Analysis of dextromethorphan and dextrorphan in decomposed skeletal tissues by microwave assisted extraction, microplate solid-phase extraction and gas chromatography- mass spectrometry (MAE-MPSPE-GCMS).

Analysis of decomposed skeletal tissues for dextromethorphan (DXM) and dextrorphan (DXT) using microwave assisted extraction (MAE), microplate solid-phase extraction (MPSPE) and gas chromatography-mass spectrometry (GC-MS) is described. Rats (n = 3) received 100 mg/kg DXM (i.p.) and were euthanized by CO2 asphyxiation roughly 20 min post-dose. Remains decomposed to skeleton outdoors and vertebral bones were recovered, cleaned, and pulverized. Pulverized bone underwent MAE using methanol as an extraction solvent in a closed microwave system, followed by MPSPE and GC-MS. Analyte stability under MAE conditions was assessed and found to be stable for at least 60 min irradiation time. The majority (>90%) of each analyte was recovered after 15 min. The MPSPE-GCMS method was fit to a quadratic response (R(2) > 0.99), over the concentration range 10-10 000 ng⋅mL(-1) , with coefficients of variation <20% in triplicate analysis. The MPSPE-GCMS method displayed a limit of detection of 10 ng⋅mL(-1) for both analytes. Following MAE for 60 min (80 °C, 1200 W), MPSPE-GCMS analysis of vertebral bone of DXM-exposed rats detected both analytes in all samples (DXM: 0.9-1.5 µg⋅g(-1) ; DXT: 0.5-1.8 µg⋅g(-1) ).

Determination of psychostimulants and their metabolites by electrochemistry linked on-line to flowing atmospheric pressure afterglow mass spectrometry.

The flowing atmospheric pressure afterglow (FAPA) ion source operates in the ambient atmosphere and has been proven to be a promising tool for direct and rapid determination of numerous compounds. Here we linked a FAPA-MS system to an electrochemical flow cell for the identification of drug metabolites generated electrochemically in order to study simulated metabolic pathways. Psychostimulants and their metabolites produced by electrochemistry (EC) were detected on-line by FAPA-MS. The FAPA source has never been used before for an on-line connection with liquid flow, neither for identification of products generated in an electrochemical flow cell. The system was optimized to achieve the highest ionization efficiency by adjusting several parameters, including distances and angles between the ion source and the outlet of the EC system, the high voltage for plasma generation, flow-rates, and EC parameters. Simulated metabolites from tested compounds [methamphetamine (MAF), para-methoxy-N-methylamphetamine (PMMA), dextromethorphan (DXM), and benzydamine (BAM)] were formed in the EC cell at various pH levels. In all cases the main products were oxidized substrates and compounds after N-demethylation. Generation of such products and their thorough on-line identification confirm that the cytochrome P450 - driven metabolism of pharmaceuticals can be efficiently simulated in an electrochemical cell; this approach may serve as a step towards predictive pharmacology using a fast and robust design.

Evaluation of two modified quick, easy, cheap, effective, rugged and safe (QuEChERS) sample preparation methods for the analysis of baclofen and gabapentin in feeds by liquid chromatography tandem mass spectrometry.

This paper presents a new analytical method for the simultaneous determination of baclofen and gabapentin in feeds based on two modified quick, easy, cheap, effective, rugged and safe (QuEChERS) sample preparation methods and liquid chromatography tandem mass spectrometry (LC-MS/MS). For the two modified QuEChERS methods, samples were first extracted with acidified acetonitrile (5.0% acetic acid, v/v) without using acetonitrile salting-out extraction. Then, the first modified QuEChERS method was established according to the original QuEChERS cleanup procedure. For the second modified QuEChERS method, the extract was evaporated to dryness and reconstituted in acetonitrile. Subsequently, the analytes in the reconstituted solution were retained by primary secondary amine (PSA) and released from PSA with 1.0% formic acid in methanol. Finally, the eluate was evaporated and dissolved in 0.1% formic acid solution/methanol (v/v, 80:20). All of the samples were analyzed by LC-MS/MS on a Waters Acquity BEH C18 column with 0.1% formic acid in water/methanol as the mobile phase with gradient elution. The matrix effect, recovery, and repeatability, within laboratory reproducibility, and the LODs and LOQs of the two modified QuEChERS sample preparation methods were investigated and compared. Comparative results showed that the second method was obviously superior to the first method.

