Determination of clozapine and its major metabolites in human plasma and red blood cells by high-performance liquid chromatography with ultraviolet absorbance detection

An isocratic high-performance liquid chromatographic (HPLC) method with UV absorbance detection is described for the quantification of clozapine (8-chloro-11-(4′-methyl)piperazino-5H-dibenzo[b,e]-1,4-diazepine) and its two major metabolites in plasma and red blood cells (RBCs). The method involves sample clean-up by liquid-liquid extraction with ethyl acetate. The organic phase was back-extracted with 0.1 M hydrochloric acid. Loxapine served as the internal standard. The analytes were separated by HPLC on a Kromasil Ultrabas C₁₈ analytical column (5 μm particle size; 250×4.6 mm I.D.) using acetonitrile-phosphate buffer pH 7.0 (48:52, v/v) as eluent and were measured by UV absorbance detection at 254 nm. The limits of quantification were 20 ng/ml for clozapine and N-desmethylclozapine and 30 ng/ml for clozapine N-oxide. Recovery from plasma or RBCs proved to be higher than 62%. Precision, expressed as % C.V., was in the range 0.6–15%. Accuracy ranged from 96 to 105%. The method's ability to quantify clozapine and two major metabolites simultaneously with precision, accuracy and sensitivity makes it useful in therapeutic drug monitoring.     

Determination of clozapine and its metabolite, N-desmethylclozapine, in serum microsamples by high-performance liquid chromatography and its application to pharmacokinetics in rats

A single solvent extraction step high-performance liquid chromatographic method is described for quantitating clozapine and its metabolite, N-desmethylclozapine, in rat serum microsamples (50 μl). The separation used a 2.1-mm I.D. reversed-phase Symmetry C₁₈ column with an isocratic mobile phase consisting of methanol–acetonitrile–28.6 mM sodium acetate buffer, pH 2.6 (10:20:70, v/v/v). The detection limit was 2.5 ng/ml for all the compounds using an ultraviolet detector operated at 230 nm. The method was used to study the pharmacokinetics of clozapine after an intravenous bolus dose (2.5 mg/kg).     

Determination of clozapine, desmethylclozapine and clozapine N-oxide in human plasma by reversed-phase high-performance liquid chromatography with ultraviolet detection

An isocratic high-performance liquid chromatography (HPLC) method with ultraviolet detection for the simultaneous determination of clozapine and its two major metabolites in human plasma is described. Analytes are concentrated from alkaline plasma by liquid–liquid extraction with n-hexane–isoamyl alcohol (75:25, v/v). The organic phase is back-extracted with 150 μl of 0.1 M dibasic phosphate (pH 2.2 with 25% H₃PO₄). Triprolidine is used as internal standard. For the chromatographic separation the mobile phase consisted of acetonitrile–0.06 M phosphate buffer, pH 2.7 with 25% phosphoric acid (48:52, v/v). Analytes are eluted at a flow-rate of 1.0 ml/min, separated on a 250×4.60 mm I.D. analytical column packed with 5 μm C₆ silica particles, and measured by UV absorbance detection at 254 nm. The separation requires 7 min. Calibration curves for the three analytes are linear within the clinical concentration range. Mean recoveries were 92.7% for clozapine, 82.0% for desmethylclozapine and 70.4% for clozapine N-oxide. C.V. values for intra- and inter-day variabilities were ≤13.8% at concentrations between 50 and 1000 ng/ml. Accuracy, expressed as percentage error, ranged from −19.8 to 2.8%. The method was specific and sensitive with quantitation limits of 2 ng/ml for both clozapine and desmethylclozapine and 5 ng/ml for clozapine N-oxide. Among various psychotropic drugs and their metabolites, only 2-hydroxydesipramine caused significant interference. The method is applicable to pharmacokinetic studies and therapeutic drug monitoring.     

