Determination of indomethacin and mefenamic acid in plasma by high-performance liquid chromatography

Indomethacin and mefenamic acid are widely used clinically as non-steroidal anti-inflammatory agents. Both drugs have also been found effective to produce closure of patent ductus arteriosus in premature neonates. A simple, rapid, sensitive and reliable HPLC method is described for the determination of indomethacin and mefenamic acid in human plasma. As these drugs are not applied together, the compounds are alternately used as analyte and internal standard. Plasma was deproteinized with acetonitrile, the supernatant fraction was evaporated to dryness and the resulting residue was reconstituted in the mobile phase and injected into the HPLC system. The chromatographic separation was performed on a C18 column (250 x 4.6 mm I.D.) using 10 mM phosphoric acid-acetonitrile (40:60, v/v) as the mobile phase and both drugs were detected at 280 nm. The calibration graphs were linear with a correlation coefficient (r) of 0.999 or better from 0.1 to 10 micrograms/ml and the detection limits were 0.06 micrograms/ml for indomethacin and 0.08 micrograms/ml for mefenamic acid, for 50-microliters plasma samples. The method was not interfered with by other plasma components and has been found particularly useful for paediatric use. The within-day precision and accuracy of the method were evaluated for three concentrations in spiked plasma samples. The coefficients of variation were less than 5% and the accuracy was nearly 100% for both drugs.     

Quantitative determination of endogenous retinoids in mouse embryos by high-performance liquid chromatography with on-line solid-phase extraction, column switching and electrochemical detection

An isocratic high-performance liquid chromatographic method for the determination of 9-cis-retinoic acid, 13-cis-retinoic acid, all-trans-retinoic acid and all-trans-retinol in mouse embryos using on-line solid-phase extraction and column switching in combination with electrochemical detection has been developed. The method was validated using retinoids in albumin solutions and 13-cis-acitretin was used as internal standard. About 370 microliters of albumin solution was injected on a 10 x 2.1-mm I.D. pre-column packed with Bondapak C18, 37-53-micron particles. The proteins were washed to waste within 5 min using as mobile phase, a 1:3 dilution of mobile phase 2, which consisted of acetonitrile-methanol-2% ammonium acetate-glacial acetic acid (79:2:16:3, v/v). Components retained on the pre-column were back-flushed to and separated on the 250 x 4.6-mm I.D. Suplex pKb-100 analytical column using mobile phase 2. The retinoids were detected electrochemically at +750 mV using a coulometric electrochemical detector. The total analysis time was about 20 min. Recoveries were in the range of 86-103%. The mass limits of detection were about 10 pg and 25 pg for the retinoic acids and all-trans-retinol, respectively. The intra-assay precision, reported as relative standard deviation, was in general better than 4% (n = 6) for the four retinoids. Inter-assay precision was in the range 3-4% (n = 10). The method was applied for determination of endogenous retinoids in 9.5 day-old mouse embryos. A 340-microliter solution containing 100 microliters of embryo homogenate (1.64 embryos) was analyzed. The concentrations of all-trans-retinol and all-trans-retinoic acid were found to be 279 pg per embryo and 75.8 pg per embryo, respectively. The amount of 13-cis-retinoic acid and 9-cis-retinoic acid was below the detection limit.     

Determination of 13-cis-3-hydroxyretinoic acid, all-trans-3-hydroxyretinoic acid and their 4-oxo metabolites in human and animal plasma by high-performance liquid chromatography with automated column switching and UV detection

A high-performance liquid chromatographic method with automated column switching was developed for the simultaneous determination of 13-cis-3-hydroxyretinoic acid, all-trans-3-hydroxyretinoic acid and their metabolites 13-cis-3-hydroxy-4-oxo-retinoic acid and all-trans-3-hydroxy-4-oxo-retinoic acid in plasma samples from man, rat, dog, rabbit and mouse. The method consists of deproteination of plasma (0.4 ml) with ethanol (1.5 ml), containing the internal standard Ro 12-7310. After centrifugation, 1.4 ml of the supernatant was directly injected onto the precolumn (PC) (4 x 4 mm) packed with LiChrospher 100 RP-18 (5 microm). Ammonium acetate (0.02%)-acetic acid-ethanol (100:3:4, v/v/v) was used as mobile phase M1A during injection, as well as to decrease the elution strength of the injection solution by on-line addition using a T-piece (M1B). After valve switching, the retained components were transferred to the analytical column (AC), separated by gradient elution and detected at 360 nm. Two coupled Purospher 100 RP-18 endcapped columns (both 250 x 4 mm) were used for the separation, together with a mobile phase consisting of acetonitrile-water-10% ammonium acetate-acetic acid, (A), 540:450:2:30 (v/v/v/v), (B), 600:350:2:30 (v/v/v/v), and (C), 950:40:2:30 (v/v/v/v). The method was linear in the range 1-500 ng ml(-1), at least, with a quantification limit of 1 ng ml(-1). The mean recoveries from human plasma were 100-107% and the mean inter-assay precision was 2.0-4.7% (range 1-500 ng ml(-1)). Similar results were obtained for animal plasma. The analytes were stable in the plasma of all investigated species stored at -20 degrees C for 3 months, at least. The method was successfully applied to clinical and toxicokinetic studies.     