A biomimetic potentiometric sensor based on molecularly imprinted polymer for the determination of memantine in tablets.

Memantine hydrochloride is one of the first novel class medications for treatment of Alzheimer's disease. In this work, a biomimetic potentiometric sensor, based on a non-covalent imprinted polymer, was fabricated for the recognition and determination of memantine in pure drug and tablet pharmaceutical form. The molecularly imprinted polymer was synthesized by precipitation polymerization, using memantine hydrochloride as a template molecule, methacrylic acid as a functional monomer, and ethylene glycol dimethacrylate as a cross-linking agent. The sensor was developed by dispersing the memantine imprinted polymer particles in dibutyl sebacate plasticizer and embedding in poly(vinyl chloride) matrix. The wide linear range (10(-5) -10(-1) M), with a near Nernstian response of 57.4 mV/decade, a limit of detection 6.0 × 10(-6) M, fast response time (~15 s) and a satisfactory long-term stability (4 months) are characterizations of the proposed sensor. The sensor showed a high selectivity and a sensitive response to the template in aqueous system. The standard electrode potentials were determined at different temperatures and used to calculate the isothermal coefficient of the electrode. It was used as indicator electrode in potentiometric determination of memantine in pharmaceutical formulations.

The establishment of a highly sensitive method in detecting ketamine and norketamine simultaneously in human hairs by HPLC-Chip-MS/MS.

An effective way to reveal the history of drug abuse is to determine the parental drug and its metabolites in hair. Here, a quantitative HPLC-Chip-MS/MS method was developed for simultaneous measurement of ketamine and its metabolite norketamine in human hair. Ketamine and norketamine were extracted from hair by acid hydrolysis, and then enriched by organic solvent extraction. The chromatographic separation was achieved in 15 min, with the drug identification and quantification by a tandem mass spectrometer. The linear regression analysis was calibrated by deuterated internal standards with a R(2) of over 0.996. The limit of detection (LOD) and the limit of quantification (LOQ) for ketamine and norketamine were 0.5 and 1 pg/mg of hair, respectively. The standard curves were linear from the value of LOQ up to 100 pg/mg of hair. The validation parameters including selectivity, accuracy, precision, stability and matrix effect were also determined. In conclusion, this method was able to reveal the present of ketamine and norketamine with less hair from the drug abusers, and which had the sensitivity of ∼1000-fold higher than the conventional method. In addition, the amount of ketamine and norketamine being detected in different hair segments would be useful in revealing the historical record of ketamine uptake in the drug abusers.

Impact of microdialysis probes on vasculature and dopamine in the rat striatum: a combined fluorescence and voltammetric study.

Measuring extracellular dopamine in the brain of living animals by means of microdialysis and/or voltammetry is a route towards understanding both normal brain function and pathology. Previous reports, however, suggest that the tissue response to implantation of devices may affect the outcome of the measurements. To address the source of the tissue response and its impact on striatal dopamine systems microdialysis probes were placed in the striatum of anesthetized rats. Images obtained by dual-label fluorescence microscopy show signs of ischemia and opening of the blood-brain barrier near the probe tracks. Opening of the blood-brain barrier was further examined by determining dialysate concentrations of carbi-DOPA, a drug that normally does not penetrate the brain. Although carbi-DOPA was recovered in brain dialysate, it did not alter dialysate dopamine levels or evoked dopamine release as measured by voltammetry near the probes. Microdialysis probes also significantly diminished the effect of intrastriatal infusion of kynurenate on extracellular dopamine levels as measured by voltammetry near the probes.