Fishing for a drug: solid-phase microextraction for the assay of clozapine in human plasma

Solid-phase microextraction (SPME) was investigated as a sample preparation method for assaying the neuroleptic drug clozapine in human plasma. A mixture of human plasma, water, loxapine (as internal standard) and aqueous NaOH was extracted with a 100-μm polydimethylsiloxane (PDMS) fiber (Supelco). Desorption of the fiber was performed in the injection port of a gas chromatograph at 260°C (HP 5890; 30 m×0.53 mm I.D., 1 μm film capillary; nitrogen–phosphorous selective detection). Fibers were used repeatedly in up to about 75 analyses. The recovery was found to be 3% for clozapine from plasma after 30 min of extraction. However, in spite of the low recovery, the analyte was well separated and the calibration was linear between 100 and 1000 ng/ml. The within-day and between-day precision was consistently about 8 to 15% at concentrations of 200 ng/ml to 1000 ng/ml. No interfering drug was found. The limit of detection was 30 ng/ml. The sample volume was 250 μl. The influence of the concentration of proteins, triglycerides and salt, i.e., changes in the matrix on the peak areas and peak-area ratios was studied. The method is not impaired by physiological changes in the composition of the matrix. Good agreement was found with a liquid–liquid extraction–gas–liquid chromatography (LLE–GLC) standard method and an on-line column-switching high-performance liquid chromatography (HPLC) method for patients’ samples and spiked samples, respectively. It is concluded that the method can be used in the therapeutic drug monitoring of clozapine because the therapeutic window of clozapine is from 350 to 600 ng/ml.     

Validated GC/MS method for the simultaneous determination of clozapine and norclozapine in human plasma. Application in psychiatric patients under clozapine treatment

A sensitive and specific GC/MS method for the determination of clozapine (CLZ) and its major metabolite norclozapine (NCLZ), in plasma has been developed, optimized and validated. Specimen preparation includes solid-phase extraction of both analytes using Bond-Elut Certify cartridge and further derivatization with TFAA. Clozapine-d8 was used as internal standard for the determination of CLZ and NCLZ. Limits of detection were 0.45 ng/mL for CLZ and 1.59 ng/mL for NCLZ, while limits of quantification were 1.37 ng/mL for CLZ and 4.8 ng/mL for NCLZ, as calculated by the calibration curves. The calibration curves were linear up to 600 ng/mL for CLZ and NCLZ. Absolute recovery ranged from 82.22% to 95.35% for both analytes. Intra- and interday accuracy was less than 7.13% and −12.52%, respectively, while intra- and interday precision was between 9.47% and 12.07%, respectively, for CLZ and NCLZ. The method covers all therapeutic range and proved suitable for the determination of CLZ and NCLZ not only in psychiatric patients but also in forensic cases with clozapine implication.     

Combining poly (methacrylic acid-co-ethylene glycol dimethacrylate) monolith microextraction and on-line pre-concentration-capillary electrophoresis for analysis of ephedrine and pseudoephedrine in human plasma and urine

A method based on poly (methacrylic acid-co-ethylene glycol dimethacrylate) (MAA-EGDMA) monolith microextraction (PMME) and field-enhanced sample injection (FESI) pre-concentration technique was proposed for sensitive capillary electrophoresis-ultraviolet (CE-UV) analysis of ephedrine (E) and pseudoephedrine (PE) in human plasma and urine. The PMME device consisted of a regular plastic syringe (1 mL), a poly (MAA-EGDMA) monolithic capillary (2 cm x 530 microm I.D.) and a plastic pinhead connecting the former two components seamlessly. The extraction was achieved by driving the sample solution through the monolithic capillary tube using a syringe pump, for the desorption step, an aliquot of organic solvent, which normally provided an excellent medium to ensure direct compatibility for FESI in CE, was injected via the monolithic capillary and collected into a vial for subsequent analysis by CZE. The best separation was achieved using a buffer composed of 0.1M phosphate electrolyte (pH 2.5) and 10% acetonitrile (v/v). The combination of both pre-concentration procedures allowed the detection limits of the analytes down to 5.3 ng/mL and 8.0 ng/mL in human plasma and urine, respectively. Excellent method of reproducibility was found over a linear range 50-5000 ng/mL in plasma and urine sample. Plasma and urine samples from volunteers receiving pseudoephedrine have also been successfully analysed.     

