A liquid chromatography-miniature mass spectrometry (LC-Mini MS) method for quantitative analysis of risperidone and 9-hydroxyrisperidone in plasma.

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a universal method for the quantitative analysis of small molecular drugs in therapeutic drug monitoring (TDM). Alternatively, liquid chromatography-miniature mass spectrometry (LC-Mini MS) is a simple operating technique for quantitative analysis. However, the wide chromatographic peaks and long retention times of TDM samples using the LC-Mini MS system deteriorated the accuracy and efficiency of quantitative analysis. Here, an optimized electrospray ionization (ESI) interface setup with a splitter valve and a capillary needle (I.D. 30 µm and O.D. 150 µm) of the LC-Mini MS system was acquired. The chromatographic peaks were narrower and smoother and the retention time was shorter for TDM compounds. Furthermore, a quantitative analysis method for risperidone and the active metabolite 9-hydroxyrisperidone in plasma was developed based on this optimal LC-Mini MS setup. The results showed that the calibration curves of risperidone and 9-hydroxyrisperidone had good linear ranges of 2-100 ng mL-1 (R2 = 0.9931) and 2-100 ng mL-1 (R2 = 0.9915), respectively. Finally, the matrix effects, recoveries and stability of risperidone and 9-hydroxyrisperidone samples were analyzed. The results satisfied the requirements of quantitative validation in routine TDM procedures.

Quantitative structure -retention relationship modeling of selected antipsychotics and their impurities in green liquid chromatography using cyclodextrin mobile phases.

Applying green chromatography methods is currently one of the challenges in liquid chromatography. Among different strategies, using cyclodextrin (CD) mobile phase modifiers was applied in this paper. CDs can form inclusion complexes with a wide variety of hydrophobic organic compounds and, consequently, affect their retention behavior. CD-containing mobile phases possess complicated complexation and adsorption equilibria so retention is dependent not only on chromatographic parameters and properties of the compound but also on properties of compound-CD complex. Docking study was used to calculate association constants of the selected antipsychotics (risperidone, olanzapine, and their impurities) and ß-CD complexes and predict which part of the molecule structure will most likely incorporate into the ß-CD cavity. Quantitative structure-retention relationship model (QSRR) for selected model substances was built employing artificial neural network (ANN) technique. Reliable QSRR model was obtained using molecular descriptors, complex association constants, and chromatographic factors. The multilayer perceptron network with 11-8-1 topology, trained with back propagation algorithm, showed the best performance. Root mean square error for training, validation, and test was 0.2954, 0.3633, and 0.4864, respectively. The correlation coefficient (R2) between experimentally obtained retention factor values [k(exp)] and values computed or predicted by ANN [k(ANN)] was 0.9962 for training, 0.9927 for validation, and 0.9829 for test, indicating good predictive ability of the model. The optimized network was used for development of green chromatography method for separation of risperidone and its related impurities, as well as olanzapine and its related impurities in a relatively short run time and with low consumption of organic modifier. The developed methods were validated in accordance with ICH Q2 (R1) quideline and all parameters fulfilled the defined criteria. The greenness of the proposed methods has also been demonstrated. Graphical Abstract Complex association constants as inputs of QSRR model in ß-cyclodextrin modified HPLC system and development of green chromatography methods.

Characterization of bonded stationary phase performance as a function of qualitative and quantitative chromatographic factors in chaotropic chromatography with risperidone and its impurities as model substances.

Numerous stationary phases have been developed with the aim to provide desired performances during chromatographic analysis of the basic solutes in their protonated form. In this work, the procedure for the characterization of bonded stationary phase performance, when both qualitative and quantitative chromatographic factors were varied in chaotropic chromatography, was proposed. Risperidone and its three impurities were selected as model substances, while acetonitrile content in the mobile phase (20-30%), the pH of the aqueous phase (3.00-5.00), the content of chaotropic agents in the aqueous phase (10-100 mM), type of chaotropic agent (NaClO4, CF3COONa), and stationary phase type (Zorbax Eclipse XDB, Zorbax Extend) were studied as chromatographic factors. The proposed procedure implies the combination of D-optimal experimental design, indirect modeling, and polynomial-modified Gaussian model, while grid point search method was selected for the final choice of the experimental conditions which lead to the best possible stationary phase performance for basic solutes. Good agreement between experimentally obtained chromatogram and simulated chromatogram for chosen experimental conditions (25% acetonitrile, 75 mM of NaClO4, pH 4.00 on Zorbax Eclipse XDB column) confirmed the applicability of the proposed procedure. The additional point was selected for the verification of proposed procedure ability to distinguish changes in solutes' elution order. Simulated chromatogram for 21.5% acetonitrile, 85 mM of NaClO4, pH 5.00 on Zorbax Eclipse XDB column was in line with experimental data. Furthermore, the values of left and right peak half-widths obtained from indirect modeling were used in order to evaluate performances of differently modified stationary phases applying a half-width plots approach. The results from half-width plot approach as well as from the proposed procedure indicate higher efficiency and better separation performance of the stationary phase extra densely bonded and double end-capped with trimethylsilyl group than the stationary phase with the combination of end-capping and bidentate silane bonding for chromatographic analysis of basic solutes in RP-HPLC systems with chaotropic agents. Graphical abstract ᅟ.