Postmortem memantine concentrations.

Postmortem fluid and tissue concentrations of memantine (Namenda), a drug recently approved for the treatment of Alzheimer's Disease by the FDA, are reported in a suspicious death. In addition, memantine concentrations considered to be incidental findings in three other cases are included to aid in the interpretation in future toxicological investigations. Memantine was extracted from biological samples by a standard liquid-liquid basic drug method followed by analysis utilizing a gas chromatograph-mass spectrometer operated in SIM mode. Blood concentrations ranged from 0.03 to 1.8 mg/L, and the liver concentration was 6.1 mg/kg.

Development and application of a validated HPLC method for the determination of gabapentin and its major degradation impurity in drug products.

A simple isocratic reversed-phase HPLC method for the determination of gabapentin and its major degradation impurity, 3,3-pentamethylene-4-butyrolactam, was developed and validated for use in the analysis of pharmaceutical tablets and capsules. Separation was achieved on a Brownlee Spheri-5 Cyano column using an acetonitrile-10 mM KH2PO4/10 mM K2HPO4 (pH 6.2) (8:92, v/v) mobile phase. The compounds were eluted isocratically at a flow rate of 1 mL/min. Both compounds were analyzed with UV detection at 210 nm. The method was validated according to USP Category I requirements for gabapentin and USP Category II for 3,3-pentamethylene-4-butyrolactam. The validation characteristics included accuracy, precision, linearity, range, specificity, limit of quantitation and robustness. Validation acceptance criteria were met in all cases. This method was used successfully for the quality assessment of four gabapentin drug products.

[Use of hyaluronic acid cleaving enzymes for absorption acceleration. Results of an in vitro study with xylazine and ketamine]. / Einsatz von Hyaluronsäure spaltenden Enzymen zur Resorptionsbeschleunigung. Ergebnisse einer In-vitro-Studie mit Xylazin und Ketamin.

The so-called "Hellabrunner Mischung", (combination of xylazine and ketamine with hyaluronidase) is frequently used for the immobilisation of wildlife animals. The enzyme hyaluronidase shall improve the distribution of the intramuscularly or subcutaneously administered compounds in the tissue and enhance their absorption. These enhancing effects of two hyaluronate lyases of bacterial origin (Streptococcus agalactiae and Streptococcus equisimilis) and a testicular hyaluronidase were compared in an in vitro test. Using the isolated perfused bovine udder, 2 ml of a solution were administered subcutaneously containing 125 mg/ml xylazine and 100 mg/ml ketamine and one of the above mentioned enzymes (150 I.U.). All three enzymes enhanced the absorption rate of xylazine and ketamine determined by measurement of the concentration in the perfusate. The bacterial hyaluronate lyases were significantly more efficient, especially during the clinically important first minutes after administration.

Glutamate receptor incorporated in a mixed hybrid bilayer lipid membrane array, as a sensing element of a biosensor working under flowing conditions.

The realization of a reliable receptor biosensor requires stable, long-lasting, reconstituted biomembranes able to supply a suitable biomimetic environment where the receptor can properly work after incorporation. To this end, we developed a new method for preparing stable biological membranes that couple the biomimetic properties of BLMs (bilayer lipid membranes) with the high stability of HBMs (hybrid bilayer membranes); this gives rise to an innovative assembly, named MHBLM (mixed hybrid bilayer lipid membrane). The present work deals with the characterization of biosensors achieved by embedding an ionotropic glutamate receptor (GluR) on MHBLM. Thanks to signal (transmembrane current) amplification, which is typical of natural receptors, the biosensor here produced detects glutamate at a level of nmol L(-1). The transmembrane current changes linearly vs glutamate up to 100 nmol L(-1), while the limit of detection is 1 nmol L(-1). In addition, the biosensor response can be modulated both by receptor agonists (glycine) and antagonists (Mg(2+)) as well, and by exploiting the biosensor response, the distribution of different kinds of ionotropic GluR present in the purified sample, and embedded in MHBLM, was also evaluated. Finally, one of the most important aspects of this investigation is represented by the high stability of the biomimetic system, which allows the use of biosensor under flowing conditions, where the solutions flow on both biomembrane faces.