Analysis of the components of Lycopus europaeus L. in body fluids during metabolism studies. Comparison of capillary electrophoresis and high-performance liquid chromatography

During pharmacokinetic studies with extracts obtained from medicinally used plants, analysis in body fluids is mainly performed by HPLC, an established separation method. In this paper high-performance capillary electrophoresis (HPCE) is investigated for its ability to separate such complex extracts. Crude extracts of Lycopus europaeus L. (Lamiaceae) are traditionally used against mild forms of hyperthyroidism. The metabolism of a 70% ethanolic extract with respect to some of its individual main components (rosmarinic and caffeic acid, luteolin-7-glucoside) and a mixture of the pure compounds were investigated using isolated perfused rat liver. After solid-phase extraction metabolites were determined using HPCE and HPLC separation techniques. A buffer solution composed of 0.05 mol l-1 Na2HPO4 at pH 7.0 with 30% acetonitrile was found to be the most suitable electrolyte for HPCE separation. The best mobile phase for isocratic HPLC was 0.03% TFA-acetonitrile (82:18, v/v). Data obtained with HPCE are in good accordance with those from HPLC; HPCE, however, is clearly more rapid and simple to perform.     

Determination of rifampicin and its main metabolite in plasma and urine in presence of pyrazinamide and isoniazid by HPLC method

A reversed phase HPLC method is described for the simultaneous estimation of rifampicin and its major metabolite desacetyl rifampicin, in the presence of isoniazid and pyrazinamide, in human plasma and urine. The assay involves simple liquid extraction of drug, metabolite and internal standard (rifapentine) from biological specimens and their subsequent separation on a C18 reversed phase column and single wavelength UV detection. In plasma as well as in urine samples, all the three compounds of interest eluted within 17 min. Using methanol-sodium phosphate buffer (pH 5.2; 0.01 M) (65:35, v/v) as mobile phase under isocratic conditions, it was established that isoniazid, pyrazinamide and ascorbic acid (added to prevent oxidative degradation of analytes) did not interfere with the analyte peaks. Recoveries (extraction efficiency) for drug were greater than 90% in both plasma and urine, whereas for metabolite the values were found to be 79 and 86% in plasma and urine, respectively. The plasma and urine methods were precise (total coefficient of variation ranged from 5 to 23%) and accurate (-7 to 5% of the nominal values) for both the analytes. Individual variance components, their estimates and their contribution to the total variance were also determined. Using the same method, unknown samples supplied by WHO were assayed and good correlations were obtained between the found and intended values. The method developed proved to be suitable for simultaneous estimation of rifampicin and desacetyl rifampicin in plasma and urine samples.     

Determination of aspirin and salicylic acid in transdermal perfusates

A high-performance liquid chromatographic (HPLC) method has been developed for the simultaneous determination of aspirin and salicylic acid in transdermal perfusates. The compounds were separated on a C8 Nucleosil column (5 microm, 250x4.6 mm) using a mobile phase containing a mixture of water-acetonitrile-orthophosphoric acid (650:350:2, v/v/v) and a flow-rate of 1 ml/min. The transdermal samples were in phosphate-buffered saline (PBS) and could be injected directly onto the HPLC system. The method was reproducible with inter-day R.S.D. values of no greater than 3.46 and 2.60% for aspirin and salicylic acid, respectively. The method was linear over the concentration range 0.2-5.0 microg/ml and had a limit of detection of 0.05 microg/ml for both compounds. For certain samples, it was necessary to ensure that no transmembrane leakage of the aspirin prodrugs had occurred. In these cases, a gradient was introduced by increasing the acetonitrile content of the mobile phase after the salicylic acid had eluted. The method has been applied to the determination of aspirin and salicylic acid in PBS following in vitro application of the compounds to mouse skin samples.     