Detection of new metabolites of risperidone in the solid tissues and body fluids obtained from two cadavers by high resolution tandem mass spectrometry.

Risperidone (Ris) is a second-generation antipsychotic that belongs to the chemical class of benzisoxazole derivatives. 9-Hydroxy (9OH-) Ris is well known among the six reported metabolites of Ris and had been examined using not only blood but also other matrices, but the other five metabolites reported such as benzisoxazole ring-cleaved Ris (c-Ris) and c-9OH-Ris had been detected only in blood, urine and feces. In the present work, large peaks of c-Ris and c-9OH-Ris were detected in the liver, kidney, cerebrum, blood, pericardial fluid, bile and urine obtained from two cadavers. There is a potential that c-Ris and c-9OH-Ris will be good markers to prove Ris consumption in forensic toxicology cases. For example, the peak ratios of c-Ris against the parent Ris in the kidney and blood were as high as 3.9 and 3.6 in cadaver 1; and 7.0 and 7.9 in cadaver 2, respectively. In addition to the previously reported six metabolites, five new metabolites such as dehydrogenated-Ris, 7-keto-Ris and three benzisoxazole ring-cleaved metabolites were disclosed in the present work, and the pathways for the totally eleven metabolites detected in human solid tissues and body fluids have also been proposed, because such pathways were neither reported nor discussed previously.

Poly (D, L-lactide) nanosuspensions of risperidone for parenteral delivery: formulation and in-vitro evaluation.

Risperidone, an "atypical" antipsychotic drug, having large scope for prolonged psychotic treatments through novel parenteral drug delivery systems. Polymeric nanoparticles suspensions containing risperidone made of poly (D, L-Lactide) were designed by nanoprecipitation method using polymeric stabilizer (Pluronic F-68 or Pluronic F-127). The prepared nanosuspensions were characterized for particle size by photon correlation spectroscopy and scanning electron microscopy. The free dissolved drug in the nanosuspension was determined by bulk equilibrium reverse dialysis bag technique. In vitro release studies were carried out using dialysis bag diffusion technique. The particle size of the prepared nanoparticles in the nanosuspensions ranged between 78-184 nm. Nanoparticles of risperidone in the nanosuspensions were obtained with high encapsulation efficiency (91-94 %). The drug release from the risperidone nanosuspension was sustained in some batches for more than 24 h with 75% drug release whereas release from risperidone solution showed release within 1.5 h. The release pattern of drug is analyzed and found to follow first order equation and Fickian diffusion kinetics. These studies suggest the feasibility of formulating risperidone loaded poly (D, L-Lactide) nanoparticles suspension for the treatment of psychotic disorders.

Determination of risperidone in tablets in the presence of its degradation products and placebo-derived constituents.

New chromatographic-densitometric assay was developed for identification and determination of risperidone in pharmaceutical formulations. Thin-layer chromatographic plates (TLC-F254) as a stationary phase and n-butanol-acetic acid-water (12:3:5 v/v/v) as a mobile phase were used for separation. Densitometric measurements were done for all constituents at lambda = 280 nm. A decrease in stability of risperidone was observed in acidic, basic and antioxidant solutions. Degradation of active pharmaceutical ingredient was consistent with first-order kinetics and unrelated to the solution. This assay is specific for risperidone. No interference of tablet origin adjuvants and degradation products were observed. Moreover, high sensitivity, limit of detection (0.22 microg/spot), limit of quantitation (0.67 microg/spot), recovery (98.2-100.82%), broad linear range (0.09 microg/spot to 1.40 microg/spot) and accuracy (1.87% RSD) are characteristic traits of the chromatographic-densitometric assay.