Simple determination of riluzole in rat brain by high-performance liquid chromatography and spectrophotometric detection.

A simple method was developed for separation and quantification of riluzole in rat brain. The analyses were performed by high-performance liquid chromatography using a C18 reversed-phase column (Hypersil ODS) with UV detection at 264 nm. The mobile phase consisted of methanol-water containing 1% triethylamine adjusted with orthophosphoric acid to pH 3.2. The retention time was 8.6 min. A simple liquid-liquid extraction with ethyl acetate was used to obtain riluzole from brain samples. The limit of quantification was 10 ng/g. The recovery was about 80%. The relationship between peak areas and concentrations was linear over the range between 0.01 and 0.8 microg/g, with r2 value over 0.99. The assay provided good reproducibility and accuracy and proved to be suitable for pharmacokinetic studies of riluzole.

Rapid quantification of the non-competitive NMDA antagonist MK-801 in canine cerebrospinal fluid and plasma by capillary gas chromatography-nitrogen phosphorus detection.

A facile and sensitive method utilizing solid-phase cartridge extraction and capillary gas chromatography (GC) with nitrogen phosphorus detection was validated for the determination of MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclo-hepten-5,10-imine maleate], a non-competitive NMDA receptor antagonist, in dog cerebrospinal fluid (CSF) and plasma. Clonidine hydrochloride was used as the internal standard (ISTD), after evaluation of several ISTD candidates. Separations were performed with an intermediate polarity fused silica capillary column, yielding typical retention times of 3.20 min for MK-801 and 4.90 min for ISTD. Plasma and CSF samples were extracted with 100 mg Bond Elut C(18) TCA Copyright cartridges to yield methanolic eluates that were evaporatively enriched before reconstitution in anhydrous ethanol prior to injection. The standard curve was validated from 1 to 100,000 ng/ml for CSF, and from 0.1 to 1,000 ng/ml for plasma. Chromatograms from naive plasma and CSF exhibited no endogenous interfering peaks. The efficiency of extraction recovery was >94%, and the intra-assay and inter-assay precision was within 9% relative standard deviation (%R.S.D.) for both fluids. MK-801 and ISTD were stable in the injection solvent at 22 degrees C for at least 48 h. The assay was applied to the toxocologic study of intrathecal MK-801 administration in the dog.

Identification of the requisite brain sites in the neuronal network subserving generalized clonic audiogenic seizures.

Comparative studies of neuronal networks that subserve convulsions in closely-related epilepsy models are revealing instructive data about the pathophysiological mechanisms that govern these networks. Studies of audiogenic seizures (AGS) in genetically epilepsy-prone rats (GEPRs) and related forms of AGS demonstrate important network similarities and differences. Two substrains of GEPRs exist, GEPR-9s, exhibiting tonic AGS, and GEPR-3s, exhibiting clonic AGS. The neuronal network for tonic AGS resides exclusively in brainstem nuclei, but forebrain sites, including the amygdala (AMG), are recruited after repetitive AGS induction. The neuronal network for clonic AGS remains to be investigated. The present study examined the neuronal network for clonic AGS in GEPR-3s by microinjecting a competitive NMDA receptor antagonist, D,L-2-amino-7-phosphonoheptanoic acid (AP7), into the central nucleus of inferior colliculus (ICc), deep layers of superior colliculus (DLSC), periaqueductal grey (PAG), or caudal pontine reticular formation (cPRF), which are implicated in tonic AGS networks. Microinjections into AMG and perirhinal cortex (PRh), which are not implicated in AGS, were also done. AGS in GEPR-3s were blocked reversibly after microinjections into ICc, DLSC, PAG or cPRF. However, AGS were also blocked by AP7 in AMG but not PRh. The sites in which AP7 blocks AGS are implicated as requisite components of the clonic AGS network, and these data support a critical role for NMDA receptors in clonic AGS modulation. The brainstem nuclei of the clonic AGS network are identical to those subserving tonic AGS. However, the requisite involvement of AMG in the clonic AGS network, which is not seen in tonic AGS, is surprising and suggests important mechanistic differences between clonic and tonic forms of AGS.