Simple high-performance liquid chromatographic method for determination of losartan and E-3174 metabolite in human plasma, urine and dialysate

A simple high-performance liquid chromatographic (HPLC) method was developed for the determination of losartan and its E-3174 metabolite in human plasma, urine and dialysate. For plasma, a gradient mobile phase consisting of 25 mM potassium phosphate and acetonitrile pH 2.2 was used with a phenyl analytical column and fluorescence detection. For urine and dialysate, an isocratic mobile phase consisting of 25 mM potassium phosphate and acetonitrile (60:40, v/v) pH 2.2 was used. The method demonstrated linearity from 10 to 1000 ng/ml with a detection limit of 1 ng/ml for losartan and E-3174 using 10 microl of prepared plasma, urine or dialysate. The method was utilized in a study evaluating the pharmacokinetic and pharmacodynamic effects of losartan in patients with kidney failure undergoing continuous ambulatory peritoneal dialysis (CAPD).     

High-performance liquid chromatography determination of mycophenolic acid and its glucuronide metabolite in human plasma

Two HPLC-UV assays are reported here: one is a rapid assay for mycophenolic acid (MPA) and the other is a simultaneous assay for MPA and its metabolite mycophenolic acid glucuronide (MPAG). For both methods, plasma samples (500 microl) with added internal standard were acidified and extracted using C18 solid-phase extraction cartridges. Chromatographic separation was achieved on a C18 Novapak column using a mobile phase consisting of methanol-0.05% orthophosphoric acid (40:60, v/v) for the rapid MPA assay and 30:70 for the simultaneous MPA and MPAG assay. The assays were linear over the ranges 0.1 to 50.0 mg/l for MPA and 2.8 to 225.8 mg/l for MPAG. Mean absolute recovery for all analytes was >99%. These methods are suitable for therapeutic drug monitoring and pharmacokinetic studies.     

Determination of naringenin and its glucuronide conjugate in rat plasma and brain tissue by high-performance liquid chromatography

An isocratic high-performance liquid chromatographic method with ultraviolet detection was utilized for the investigation of the pharmacokinetics of naringenin and its glucuronide conjugate in rat plasma and brain tissue. Plasma and brain tissue were deproteinized by acetonitrile, then centrifuged for sample clean-up. The drugs were separated by a reversed-phase C18 column with a mobile phase consisting of acetonitrile-orthophosphoric acid solution (pH 2.5-2.8) (36:64, v/v). The detection limits of naringenin in rat plasma and brain tissue were 50 ng/ml and 0.4 microg/g, respectively. The glucuronide conjugate of naringenin was evaluated by the deconjugated enzyme beta-glucuronidase. The naringenin conjugation ratios in rat plasma and brain tissue were 0.86 and 0.22, respectively, 10 min after naringenin (20 mg/kg, i.v.) administration. The mean naringenin conjugation ratio in plasma was approximately four fold that in brain tissue.     

Simultaneous determination of guanine, uric acid, hypoxanthine and xanthine in human plasma by reversed-phase high-performance liquid chromatography with amperometric detection

A reversed-phase high-performance liquid chromatographic method with amperometric detection is described for the separation and quantification of uric acid, guanine, hypoxanthine and xanthine. The isocratic separation of a standard mixture of the compounds was achieved in 5 min on a Spherisorb 5 C18 reversed-phase column, with a mobile phase of NaH2PO4 (300 mmol dm-3, pH 3.0)-methanol-acetonitrile-tetrahydrofuran (97.8 + 0.5 + 1.5 + 0.2). Uric acid, guanine, hypoxanthine and xanthine were completely separated, with detection limits in the range 2-20 pmol per injection. The effect of pH and the composition of the mobile phase on the separation are described. The hydrodynamic voltammograms of these compounds were recorded at a glassy carbon electrode. The linear range of the calibration graph for each compound was: uric acid, 1-5000 mumol dm-3; guanine, 0.5-2000 mumol dm-3; hypoxanthine, 0.1-500 mumol dm-3 and xanthine, 0.5-5000 mumol dm-3. The within- and between-day precision was good. The uric acid and hypoxanthine content in human plasma was measured using the proposed method. Good recoveries of uric acid (97.9-103%), hypoxanthine (98.0-99.2%), guanine (96.0-98.3%) and xanthine (96.0-102%) were obtained from human plasma. The results of electrochemical detection were in good agreement with those of UV detection.     