Determination of clozapine,and its metabolites,N-desmethylclozapine and clozapine N-oxide in dog plasma using high-performance liquid chromatography

Clozapine and its two major metabolites, N-desmethylclozapine and clozapine N-oxide were quantified using a high-performance liquid chromatographic method with UV detection in dog plasma following a single dose of clozapine. The analysis was performed on a 5-micrometer Hypersil CN (CPS-1; 250x4.6 mm) column. The mobile phase consisted of acetonitrile-water-1 M ammonium acetate (50:49:1, v/v/v), which was adjusted to pH 5.0 with acetic acid. The detection wavelength was 254 nm. A liquid-liquid extraction technique was used to extract clozapine and its metabolites from dog plasma. The recovery rates for clozapine, N-desmethylclozapine, and the internal standard (I.S.) were close to 100% using this method. The recovery rate for clozapine N-oxide (62-66%) was lower as expected because it is more polar. The quantitation limits for clozapine, clozapine N-oxide, and N-desmethylclozapine were 0.11, 0.05 and 0.05 microM, respectively. Intra-day reproducibility for concentrations of 0.1, 1.0 and 5.0 microM were 10.0, 4.4 and 4.2%, respectively, for N-oxide; 11.2, 4.3 and 4.9%, respectively, for N-desmethylclozapine; and 10.8, 2.2 and 4.9%, respectively, for clozapine. Inter-day reproducibility was <15% for clozapine N-oxide, <8% for N-desmethylclozapine and <19% for clozapine. This simple method was applied to determine the plasma concentration profiles of clozapine, N-desmethylclozapine and clozapine N-oxide in dog following administration of a 10 mg/kg oral dose of clozapine.     

High-performance liquid chromatographic method with diode array detection to identify and quantify atypical antipsychotics and haloperidol in plasma after overdose

For toxicological purposes, an HPLC assay was developed for the simultaneous determination of haloperidol and atypical antipsychotics (risperidone, 9-hydroxyrisperidone, olanzapine, clozapine) in human plasma. After a double-step liquid-liquid extraction, compounds were separated on a C(8) column eluted with a gradient of acetonitrile and phosphate buffer 50 mM pH 3.8. A sequential ultraviolet detection was used (260, 280 and 240 nm). Calibration curves were linear in the range 10-1000 ng/ml. The limits of quantification were 5 ng/ml for all drugs. Average accuracy at four concentrations ranged from 93 to 109%. Both inter- and intra-day variation coefficients were lower than 11% for all drugs. This simple and rapid method (run time<15 min) is currently used for poison management.     

High sensitivity analysis of oxprenolol in urine by capillary electrophoresis with C18 packed on-line preconcentrator

High sensitivity analysis of oxprenolol in spiked human urine has been performed by capillary zone electrophoresis (CZE) in ammonium formate buffer pH 2.5 using an uncoated capillary with 1cm length C18 on-capillary preconcentrator at the inlet side. The preconcentrator was fabricated in laboratory using the packing method and not encapped C18 5 microm particles as stationary phase material. The packed path was retained into the capillary by sintered stationary phase frits. Before running the CZE analysis, the oxprenolol was eluted from the preconcentrator by injecting a short plug of acetonitrile/water mixtures. With respect to classical CZE, the use of on-line preconcentrator widely increased the method sensitivity allowing the detection of the drug at 0.5 ng/mL (injected concentration). The method showed a linear response in the range of 1-150 ng/mL oxprenolol standard compound. The intra-day repeatability (n = 11) R.S.D. values for migration time, peak area and normalized peak area were 0.72%, 3.96% and 3.66%, respectively, while inter-day repeatability (n = 5 days) R.S.D. values were 2.74%, 9.41% and 9.83%, respectively. The method was successfully applied to the analysis of oxprenolol in extracted urine spiked at 250 pg/mL (oxprenolol LOQ concentration in urine).     

Determination of josamycin in rat plasma by capillary electrophoresis coupled with post-column electrochemiluminescence detection

A novel determination method for josamycin (JOS) based on capillary electrophoresis-electrochemiluminescence detection has been described. In this study, platinum disk electrode (300 microm in diameter) was used as a working electrode and the conditions affecting separation and detection were investigated in detail. Under optimal condition: 40 cm separation capillary (75 microm i.d.); 1.25 V applied potential on the Pt disc of the ECL detector cell; 5 mM Ru(bpy)3(2+) and 50mM phosphate buffer (pH 7.5) in the detection cell; 12 kV separation voltage; 8s injection time; 10 kV injection voltage and 15 mM running buffer (pH 7.5), calibration curve was linear over the range from 10 ng/mL to 5.0 microg/mL with a detection limit of 3.1 ng/mL at a signal-to-noise ratio of 3. The method can be successfully applied for the determination of josamycin in rat plasma in 6 min and the extraction recoveries with spiked plasma samples were over 92%.     

