine isolation and the results of analysis of individual samples is proposed.

An LC-MS/MS method for the determination of the atypic neuroleptic clozapine and its two main metabolites norclozapine and clozapine-N-oxide has been developed and validated for serum and urine. After addition of d4-clozapine as deuterated internal standard a fast single-step liquid-liquid extraction under alkaline conditions and with ethyl acetate as organic solvent followed. The analytes were chromatographically separated on a Synergi Polar RP column using gradient elution with 1 mM ammonium formate and methanol. Data acquisition was performed on a QTrap 2000 tandem mass spectrometer in multiple reaction monitoring mode with positive electrospray ionization. Two transitions were monitored for each analyte in order to fulfill the established identification criteria. The validation included the determination of the limits of quantification (1.0 ng/mL for all analytes in serum and 2.0 ng/mL for all analytes in urine), assessment of matrix effects (77% to 92% in serum, 21 to 78% in urine) and the determination of extraction efficiencies (52% to 85% for serum, 59% to 88% for urine) and accuracy data. Imprecision was <10%, only the quantification of norclozapine in urine yielded higher relative standard deviations (11.2% and 15.7%). Bias values were below ±10%. Dilution of samples had no impact on the correctness for clozapine and norclozapine in both matrices and for clozapine-N-oxide in serum. For quantification of clozapine-N-oxide in urine a calibration with diluted calibrators has to be used. Calibration curves were measured from the LOQ up to 2,000 ng/mL and proved to be linear over the whole range with regression coefficients higher than 0.98. The method was finally applied to several clinical serum and urine samples and a cerebro-spinal fluid sample of an intoxicated 13-month-old girl.

A simple method has been proposed for the determination of clozapine (CLZ) and chlorpromazine (CPZ) in human urine by dispersive liquid-liquid microextraction (DLLME) in combination with high-performance liquid chromatography-ultraviolet detector (HPLC-UV). All important variables influencing the extraction efficiency, such as pH, types of the extraction solvent and the disperser solvent and their volume, ionic strength and centrifugation time were investigated and optimized. Under the optimal conditions, the limit of detection (LODs) and quantification (LOQs) of the method were 13 and 39 ng/mL for CLZ, and 2 and 6 ng/mL for CPZ, respectively. The relative standard deviations (RSDs) of the targets were less than 5.1% (C=0.100 μg/mL, n=9). Good linear behaviors over the tested concentration ranges were obtained with the values of R (2)>0.999 for the targets. The absolute extraction efficiencies of CLZ and CPZ from the spiked blank urine samples were 98.3% and 97.8%, respectively. The applicability of the technique was validated by analyzing urine samples and the mean recoveries for spiked urine samples ranged from 93.3% to 105.0%. The method was successfully applied for the determination of CLZ and CPZ in real human urine.

Liquid-liquid microextraction using the water immiscible ionic liquid, 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, EMIM NtfO₂, for the concentration and cleanup of basic compounds for analysis by CE has been investigated. Using an electrolyte comprising 1 mol/L alanine and 3 mol/L acetic acid, EMIM NtfO₂ could be directly injected into the capillary after liquid phase extraction. Using the basic dye chryisoidine, sensitivity enhancements approaching 1000-fold were obtained by mixing 20 μL of EMIM NtfO₂ with 1500 μL of aqueous sample, leaving only 5 μL of the undissolved ionic liquid which was used for injection into the CE. Lower more repeatable enhancement factors of 200-fold were obtained with slightly larger initial 25 μL volumes of EMIM NtfO₂ due to the larger residual volume of ionic liquid which made handling easier. This could be extended to basic pharmaceuticals, and the extraction of clozapine and its two active metabolites, nor-clozapine and clozapine-N-oxide, was demonstrated from urine with enrichment factors greater than 100 obtained. Handling of potentially more dangerous samples, such as serum, through in-vial extraction of clozapine and its metabolites and direct injection of the ionic liquid layer was also demonstrated with enhancements in sensitivity of 80. Limits of detection from 3 to 11 μg/L and 6 to 55 μg/L were obtained from urine and serum, respectively, which are sufficiently low to be useful for the determination of these pharmaceuticals clinically for therapeutic drug monitoring and for forensic toxicology., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Nanostructure initiator mass spectrometry (NIMS) is a recently introduced matrix-free desorption/ionization platform that requires minimal sample preparation. Its application to xenobiotics and endogenous metabolites in tissues is demonstrated, where clozapine and N-desmethylclozapine were observed from mouse and rat brain sections. It has also been applied to direct biofluid analysis where ketamine and norketamine were observed from plasma and urine. Detection of xenobiotics from biofluids was made even more effective using a novel NIMS on-surface extraction method taking advantage of the hydrophobic nature of the initiator. Linear response and limit of detection were also evaluated for xenobiotics such as methamphetamine, codeine, alprazolam, and morphine, revealing that NIMS can be used for quantitative analysis. Overall, our results demonstrate the capacity of NIMS to perform sensitive, simple, and rapid analyses from highly complex biological tissues and fluids.