Identification of metabotropic glutamate receptor antagonists using an automated high-throughput screening system.

Antagonists to the human metabotropic glutamate receptor subtype 5a(mGluR(5a)) have been implicated as potential therapeutics for the treatment of a variety of nervous system disorders, including pain, anxiety, and Parkinson's disease. To discover novel antagonists to the mGluR(5a), a functional assay measuring agonist-induced intracellular calcium release was developed. The assay was used for the high-throughput screening of a large collection of compounds in single wells using a fully automated robotic platform. Primary high-throughput screening hits were subjected to a combination of data analysis and counterscreening assays to identify several compounds with both efficacy and selectivity for the metabotropic glutamate receptor target.

Topiramate selectively decreases intracortical excitability in human motor cortex.

PURPOSE: Topiramate (TPM) is a novel drug with broad antiepileptic effect in children and adults. In vitro studies suggest activity as sodium-channel blocker, as gamma-aminobutyric acid type A (GABAA)-receptor agonist and as non-N-methyl-D-aspartate (NMDA)-glutamate receptor antagonist. METHODS: With transcranial magnetic stimulation (TMS), we evaluated which of the mechanisms of action of TPM detected in vitro are relevant for the modulation of human motor cortex excitability. In a double-blind, placebo-controlled, crossover study design, we investigated the effect of single oral doses of 50 mg and 200 mg TPM on motor thresholds, cortical silent period (CSP), and on intracortical inhibition (ICI) and intracortical facilitation (ICF) in 20 healthy subjects. RESULTS: A significant dose-dependent increase of ICI was noticed after 200 mg TPM as compared with placebo at short interstimulus intervals of 2 to 4 ms. TPM had no effect on motor thresholds or the CSP. CONCLUSIONS: We conclude that a single dose of TPM selectively increases ICI by GABAAergic and/or glutamatergic mechanisms without a relevant influence on measures, depending on ion-channel blockade or GABAB-receptor activity. The decrease of intracortical excitability (as measured by ICI and ICF) caused by TPM may correlate with its lack of proconvulsive potential in idiopathic generalized epilepsy, because drugs without this action or with less pronounced action may exacerbate seizures in this condition.

Brain penetration and in vivo recovery of NMDA receptor antagonists amantadine and memantine: a quantitative microdialysis study.

PURPOSE: To determine free brain concentrations of the clinically used uncompetitive NMDA antagonists memantine and amantadine using microdialysis corrected for in vivo recovery in relations to serum, CSF and brain tissue levels and their in vitro potency at NMDA receptors. METHODS: Microdialysis corrected for in vivo recovery was used to determine brain ECF concentrations after steady-state administration of either memantine or amantadine. Additionally CSF, serum, and brain tissue were analyzed. RESULTS: Following 7 days of infusion of memantine or amantadine (20 and 100 mg/kg/day respectively) whole brain concentrations were 44-and 16-fold higher than free concentrations in serum respectively. The free brain ECF concentration of memantine (0.83 +/- 0.05 microM) was comparable to free serum and CSF concentrations. In case of amantadine, it was lower. A higher in vivo than in vitro recovery was found for memantine. CONCLUSIONS: At clinically relevant doses memantine reaches a brain ECF concentration in range of its affinity for the NMDA receptor and close to its free serum concentration. This is not the case for amantadine and different mechanisms of action may be operational.

Screening of receptor antagonists using agonist-activated patch clamp detection in chemical separations.