Non-destructive quantitative analysis of risperidone in film-coated tablets.

A simple, non-destructive, methodology based on FT-Raman spectroscopy was developed for the quantitative analysis of risperidone in commercially available film-coated tablets. A simple linear regression model was constructed based on standard tablets, prepared using the same manufacturing process as the commercially available. The tablets contained 0.27, 0.54, 1.08, 1.62, 2.16, 3.24 and 4.32 wt% risperidone. The most prominent Raman vibration of the active pharmaceutical ingredient at 1533 cm(-1), recorded using a home-made rotating system, was plotted against concentration. The model was tested on commercial film-coated tablets. The results were compared against those obtained by application of HPLC on the same samples.

Long-term therapeutic drug monitoring of risperidone and olanzapine identifies altered steady-state pharmacokinetics: a clinical, two-group, naturalistic study.

BACKGROUND AND OBJECTIVE: Conflicting therapeutic drug monitoring (TDM) results have been reported for risperidone and olanzapine. This study set out to examine the long-term pharmacokinetics of risperidone and olanzapine in a locked skilled nursing facility where medication administration was controlled by nursing staff. METHODS: TDM was performed in a long-term treatment protocol for risperidone and olanzapine in 67 refractory chronic schizophrenic patients in a locked, skilled nursing facility. TDM was performed 632 times in the risperidone group of 34 patients and 563 times in the olanzapine group of 33 patients. The logarithm of plasma concentrations were analysed through time by piecewise linear mixed model regressions adjusted for the logarithm of dose. RESULTS: We found risperidone plasma concentration/dose ratio (C/D) accumulation peaks of 49% at 2 months (from baseline concentration) and 9-hydroxy-risperidone and total moiety C/D accumulation peaks of 66% and 55% above the 2-month level at 6 months, which are somewhat similar to those found in our prior study that included a subset of data points analysed here. The risperidone conversion to 9-hydroxy-risperidone by cytochrome P450 (CYP) 2D6 suggests CYP2D6 inhibition or DNA down-regulation in the first 2 months. Olanzapine showed a C/D accumulation peak at 4 months of 31% above baseline, and a slower increase to 47% above baseline at 18 months with no clear plateau. CONCLUSION: We identified five potential perturbations in the pharmacokinetics of risperidone and olanzapine that could potentially lead to adverse drug reactions. These long-term effects would not be captured by a standard 5-day pharmacokinetic TDM developmental testing model for antipsychotics, and a new model for characterizing variation in C/D by time course is therefore proposed. The time course of the accumulations identified suggests that both CYP inhibition and DNA regulatory mechanisms may be involved in the metabolism of these drugs. Long-term TDM can optimize treatment with risperidone and olanzapine and antipsychotics in general.

High throughput µ-SPE based elution coupled with UPLC-MS/MS for determination of eluxadoline in plasma sample: Application in pharmacokinetic characterization of PLGA nanoparticle formulations in rats.

Eluxadoline is a novel µ- and κ-opioid receptor (OR) agonist and δ-OR antagonist, recently approved as a first line therapy for the treatment of irritable bowel syndrome. Due to abuse potential, poor bioavailability and high intersubject variability, a sensitive and reliable assay is prerequisite for its determination in biological samples. This work first time report the development and validation of UPLC-MS/MS assay for determination of eluxadoline in rat plasma sample using risperidone as an internal standard (IS). A high-throughput 96-well plate format µ-SPE technique was used for plasma sample extraction. The extracted samples were separated on Acquity BEH™ C18 column (100×2.1mm, 1.7µm) using mobile phase elution of acetonitrile: 20mM ammonium acetate (80:20, v/v) at a flow rate of 0.3mLmin-1. The precursor to product ion transition of m/z 570.16→118.12 (qualifier), 570.16→171.08 (quantifier) for eluxadoline, and m/z 411.18→191.07 for IS were used for MRM monitoring. The calibration curves were linear in concentration range of 0.15-50ngmL-1 with LOD and LOQ of 0.07 and 0.15ngmL-1, respectively. The validation results satisfied the criteria of USFDA and SWGTOX guidelines and were within the acceptable limit. Finally, the method was successfully applied in bioavailability enhancement study of the newly developed PLGA nanoparticles and Eudragit coated PLGA nanoparticles of eluxadoline in rats.