The adsorptive and electrochemical behaviors of clozapine (CLZ) were investigated on a glassy carbon electrode that was electrochemically treated by anodic oxidation at +1.8 V, following potential cycling in the potential range from -0.8 to 1.0 V vs. Ag/AgCl reference electrode. Based on the obtained electrochemical results, an electrochemical-chemical (EC) mechanism was proposed to explain the electrochemical oxidation of CLZ. The resulting electrochemically pretreated glassy carbon electrode (EPGCE) showed good activity to improve the electrochemical response of the drug. CLZ was accumulated in a phosphate buffer (pH 6) at a certain time, and then determined by differential pulse voltammetry. The anodic and cathodic peak currents showed a linear function in the concentration ranges of 0.1 - 1, 1 - 10 and 10 - 100 microM with various accumulation times. The proposed method was successfully used for the determination of CLZ in pharmaceutical preparations. The preconcentration medium-exchange approach was utilized for the selective determination of the drug in spiked urine samples with satisfactory results. The recovery levels of the method reached 96% (RSD, 1.8%) and 90% (RSD, 2.8%) for urine and plasma samples, respectively.

Azaleptine detection and quantitation in cadaveric material in forensic-chemical tests using thin-layer chromatography, UV-spectrophotometry, gas chromatography-mass spectrometry are outlined.

Five spectrophotometric methods and one fluorimetric method have been developed and validated for the analysis of clozapine. The spectrophotometric methods were based on the charge-transfer complexation reaction between clozapine as electron donor and each of iodine as sigma-acceptor or 7,7,8,8-tetracyanoquinondimethane (TCNQ), 2,3-dichloro-5,6-dicyano-1,4-benzo-quinone (DDQ), tetracyanoethane (TCNE), and p-chloranilic acid (pCA) as pi-acceptors. The obtained complexes were measured spectrophotometrically at 365, 843, 460, 414, and 520 nm for iodine, TCNQ, DDQ, TCNE, and pCA, respectively. The fluorimetric method was based on the oxidation of clozapine in the presence of perchloric acid by cerium (IV), and subsequent measuring the fluorescence of the produced cerium (III) fluorimetrically at lambda(excitation) 260 and lambda(emission) 355 nm. Under the optimum assay conditions, Beer's law was obeyed at concentrations ranged from 4-200 microg mL(-1) for the spectrophotometric methods and from 24-250 ng mL(-1) for the fluorimetric method. The limits of detection for the spectrophotometric methods were 1.12, 1.76, 2.22, 0.95, and 13.26 microg mL(-1) for iodine, TCNQ, DDQ, TCNE, and pCA, respectively. The limit of detection for the fluorimetric method was 6.69 ng mL(-1). The proposed methods were successfully applied to the analysis of clozapine in tablets with good recoveries. The fluorimetric method could also be applied to the analysis of clozapine in spiked urine samples. The molar ratios and the reaction mechanisms were investigated.

Acute poisonings by medical, narcotic substances and alcohol are actual in Russia in the recent years. Comparison of analytic facilities of modern analytical techniques: chromatographic (HPLC, GC, GC-MS) and immuno-chemical (FPIA) in clinical toxicology for urgent diagnostics, assessment of the severity of acute poisoning and the efficacy of the treatment in patients with acute poisonings by psychotropic drugs, narcotics and alcohol have been done. The object of the study were serum, blood, urine of 611 patients with acute poisonings by amitriptyline, clozapine, carbamazepine, opiates and also alcohol. Threshold concentrations (threshold, critical and lethal) of the toxicants and their active metabolites which corresponded to different degrees of poisoning severity have been determined. The most comfortable and informative screening method for express diagnostics and assessment of severity of acute poisonings by psychotropic drugs and narcotics showed the HPLC with using automatic analyzers. FPIA using the automatic analyzer could be applied for screening studies, if group identification is enough. GC-FID method is advisable in case of poisoning by medical substances and narcotics in view of repeated investigation for assessment of the efficacy of the therapy. GC-MS could be advisable for confirming the results of other methods. GC-TCD possess high sensitivity and specificity and is optimal for express differential diagnostics and quantitative assessment of acute poisoning by ethanol and other alcohols.