Quantitative analysis of pyoluteorin in anti-fungal fermentation liquor of Pseudomonas species by capillary zone electrophoresis with UV-vis detector

This paper investigated potential utility of capillary zone electrophoresis (CZE) for very succinct but robust quantitative analysis of pyoluteorin (Plt) in anti-fungal fermentation liquor of Pseudomonas species. The experimental conditions for the separation and quantification of Plt were optimized at first. The optimized conditions are: 80 mmol/L pH 8.40 Gly-NaOH buffer, 51 cm total length (42 cm effective) and 75 microm I.D. capillary, 230 nm wavelength, 25 kV, 13 mbar 10s pressure sample injection and 24 degrees C air-cooling. Under the optimized conditions, the migration times of Plt and the internal standard phenobarbital are 2.09 and 2.49 min, respectively, the linear response of Plt concentration ranges from 5.0 to 1000 microg/mL with high correlation coefficient (r=0.99977, n=9), the limits of detection (LOD) and quantification (LOQ) for Plt are 0.66 and 2.2 microg/mL, the precision values (expressed as R.S.D.) of intra- and inter-day are 1.19-1.94% and 1.55-6.21%, respectively, the recoveries of Plt at three concentration levels of 750, 250 and 50 microg/mL range from 90.31% to 97.85% and to 98.96%, respectively. The developed method can be well used for the quantification of Plt in the fermentation liquor.     

High-speed microchip electrophoresis method for the separation of (R,S)-naproxen

In this research, a capillary electrophoretic method for the fast enantiomeric resolution of (R,S)-naproxen was investigated. Method development involved variation of applied potential, buffer concentration, buffer pH, and cyclodextrin concentration. The optimum electrophoretic separation conditions were 110 mM sodium acetate run buffer (pH 6.0), 30 mM methyl-beta-cyclodextrin, 20% (v/v) acetonitrile, 25 degrees C. The total length of capillary was 48 cm, (50 microm I.D.) with ultra violet (UV) detection at 232 nm. Using these conditions, the number of theoretical plates was close to one million (896,000/m). The possibility of achieving a fast chiral separation of (R,S)-naproxen on a microchip of 2.5 cm in length was investigated. Complete enantiomeric resolution of naproxen was achieved in less than 1 min, on this microchip platform, with linear imaging UV detection. This system had the advantage of real-time separation monitoring, so that enantiomeric resolution could be visually observed, and high-speed chiral analysis was realized. The microchip electrophoresis (MCE) separation was compared with the capillary electrophoresis (CE) separation with regards to speed, efficiency, separation platform, and precision. This work highlights the potential of CE and MCE in future chiral separations.     

Effect of chronic infusion of olanzapine and clozapine on food intake and body weight gain in male and female rats

Many antipsychotics cause weight gain in humans, but usually not in rats, when injected once or twice daily. Since blood antipsychotic half-lives are short in rats, compared to humans, chronic administration by constant infusion may be necessary to see consistent weight gain in rats. Male and female rats were implanted with mini-pumps for constant infusion of olanzapine (5 mg/kg/day), clozapine (10 mg/kg/day) or vehicle for 11 days. Food intake and body weight were measured; blood drug levels were measured by HPLC. Olanzapine increased food intake and body weight in female, but not male rats. Serum olanzapine concentrations were 30-35 ng/ml. Clozapine had no effect on food intake or body weight in female or male rats. Serum clozapine concentrations were about 75 ng/ml. Single-dose pharmacokinetic analysis revealed a serum terminal half-life of 1.2-1.5 h for each drug, with no sex differences. Despite the fact that olanzapine and clozapine promote weight gain in humans, these drugs appear to have minimal effects on body weight and food intake in rats, except for a modest effect of olanzapine in female rats, even though therapeutic levels of olanzapine are achieved in serum during chronic infusion. Hence, the rapid clearance of drug following single administration in previous studies cannot explain the weak or absent effects of antipsychotics on weight gain in this species. The rat thus appears to be an inadequate model of weight gain produced by some antipsychotics in humans.     