Analytical and semipreparative enantioseparation of 9-hydroxyrisperidone, the main metabolite of risperidone, using high-performance liquid chromatography and capillary electrophoresis. Validation and determination of enantiomeric purity.

The HPLC semipreparative enantioseparation of 9-hydroxyrisperidone (9-OHRisp) was studied by optimizing various experimental conditions: the nature of the chiral stationary phase (CSP), mobile phase composition, temperature and analyte loading. This semipreparative enantioseparation was successfully completed using the polysaccharide Chiralcel OJ chiral stationary phase and a n-hexane/ethanol/methanol (50/35/15, v/v/v) ternary mobile phase. To assess the enantiomeric purity of both isolated isomers, three analytical methods using UV detection were developed and validated: one CE method using dual cyclodextrin mode and two HPLC methods using either the Chiralcel OJ CSP in normal-phase mode or the alpha-acid glycoprotein (alpha-AGP) CSP in reversed-phase mode. The three methods make it possible to obtain excellent enantioseparations (R(s) >3) with analysis times lower than 15 min, and the calculated limits of detection allow for the determination of minor enantiomeric impurities (0.1%). Enantiomeric purity obtained for dextrorotatory and levorotatory enantiomers was superior to 99.9% and equal to 98.9%, respectively, which proved the success of the semipreparative enantioseparation. A brief comparison of the performances of the analytical methods completes this work.

Sensitive liquid chromatography/tandem mass spectrometry method for the simultaneous determination of olanzapine, risperidone, 9-hydroxyrisperidone, clozapine, haloperidol and ziprasidone in rat brain tissue.

One prerequisite for therapeutic effects of psychiatric drugs is the ability to pass the blood brain barrier. Hence, it is important to know the concentration of antipsychotic drugs in brain tissue. In general, determinations of lipophilic compounds from lipophilic matricies such as the brain are a challenge. Here we have adapted a plasma assay for antipsychotics for the target organ the brain. Using modified sample preparation and chromatographic strategies, the analytes were extracted from rat brain homogenate and analyzed by LC-MS/MS. The method used a Waters Atlantis dC-18 (30 mm x 2.1 mm i.d., 3 microm) column with a mobile phase of acetonitrile/5 mM ammonium formate (pH 6.1 adjusted with formic acid) and gradient elution. All analytes were detected in positive ion mode using multiple-reaction monitoring. The method was validated and the linearity, lower limit of quantitation, precision, accuracy, recoveries, specificity and stability were determined. This method was then successfully used to quantify the rat brain tissue concentration of the analytes after chronic treatment with these antipsychotic drugs.

Optimization of the separation of some psychotropic drugs and their respective metabolites by liquid chromatography.

A chemometric procedure is described to optimize the separation of some drugs used in the treatment of psychotic disorders: haloperidol, levomepromazine, risperidone, venlafaxine, carbamazepine and their main metabolites: reduced haloperidol, 9-hydroxy risperidone, desmethyl levomepromazine, desmethyl venlafaxine. The purpose of the procedure is the unambiguous identification and detection in biological fluids. Isocratic reversed-phase liquid chromatography with diode array detection was utilized. An experimental design methodology was carried out in which the experimental response is selectivity. In this way the designs for mixture compounds and for process variables (five variables) was performed which produced 36 experiments to carry out. The desirability function was used to select optimum separation conditions. The procedure provides a chromatogram of well separated solutes.

Structural studies of impurities of risperidone by hyphenated techniques.

During the impurity profile of risperidone (RSP), a more polar impurity (RSP-1) and a more non-polar impurity (RSP-2) were detected in LC-MS with respect to risperidone. These impurities were isolated, enriched and were subjected to mass and NMR spectral studies. Based on the spectral data, RSP-1 and RSP-2 were characterized as risperidone N-oxide and 9-methylene risperidone, respectively. The formation of these impurities is rationalized. The structures of both the impurities were unambiguously confirmed by single crystal XRD Studies.

The redistribution of selected psychiatric drugs in post-mortem cases.