Clozapine is a tricyclic dibenzodiazepine derivative used commonly in the treatment of schizophrenia. Severe side effects of overdose have been described in children. This report describes the unintentional ingestion of clozapine by a 10-y-o girl, presenting dramatic changes of mental status and progressive alteration of consciousness with a Glasgow Coma Scale drop from 12 to 7. Urine analysis revealed 500 microg clozapine/L 24 h after ingestion. Full resolution of symptoms occurred after approximately 55 h with no sequels or alterations at follow-up. Awareness must be taken when administering atypical antipsychotics such as clozapine to children because of the severe intoxication effects cases reported.

This report describes a fatal intoxication with two different drugs: clozapine and orphenadrine. A 38-year-old man was found dead in the bedroom of his residence. Near the body were found various empty pharmaceutical boxes, employed in schizophrenic therapy, two of them containing clozapine and orphenadrine. High concentrations of clozapine and orphenadrine detected in blood, urine and gastric content were related to death. These compounds were identified and quantitated by liquid-liquid extraction followed by gas chromatographic/mass spectrometry (GC/MS) analysis.

In experiments with expressed human UDP-glucuronosyltransferase 1A4 (UGT1A4), the antipsychotic clozapine proved to be conjugated to two different glucuronides, one of which was identified as the quaternary ammonium glucuronide derivatized at the N-methylpiperazine group; this compound had previously been isolated from patient urine. An additional glucuronide produced in larger quantity was assumed to be conjugated at the secondary nitrogen of the central ring to form 5-N-glucuronide, but this was not proven. The analogous olanzapine 10-N-glucuronide was found to make a major contribution to urinary metabolites in human volunteers. In the present investigation, tertiary 5-N-glucuronides were isolated from incubations of clozapine and desmethylclozapine with human liver microsomes fortified with UDP-glucuronic acid, and their structures were confirmed by mass and (1)H NMR spectrometry. The same conjugates could also be purified from patient urine. Their approximate quantities in urine from four patients ranged between 0.1 and 0.5% of the dose, as did those of the quaternary ammonium glucuronide of clozapine. Analogous to olanzapine 10-N-glucuronide, the tertiary clozapine 5-N-glucuronide was resistant toward enzymatic hydrolysis but was labile under acidic conditions.

Biotransformation products of the atypical neuroleptic clozapine were isolated from urine samples of three schizophrenic patients by solid-phase extraction, liquid-liquid extraction for the separation of unpolar and polar metabolites, and thin-layer chromatography followed by final purification by high-performance liquid chromatography. Their structures were elucidated by mass spectrometry and (1)H NMR spectroscopy and in some cases by enzymatic deconjugation. Besides the known metabolites desmethylclozapine, clozapine N-oxide, 8-deschloro-8-hydroxyclozapine, and 8-deschloro-8-hydroxydesmethylclozapine, the unpolar fraction contained 7-hydroxyclozapine and a compound in which the piperazine ring of clozapine was partially degraded to an ethylenediamine derivative. Novel metabolites identified in the polar fraction were the sulfate and glucuronide conjugates of 7-hydroxyclozapine N-oxide, 8-deschloro-8-hydroxyclozapine-O-glucuronide, and the O-glucuronide of N-hydroxydesmethylclozapine; further conjugates were tentatively identified as 9-hydroxydesmethylclozapine-O-sulfate and 6-hydroxyclozapine-O-sulfate. In addition, the previously described conjugates 7-hydroxydesmethylclozapine-O-sulfate, 7-hydroxyclozapine-O-glucuronide and -O-sulfate, 8-deschloro-8-hydroxydesmethylclozapine-O-glucuronide, and the quaternary ammonium glucuronide of clozapine were detected.

A sensitive reduction peak of clozapine is obtained by single-sweep oscillopolarography in 0.1 mol.L-1 H2SO4 solution. The peak potential is -0.95 V (vs. SCE). The peak current is directly proportional to the concentration of clozapine with a detection limit of 2.0 x 10(-8) mol.L-1. The voltammetric behaviour of clozapine and its mechanism of electrode reaction have been studied by linear-sweep and cyclic voltammetry and pulse polarography etc. The reduction process is irreversible with adsorptive characteristics. The number of electrons transferred n is 2. The electron transfer coefficient alpha is 0.62. Its behaviour obeys Langmuir adsorption isotherm. The adsorption coefficient beta is 1.91 x 10(6) L.mol-1. The energy of adsorption delta G0 = -35.83 kJ.mol-1. The method has been applied to the determination of clozepine in tablet and urine.