Biocompatible in-tube solid-phase microextraction coupled to HPLC for the determination of angiotensin II receptor antagonists in human plasma and urine

A poly (methacrylic acid-ethylene glycol dimethacrylate, MAA-EGDMA) monolithic capillary was used for the in-tube solid-phase microextraction (in-tube SPME) of several angiotensin II receptor antagonists (ARA-IIs) from human plasma and urine. Under the optimized extraction condition, the protein component of the biological sample was flushed through the monolithic capillary, while the analytes were successfully trapped. Coupled to HPLC with fluorescence detection, this on-line in-tube SPME method was successfully applied for the determination of candesartan, losartan, irbesartan, valsartan, telmisartan, and their detection limits were found to be 0.1-15.3ng/mL and 0.1-15.2ng/mL in human plasma and urine, respectively. The method was linear over the range of 0.5-200ng/mL for telmisartan, 5-2000ng/mL for candesartan and irbesartan, 10-2000ng/mL for valsartan, and 50-5000ng/mL for losartan with correlation coefficients being above 0.9985 in plasma sample and above 0.9994 in urine sample. The method reproducibility was evaluated at three concentration levels, resulting in the R.S.D. <7%. The poly (MAA-EGDMA) monolithic capillary was demonstrated to be robust and biocompatible by using direct injections of biological samples.     

Quantification of meropenem in plasma and cerebrospinal fluid by micellar electrokinetic capillary chromatography and application in bacterial meningitis patients

A high-performance micellar electrokinetic capillary chromatography (MEKC) has been demonstrated for the determination of meropenem in human plasma and in cerebrospinal fluid (CSF) and application in meningitis patients after intravenous (IV) administration. Plasma sample was pretreated by means of solid-phase extraction (SPE) on C(18) cartridge and CSF sample was by direct injection without sample pretreatment, with subsequent quantitation by MEKC. The separation of meropenem was carried out in an untreated fused-silica capillary (40.2 cm x 50 microm I.D., effective length 30 cm) and was performed at 25 degrees C using a background electrolyte consisting of Tris buffer (40 mM, pH 8.0) solution with sodium dodecyl sulfate (SDS) as the running buffer and on-column detection at 300 nm. Several parameters affecting the separation and sensitivity of the drug were studied, including pH, the concentrations of Tris buffer and surfactant. Using cefotaxime as an internal standard (IS), the linear ranges of the method for the determination of meropenem in plasma and in CSF were all over 0.5-50 microg/mL; the detection limits (signal-to-noise ratio=3) of meropenem in plasma and in CSF were 0.2 microg/mL and 0.3 microg/mL, respectively.     

Development and validation of a liquid chromatography-tandem mass spectrometry method for the determination of xanthinol in human plasma and its application in a bioequivalence study of xanthinol nicotinate tablets

A sensitive, rapid liquid chromatographic-electrospray ionization mass spectrometric method for the determination of xanthinol in human plasma was developed and validated. Xanthinol nicotinate in plasma (0.5mL) was pretreated with 20% trichloroacetic acid for protein precipitation. The samples were separated using a Lichrospher silica (5mum, 250mmx4.6mm i.d.). A mobile phase of methanol-water containing 0.1% formic acid (50: 50, v/v) was used isocratically eluting at a flow rate of 1mL/min. Xanthinol and its internal standard (IS), acyclovir, were measured by electrospray ion source in positive selected reaction monitoring mode. The method demonstrated that good linearity ranged from 10.27 to 1642.8ng/mL with r=0.9956. The limit of quantification for xanthinol in plasma was 10.27ng/mL with good accuracy and precision. The mean plasma extraction recovery of xanthinol was in the range of 90.9-100.2%. The intra- and inter-batch variability values were less than 4.8% and 7.9% (relative standard deviation, R.S.D.), respectively. The established method has been successfully applied to a bioequivalence study of two xanthinol nicotinate tablets for 20 healthy volunteers.     

Validation of a rapid micellar electrokinetic capillary chromatographic method for the simultaneous determination of isoniazid and pyridoxine hydrochloride in pharmaceutical formulation