The post-mortem redistribution of a number of psychiatric drugs was investigated. A portion of liver, the gastric contents and blood collected from heart and femoral sites was obtained from 13 cases and analyzed by liquid chromatography-mass spectrometry. Drugs detected included five selective serotonin reuptake inhibitors; venlafaxine, a serotonin/noradrenaline reuptake inhibitor; and risperidone, an atypical antipsychotic. Heart blood concentrations were significantly higher (3.4-fold on average) than those measured in femoral blood when results from all drugs were included together. The range for parent drug concentrations in these two blood specimens was 0.5-6.2. There was no significant correlation of the post-mortem interval, the liver concentration and content of drugs in the gastric contents to the heart:femoral blood concentration ratio. These data serve to demonstrate that variable increases in blood concentration occur post-mortem and limit the interpretative value of such toxicological data.

High performance liquid chromatographic and thin layer densitometric methods for the determination of risperidone in the presence of its degradation products in bulk powder and in tablets.

Two reproducible stability indicating methods were developed for the determination of risperidone (RISP) in presence of its degradation products in pure form and in tablets. The first method was based on reversed phase high performance liquid chromatography (HPLC), on Lichrosorb RP C 18 column (250 mm i.d., 4 mm, 10 microm), using methanol:0.05 M potassium dihydrogen phosphate pH 7 (65:35 (v/v)) as the mobile phase at a flow rate of 1 ml min(-1) at ambient temperature. Quantification was achieved with UV detection at 280 nm over a concentration range of 25-500 microg ml(-1) with mean percentage recovery of 99.87 +/- 1.049. The method retained its accuracy in the presence of up to 90% of RISP degradation products. The second method was based on TLC separation of RISP from its degradation products followed by densitometric measurement of the intact drug spot at 280 nm. The separation was carried out on aluminum sheet of silica gel 60F254 using acetonitrile:methanol:propanol:triethanolamine (8.5:1.2:0.6:0.2 (v/v/v/v)), as the mobile phase, over a concentration range of 2-10 microg per spot and mean percentage recovery of 100.1 +/- 1.18. The two methods were simple, precise, sensitive and could be successfully applied for the determination of pure, laboratory prepared mixtures and tablets. The results obtained were compared with the manufacturer's method.

Quantification of risperidone and 9-hydroxyrisperidone in plasma and saliva from adult and pediatric patients by liquid chromatography-mass spectrometry.

A robust and validated LC-MS-MS quantitative method, using column switching and mutiple reaction monitoring was developed for the analysis of risperidone (RIS) and 9-hydroxyrisperidone in human plasma and saliva. The analytical range was 1-100 ng/ml. The method used 25 microl of sample precipitated with 75 microl of acetonitrile containing internal standard (R068808). Analyses were conducted on a PE Sciex API-III + triple quadrupole mass spectrometer fitted with a Turbo IonSpray source. The method was validated for human plasma using EDTA as the anticoagulant and cross-validated to heparinized human plasma and saliva. The recoveries of risperidone and 9-hydroxyrisperidone were 90-93 and 89-93%, respectively. The validated method was applied to clinical samples to study risperidone and 9-hydroxyrisperidone concentrations in plasma and saliva. Risperidone and 9-hydroxyrisperidone appear in the saliva of patients treated with risperidone. Their detection/quantification in saliva provides evidence for recent adherence with therapy.

Sensitive chemiluminescence assay for risperidone in pharmaceutical preparations.

A sensitive chemiluminescence (CL) method to determine the antipsychotic risperidone (RSP) is proposed, based on the catalytic effect of risperidone on the CL reaction between luminol and hydrogen peroxide in flow system. The increment of CL intensity was correlated with risperidone concentration in the range of 10pg ml(-1) to 1.0ng ml(-1) with a relative standard deviation of less than 5.0% (n = 5); and a limit of detection of 4pg ml(-1) (3sigma). At a flow rate of 2.0ml min(-1), the flow injection CL method exhibited both high sensitivity and excellent selectivity giving a throughput of 120 samples per hour. The proposed method was applied successfully to the determination of risperidone in pharmaceutical preparations.

Identification and characterization of major degradation products of risperidone in bulk drug and pharmaceutical dosage forms.

Acid, base and oxygen stability of risperidone, a novel anti-psychotic drug, has been evaluated storing the sample in solution phase. One of the major degradation products has been identified and characterized by using techniques namely IR, MS and NMR after isolation by preparative LC. The other major degradation product has been identified with help of MS/MS data and by co-eluting in analytical LC with the available standard. The effect of acid and base resulted in the formation of hydroxy risperidone and the effect of oxygen lead to the formation of N-oxide of risperidone. The two major degradation products in the dosage forms were also characterized as 9-hydroxy risperidone and N-oxide of risperidone, after enrichment through preparative LC, by LC-MS/MS and HPLC. Structural elucidation of degradation product leading to the formation of N-oxide of risperidone is discussed in detail.