Clozapine is an atypical neuroleptic that is increasingly used for the treatment of schizophrenia. An automated method was developed for the routine quantification of clozapine and its major metabolites, N-desmethylclozapine and clozapine N-oxide, in human serum and urine by column switching and online high-performance liquid chromatography with ultraviolet detection. The method included adsorption of clozapine and its metabolites on a cyanopropyl-coated clean-up column (10 microns; 10 mm x 4.0 mm ID), washing interfering serum constituents to waste by deionized water, and, after column switching, separation on C18 ODS Hypersil reversed-phase material (5 microns; 250 mm x 4.6 mm ID). The compounds of interest were separated and eluted in fewer than 20 minutes, using a mobile phase consisting of 37.5 acetonitril:62.5 water, containing 0.4% (vol/vol) tetramethylethylenediamine and adjusted to pH 6.5 with concentrated acetic acid. Ultraviolet-detection was performed at 254 nm. The determinations exhibited linearity between detector signal and drug concentrations in a range from 5 ng/ml to 50 micrograms/ml. As little as 10 ng/ml of clozapine and 20 or 30 ng/ml of the metabolites was quantifiable. Interferences with other psychotropic drugs, serum, or urine constituents were negligible. The automated procedure enables the analysis of clozapine and metabolites in serum or urine in less than 1 hour.

The metabolic pathways of clozapine (CZ, Clozaril (Novartis Pharmaceuticals Corporation, East Hanover, NJ), 8-chloro-11-(4-methyl-1-piperazinyl)-5H-dibenzo [b,e][1,4]diazepine, a tricyclic benzodiazepine neuroleptic which has a reduced risk of unwanted neurological effects, were determined in normal male volunteers after a single oral dose of 50 mg of [14C]CZ. There was no radio-activity in exhaled breath, and excretion of total radioactivity was approximately 50% in urine and 30% in feces; parent CZ was a minor component in the excreta. The metabolic profiles were determined in urine and feces using HPLC coupled with radioactivity monitoring. The major metabolic pathways were demethylation, oxidation of the aromatic ring in the 7- and 8-positions, and conjugation. The major urinary components were 8-hydroxy-deschloro-DCZ (desmethylCZ) and its glucuronide, 7-hydroxy-8-chloro-DCZ sulfate and CZ-NO (clozapine N-oxide). Minor amounts of CZ, 7-hydroxy-8-chloro-CZ glucuronide and DCZ were also present. In feces the major component was CZ-N-glucuronide. Urinary excretion of CZ-NO was more rapid than the products of aromatic ring hydroxylation and conjugation.

Case Report: A rare case of clozapine intoxication of a 23-year-old woman, who intended suicide by its overdose, but recovered under medical treatments, is presented., Method: Gas chromatography/mass spectrometry and gas chromatography with nitrogen phosphorus detection were employed for its identification and quantitation, respectively., Results: Clozapine serum concentration reached 3.62 micrograms/mL 3 h after ingestion. The urine concentration 32 h after ingestion was 4.28 micrograms/mL.

The metabolism of clozapine N-oxide was investigated in the rat (n = 6) after a single oral dose of 20 mg kg-1. The organic extracts of rat urine were separated by conventional high performance liquid chromatography (HPLC) and individual collected fractions were analyzed by micro-column electrospray HPLC/mass spectroscopy. The compounds identified in rat urine were clozapine N-oxide, clozapine, N-desmethylclozapine, 8-deschloro-8-hydroxyclozapine, 8-deschloro-8-thiomethylclozapine, N-desmethylclozapine, 8-deschloro-8-hydroxyclozapine, 8-deschloro-8-thiomethylclozapine, N-desmethyl-8-deschloro-8-thiomethylclozapine and 8-deschloro-8-methylsulfinylclozapine. With the exception of the unchanged clozapine N-oxide, no other metabolite containing a N-oxide functional group could be found, the concentrations of clozapine N-oxide, clozapine and N-desmethylclozapine excreted from rat urine were determined utilizing a conventional HPLC procedure with UV detection. The recoveries of these three analytes reported as the percentage of the dosage from the 0.24 h urine are 0.93 +/- 0.54%, 0.06 +/- 0.03% and 0.01 +/- 0.006% respectively.