We present a capillary electrophoresis-patch clamp detection system optimized for screening of antagonists and inhibitors of ligand-gated ion channels. In this system, highly selective receptor agonists are delivered through the electrophoresis capillary to the cell surface where they continuously activate a receptor, resulting in increased steady-state transmembrane currents. Thus, receptor selection and biosensor functionality is simply achieved by selection of an appropriate agonist. The antagonists are fractionated in the same electrophoresis capillary and inhibit the agonist-evoked response, resulting in transiently decreased steady-state transmembrane currents. Specifically, a mixture containing 6-cyano-7-nitroquinoxaline-2,3-dione, that reversibly blocks alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and kainate receptors, and 6,7-dichloro-3-hydroxy-2-quinoxaline-carboxylate, a broad-spectrum glutamate receptor antagonist, were separated and detected by kainate-activated patch-clamped interneurons freshly dissociated from rat brain olfactory bulb. In addition, Mg2+ that reversibly blocks the N-methyl-D-aspartate receptor in a voltage-dependent way was detected using the same cell detector system when activated by N-methyl-D-aspartate and the co-agonist glycine. The presented method offers new possibilities for drug screening and for identifying endogenous receptor antagonists and to determine their mode of action on any ionotropic receptor system of interest.

Determination of several N-methyl-D-aspartate receptor blockers in plasma and brain by a selective high-performance liquid chromatographic method with column switching.

A general procedure is presented for the determination of several N-methyl-D-aspartate (NMDA) receptor open-channel and subtype-selective blockers, which have been evaluated and developed as neuroprotective drugs for the treatment of brain stroke and trauma. The method involves deproteination of plasma with ethanol, or homogenization of brain samples in ethanol, dilution of the supernatant with ammonium acetate and direct injection into an HPLC column-switching system. Although the investigated NMDA receptor blockers are all tertiary amines, they have quite different structures. However, they are all concentrated on the first column (Purospher RP-18, 125 x 4 mm), whereas polar interfering compounds are washed out with 1% ammonium acetate-acetic acid-acetonitrile (100:1:5, v/v/v). Due to the special selectivity of the Purospher RP-18 material, the analytes and the internal standard are then selectively eluted with 25% acetonitrile (without any buffer in the mobile phase) and transferred to the analytical column (Superspher 60 RP-select B, 250 x 4 mm), where they are separated by gradient elution and detected by UV or fluorescence detection. The low degree of interference allowed the development of sensitive methods with quantification limits of 5 ng/ml for animal plasma (0.4 ml used), 0.5 ng/ml for human plasma (1 ml used) and 50 ng/g for brain tissue (200 mg used).

High-performance liquid chromatographic analysis of (S)-alpha-amino-5-phosphonomethyl[1,1'-biphenyl]-3-propanoic acid (EAB 515) in brain and blood microdialysate (on-line) and in plasma ultrafiltrate of freely moving rats.

(S)-alpha-Amino-5-phosphonomethyl[1,1'-biphenyl]-3-propanoic acid (EAB 515, I), a competitive antagonist of the N-methyl-D-aspartate receptor, has significant pharmacological activity in the central nervous system (CNS). An extremely sensitive and selective analytical method was developed for the simultaneous analysis of I and its hydroxylated analog (RDC, II) in the microdialysate (MD) and plasma ultrafiltrate (UF) of rats. Microdialysis was used for in vivo sampling of unbound drug in the CSF, cortical extracellular fluid and in the blood of freely moving rats. Compound II was used for retrodialysis-based in vivo calibration of microdialysis probes to estimate the recovery of I. Compound I, being extremely hydrophilic with a high degree of ionization at the physiological pH of 7.4, has limited access to the brain regions. This, combined with its low microdialysis recovery, made the estimation of low brain concentrations of I a challenge. The analytes in MD and UF were separated (within 5 min) by reversed-phase HPLC on a 250 x 4.6 mm I.D. Maxsil 5 microns RP-2 column, and fluorescence of the eluent was monitored at 255 nm (lambda ex) and 320 nm (lambda em). A 0.09% (v/v) aqueous solution of trifluoroacetic acid (1 ml/min) was used as the mobile phase. The response for I in MD and UF samples was linear from 5 to 2000 ng/ml and from 20 to 10,000 ng/ml, respectively. The between-run (n = 6) and within-run (n = 3) variability of the assay was < 15%. Plasma-protein binding of I (fu = 0.68) was determined to be linear from 0.1 to 10 micrograms/ml. The analytical sensitivity, precision and accuracy of this method was suitable for the characterization of the pharmacokinetics and the CNS distribution of I, following administration of intravenous (i.v.) infusion, single i.v. bolus and multiple i.v. bolus doses of I to freely moving rats, with continuous microdialysate sampling of multiple tissues and simultaneous on-line HPLC analysis. Pharmacokinetic parameters for I, as determined from concentrations in blood MD samples with on-line analysis, were in good agreement with those estimated from concentrations in the UF of plasma samples obtained by conventional sampling.