LC-MS/MS of some atypical antipsychotics in human plasma, serum, oral fluid and haemolysed whole blood.

Therapeutic drug monitoring (TDM) of atypical antipsychotics is common, but published methods often specify relatively complex sample preparation and analysis procedures. The aim of this work was to develop and validate a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the analysis of amisulpride, aripiprazole and dehydroaripiprazole, clozapine and norclozapine, olanzapine, quetiapine, risperidone and 9-hydroxyrisperidone, and sulpiride in small (200 µL) volumes of plasma or serum for TDM purposes. The applicability of the method as developed to haemolysed whole blood and to oral fluid was also investigated. Analytes and internal standards were extracted into butyl acetate:butanol (9+1, v/v) and a portion of the extract analysed by LC-MS/MS (100 mm × 2.1 mm i.d. Waters Spherisorb S5SCX; eluent: 50 mmol/L methanolic ammonium acetate, pH* 6.0; flow-rate 0.5 mL/min; positive ion APCI-SRM, two transitions per analyte). Assay calibration (human plasma, oral fluid, and haemolysed whole blood calibration solutions) was performed by plotting the ratio of the peak area of the analyte to that of the appropriate internal standard. Assay validation was as per FDA guidelines. Assay calibration was linear across the concentration ranges studied. Inter- and intra-assay precision and accuracy were within 10% for all analytes in human plasma. Similar results were obtained for oral fluid and haemolysed whole blood, except that aripiprazole and dehydroaripiprazole were within 15% accuracy at low concentration (15 µg/L) in oral fluid, and olanzapine inter-assay precision could not be assessed in these matrices due to day-by-day degradation of this analyte. Recoveries varied between 16% (sulpiride) and 107% (clozapine), and were reproducible as well as comparable between human plasma, human serum, calf serum and haemolysed whole blood. For oral fluid, recoveries were reproducible, but differed slightly from those in plasma suggesting the need for calibration solutions to be prepared in this medium if oral fluid is to be analysed. LLOQs were 1-5 µg/L depending on the analyte. Neither ion suppression/enhancement, nor interference from some known metabolites of the antipsychotics studied has been encountered. The method has also been applied to the analysis of blood samples collected post-mortem after dilution (1+1, 1+3; v/v) in analyte-free calf serum.

Stability of some atypical antipsychotics in human plasma, haemolysed whole blood, oral fluid, human serum and calf serum.

Long-term stability data of atypical antipsychotics in different matrices are not widely available. The aim of this work was to assess the stability of amisulpride, aripiprazole and dehydroaripiprazole, clozapine and norclozapine, olanzapine, quetiapine, risperidone and 9-hydroxyrisperidone, and sulpiride in human EDTA plasma, heparinised haemolysed human whole blood, oral fluid, human serum, and newborn calf serum stored in tightly capped plastic containers under a range of conditions. Measurements were performed by LC-MS/MS. Analyte instability was defined as a deviation of 15% or greater from the expected concentration. All analytes were stable following 3 freeze-thaw cycles in human plasma, and were stable in this matrix for at least 5 days at ambient temperature (olanzapine, 3 days); 4 weeks at 2-8°C (olanzapine, 2 weeks), and 2 years at -20°C (except for dehydroaripiprazole, olanzapine, and quetiapine, 1 year). In human serum, aripiprazole, dehydroaripiprazole, norclozapine, olanzapine, quetiapine, risperidone, 9-hydroxyrisperidone, and sulpiride were unstable after 5 days at ambient temperature, 3 weeks at 2-8°C, and 9 months at -20°C. Olanzapine was unstable in whole blood and oral fluid under most conditions studied, although prior addition of ascorbic acid had a moderate stabilising effect. All other analytes were stable in whole blood and oral fluid for at least 2 days at ambient temperature, 1 week at 2-8°C, and 2 months at -20°C (clozapine and norclozapine, 1 month whole blood). These results confirm that plasma (EDTA anticoagulant) is the sample of choice for TDM of atypical antipsychotics. Delayed (more than 1 week) analysis of patient samples should be undertaken with caution, especially with serum and with haemolysed whole blood. With olanzapine, only plasma collected and stored appropriately is likely to give reliable quantitative results.