1. The phase II metabolites in the bile, urine and faeces of rat dosed with clozapine were investigated by means of electrospray mass spectrometry (ESMS) in both positive and negative ion modes. 2. When operated at a cone voltage of 45 V, this soft ionization technique permitted the detection of quasi molecular ions of both sulphate and glucuronide conjugates of hydroxylated phase I metabolites of clozapine. With the cone voltage set at 90 V, however, the ESMS also contained highly diagnostic ions resulting from the loss of 80 Daltons (sulphur trioxide) or 176 Daltons (the glucuronide moiety) from sulphates and O-glucuronides respectively. 3. A sufficient quantity of one metabolite was isolated from rat bile to permit further analysis by 1H-nmr. This metabolite, which was also found in rat urine, was proved to be 7-O-glucuronyl-7-hydroxyclozapine. The analogous sulphate metabolite was also identified in bile by ESMS. 4. Correspondingly glucuronide and sulphate conjugates of a hydroxylated N-desmethyl clozapine were similarly detected in rat bile. There was insufficient material to permit analysis by 1H-NMR, but it appears likely that conjugation was also at the 7-position of N-desmethylclozapine. 5. Finally, the sulphate conjugate of a hydroxy dechlorinated derivative of clozapine was identified by ESMS in both urine and bile. By analogy with a previous report of a similar metabolite in man, the metabolite was tentatively identified as 8-hydroxy-8-deschloroclozapine.

Studies were undertaken to determine if clozapine-N-oxide, the principal urinary metabolite of the antipsychotic agent clozapine, may interfere with the gas chromatographic-mass spectrometric bioanalysis of clozapine. Following injection of clozapine-N-oxide onto a (5% phenyl)methylpolysiloxane capillary column operated at 250 degrees C, significant on-column reduction of clozapine-N-oxide to the parent drug occurred. Accordingly, preparation of biological samples for clozapine determination by gas chromatography should avoid conditions which reportedly co-extract the N-oxide to assure no artifactual contribution of this metabolite in the detection of clozapine.

In a previous study we showed that the disposition of clozapine after a single oral dose is unrelated to either debrisoquine or S-mephenytoin hydroxylation polymorphism. The same 14 healthy subjects studied in that investigation were given 150 mg of caffeine. The reciprocal of plasma clozapine AUC (0,24), was correlated with an index of the N3-demethylation of caffeine (rs = 0.84; P = 0.0024), used as a measure of cytochrome P4501A2 (CYP1A2) activity. N1- and N7-demethylation indices of caffeine also reflect CYP1A2 activity and were also correlated with clozapine clearance (rs = 0.89 and 0.85; P = 0.0013 and 0.0023; respectively). No significant relationships with xanthine oxidase and N-acetyl transferase activity, also assessed by a caffeine test, were found. This study suggests that clozapine is metabolised by CYP1A2 to a major extent.

Clozapine N(+)-glucuronide was detected in the urine of five patients chronically treated with clozapine. Identification was made on the basis that the material isolated from urine had the same high-performance liquid chromatographic retention time and positive electrospray mass spectra as that of an authentic reference standard of clozapine N(+)-glucuronide. These results indicate that electrospray mass spectrometry is a valuable technique in the analysis of low-molecular-weight biologically derived N(+)-glucuronide metabolites.

The neuroleptic drug clozapine is used in the treatment of schizophrenia and is characterized by not having the extrapyramidal side-effects usually shown by neuroleptics. Unfortunately clozapine has other side-effects, which limit its use. This study presents methods for the analysis of clozapine and desmethylclozapine in whole blood and tissue. Case histories and pathology findings are described for 3 autopsy cases with fatal concentrations of clozapine, 5 with toxic concentrations and 2 with therapeutic concentrations together with the concentrations found in a living person.

A rapid, specific and sensitive method using reversed phase HPLC for the simultaneous determination of clozapine and its two metabolites in serum and urine has been developed. The mobile phase was a mixture of 67% (v/v) methanol in water containing 0.4% tetramethylethylenediamine and 0.32% acetic acid (pH 5.5). The influence of methanol content, the pH of the mobile phase and the effect of adding alkylammonium ions as peak tailing reducer in the mobile phase have been investigated. The solvent for extracting clozapine from serum and urine was ether. 50 microliters of 0.25 M H2SO4 solution was used to redissolve the dry residue to eliminate the endogenous compounds which could otherwise be eluted together with clozapine from the HPLC column. The analysis of a single sample was accomplished within half an hour. The identities of the chromatographic peaks of clozapine and its N-demethyl metabolite collected from the patient urine sample were confirmed by mass spectrometry. The method is sufficiently sensitive (5 ng/ml) and reproducible (CV 2.9%-6.7%) for clinical and pharmacokinetic studies, and preliminary results in these respects are presented.