Stereoselective determination of unchanged and glucuroconjugated eliprodil, a new anti-ischaemic drug, in human plasma and urine by precolumn derivatization and column-switching high-performance liquid chromatography with fluorescence detection.

An HPLC method was developed and validated for the determination in human plasma and urine of the enantiomers of eliprodil, (+/-)-alpha-(4-chlorophenyl)-4[(4-fluorophenyl) methyl]piperidine-1-ethanol hydrochloride, a new anti-ischaemic agent administered as a racemate. Both enantiomers are present in human plasma in unchanged and glucuroconjugated form, whereas only the glucuroconjugated form is excreted into urine; as a consequence, such metabolites in human plasma and urine should be submitted to enzymatic deconjugation with beta-glucuronidase (Escherichia coli) before being extracted. The general method involves a liquid-liquid extraction of eliprodil and internal standard from alkalinized plasma or urine with n-hexane, evaporation of the organic phase and derivatization with (S)-(+)-naphthylethyl isocyanate to give carbamate diastereoisomeric derivatives of (S)-(+)- and (R)-(-)-eliprodil and internal standard; after evaporation of the derivatizing mixture and dissolution of the residue in a small volume of phosphate buffer-acetonitrile (60:40, v/v), an aliquot is injected into a column-switching HPLC system. The derivatized sample extract is purified on a precolumn filled with C8-bonded silica material, which is flushed with acetonitrile-water, then diastereoisomers of eliprodil and the internal standard are automatically transferred by the mobile phase to the analytical column. The analytical column is a C8 type, specially deactivated for basic compounds, the mobile phase is 0.025 M phosphate buffer (pH 2.6)-methanol-acetonitrile (42:2:56) at a flow-rate of 1.2 ml min-1 and fluorimetric detector operating at lambda ex = 275 nm and lambda em = 336 nm is used. The retention times, under these conditions, are about 16 and 17 min for (S)-(+)- and (R)-(-)-eliprodil diastereoisomers, respectively, and about 19 min for the first-eluted diastereoisomer of the internal standard. During the analysis time, the precolumn, reset in a different path from that of the analytical column, is back-flushed with different solvents, then re-equilibrated with acetonitrile-water before the next injection. Linearity in plasma, for unchanged eliprodil enantiomers, was assessed in the range 0.15-10 ng ml-1 and for total eliprodil enantiomers (unchanged + conjugated) in the range 0.75-500 ng ml-1; the limit of quantitation (LOQ) is 0.15 ng ml-1 for each unchanged enantiomer and 0.75 ng ml-1 for each total enantiomer. Linearity was also assessed in urine for total (conjugated) eliprodil enantiomers in the range 50-25 000 ng ml-1; the LOQ is 50 ng ml-1 for each enantiomer. The intra- and inter-day precision and accuracy of the method were investigated in plasma and urine and found to be satisfactory for pharmacokinetic studies. The method has been extensively used in pharamcokinetic studies in man treated with a 20-mg dose of eliprodil racemate and some results of this application are reported.