An efficient and reliable micellar electrokinetic capillary chromatography (MEKC) method has been developed for the simultaneous determination of isoniazid (ISO) and pyridoxine hydrochloride (PYR) in pharmaceutical formulations. A chemometric two level full factorial design approach was used to search for the optimum conditions of separation. Three parameters were selected for this study: the buffer pH, the buffer concentration and sodium dodecyl sulphate (SDS) concentrations. Resolution, peak symmetry and analysis time were established as response. The two analytes were separated within 6 min with the optimized conditions: 50 mM borate buffer, 25 mM SDS pH 7.8, 35 degrees C, at 50 mbar 4s injection and 30 kV by using a fused silica capillary (72 cm effective length, 50 microm i.d.). The detection wavelength was set to 205 nm. Meloxicam was used as internal standard. The method was validated with respect to stability, linearity range, limit of quantitation and detection, precision, accuracy, specificity and robustness. The detection limits of the method were 1.0 microg mL(-1) for ISO and 0.40 microg mL(-1) for PYR and the method was linear at least in the range of 3.0-100 microg mL(-1) for ISO and 1.0-100 microg mL(-1) for PYR with excellent correlation coefficients (0.9995 for ISO and 0.9998 for PYR). Relative standard deviations (R.S.D.s) of the described method ranged between 0.54 and 2.27% for intra-day precision and between 0.65 and 2.69% for inter-day precision. The developed method was applied to the tablet form of ISO and PYR-containing the pharmaceutical preparations and the data were compared with obtained from the standard addition method. No statistically significant difference was found.     

Simultaneous determination of AMN107 and Imatinib (Gleevec, Glivec, STI571) in cultured tumour cells using an isocratic high-performance liquid chromatography procedure with UV detection

A reversed phase high-performance liquid chromatographic (HPLC) method with UV detection was developed for the simultaneous determination of imatinib (Gleevec, Glivec, STI571) and AMN107 in cultured tumour cells, using clozapine as an internal standard. The compounds of interest were extracted by liquid-liquid extraction using TOXI-TUBES((R)) A extraction tubes. Chromatographic separation was performed on a Phenomenex Gemini C18 reversed phase column (150 mm x 2.0 mm, 5 microm particle size), using a mixture of 65% CH(3)OH (methanol) and 35% NH(4)Ac (Ammonium acetate) buffer (20mM, pH 10). Separation was achieved under isocratic conditions at a flow rate of 0.5 ml/min. Imatinib, clozapine and AMN107 are detected by UV detection at 260 nm. Calibration curves were linear from 50 to 7500 ng/ml with correlation coefficients (r(2)) better than 0.998. The limit of quantitation (LOD) was 50 ng/ml. The method has been successfully applied to a cellular kinetics study.     

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

The characteristics of automated on-line solid phase extraction with liquid chromatography-mass spectrometry (SPE-LC-MS) are very amenable for flexibility and throughput in therapeutic drug monitoring (TDM). We demonstrate this concept of automated, on-line SPE-LC-MS for the analysis of clozapine and metabolites (desmethylclozapine and clozapine-N-oxide) in serum. Method development, optimisation and validation are described and a comparison with previously published methods for the determination of clozapine and metabolites in serum and plasma is made. Optimisation of chromatographic and SPE conditions for increased throughput resulted in SPE-LC-MS cycle times of only about 2.2 min, demonstrating the great potential of automated on-line SPE-LC-MS for TDM. The new method is shown to be clearly favourable, in particular in terms of ease of sample handling, throughput and detection limits. Recovery is essentially quantitative. Detection limits are at about 0.15-0.3 ng ml(-1), depending on the ionisation source used. Calibration follows a quadratic model for clozapine and its N-oxide and a linear model for the desmethyl metabolite (all cases: R > 0.99). Accuracy, evaluated at three concentration levels spanning the whole therapeutic range, shows that bias is less than 10%. Precision (intra - and inter assay) ranges from about 5% R.S.D. at the high end of the therapeutic range (700-1,000 ng ml(-1)) to about 20% R.S.D. (OECD defined limit) at the lower limit of quantitation ( approximately 50 ng ml(-1)). The lower limit of quantitation is well below the low end of the therapeutic range at 350 ng ml(-1).     

Simultaneous determination of olanzapine,clozapine and demethylated metabolites in serum by on-line column-switching high-performance liquid chromatography

An automated method for simultaneous routine quantification of the antipsychotic drugs clozapine, olanzapine and their demethylated metabolites is described. The method included adsorption on a cyanopropyl (CPS) coated clean-up column (10 μm; 10×2.0 mm I.D.), washing off interfering serum constituents to waste, and separation on C18 ODS Hypersil reversed phase material (5 μm; 250×4.6 mm I.D.) using acetonitrile–water–tetramethylethylenediamine (37:62.6:0.4, v/v/v) adjusted to pH 6.5 with concentrated acetic acid. UV-detection was performed at 254 nm. The limit of quantification was 10–20 ng/ml. Relative day to day standard variations ranged between 4.5 and 13.5%. The method is suitable for routine monitoring of olanzapine and clozapine including their demethylated metabolites.