Delivery of salbutamol to nonventilated preterm infants by metered-dose inhaler, jet nebulizer, and ultrasonic nebulizer

An isocratic reversed-phase high-performance liquid chromatographic method with ultraviolet detection at 230 nm has been developed for the determination of paclitaxel in human plasma. Plasma samples were prepared by a selective one-step liquid-liquid extraction involving a mixture of acetonitrile-n-butyl chloride (1:4, v/v). Paclitaxel and the internal standard docetaxel were separated using a column packed with ODS-80A material, and a mobile phase consisting of water-methanol-tetrahydrofuran-ammonium hydroxide (37.5:60:2.5:0.1, v/v). The calibration graph for paclitaxel was linear in the range 10-500 ng/ml, with a lower limit of quantitation of 10 ng/ml, using 1 ml plasma samples. The extraction recoveries of spiked paclitaxel and docetaxel to drug-free human plasma were 89.6+/-8.52 and 93.7+/-5.0%, respectively. Validation data showed that the assay for paclitaxel is sensitive, selective, accurate and reproducible. The assay has been used in a single pharmacokinetic experiment in a patient to investigate the applicability of the method in vivo.     

Determination of ciprofloxacin levels in chinchilla middle ear effusion and plasma by high-performance liquid chromatography with fluorescence detection

An isocratic high-performance liquid chromatographic method has been developed to determine ciprofloxacin levels in chinchilla plasma and middle ear fluid. Ciprofloxacin and the internal standard, difloxacin, were separated on a Keystone ODS column (100 x 2.1 mm I.D., 5 microns Hypersil) using a mobile phase of 30 mM phosphate buffer (pH 3), 20 mM triethylamine, 20 mM sodium dodecyl sulphate-acetonitrile (60:40, v/v). The retention times were 3.0 min for ciprofloxacin and 5.2 min for difloxacin. This fast, efficient protein precipitation procedure together with fluorescence detection allows a quantification limit of 25 ng/ml with a 50 microliters sample size. The detection limit is 5 ng/ml with a signal-to-noise ratio of 5:1. Recoveries (mean +/- S.D., n = 5) at 100 ng/ml in plasma and middle ear fluid were 89.4 +/- 1.2% and 91.4 +/- 1.6%, respectively. The method was evaluated with biological samples taken from chinchillas with middle ear infections after administering ciprofloxacin.     

Chromatographic studies on the isolation of peroxydisulphate oxidation products of primaquine

Eight compounds from peroxydisulphate oxidation of primaquine were fractionated on Bio-Gel P-2 column using water as an eluent. A HPLC method employing acetonitrile-methanol-1 M perchloric acid-water (30:7:1:95, v/v) as a mobile phase at 1.0 ml/min on microBondapak reversed-phase column and UV detection at 254 nm was developed for the separation and identification of different oxidation products of primaquine. A combination of Bio-Gel chromatography with reversed-phase HPLC was found to be the most suitable analytical technique for the semipreparative isolation of various products formed from the oxidation. Two oxidation products that were isolated had three or four times higher gametocytocidal activity as compared to primaquine.     

Genetic polymorphisms in glutathione S-transferase mu and theta, N-acetyltransferase, and CYP1A1 and risk of gliomas

The simultaneous determination of betamethasone dipropionate (BD) and salicylic acid (SA) in both ointment and topical solution was developed using high-performance liquid chromatography (HPLC). The method was standardized using a LiChrospher 100 RP-18 (125 x 4 mm, 5 microns) column, acetonitrile-tetrahydrofuran-acetic acid 1% (25:20:55 v/v), apparent pH 3.3, as mobile phase, and UV detection at 254 nm. The peak area response versus concentration was linear in a concentration range from 5.0 to 50.0 micrograms/ml of BD and from 20.0 to 200.0 micrograms/ml of SA. The correlation coefficients were 0.9997 for BD and 0.9987 for SA, and the relative standard errors of estimates were 1.38% for BD and 3.27% for SA. The coefficient of variation and the recovery average were, respectively, 0.41-1.15% and 100.09% for BD, and 0.57-0.95% and 99.79% for SA.     

Determination of artemether and its major metabolite, dihydroartemisinin, in plasma using high-performance liquid chromatography with electrochemical detection

A rapid, selective, sensitive and reproducible HPLC with reductive electrochemical detection for quantitative determination of artemether (ART) and its plasma metabolite, dihydroartemisinin (DHA: alpha and beta isomers) in plasma is described. The procedure involved the extraction of ART, DHA and the internal standard, artemisinin (ARN) with dichloromethane-tert.-methylbutyl ether (1:1, v/v) or n-butyl chloride-ethyl acetate (9:1, v/v). Chromatographic separation was performed with a mobile phase of acetonitrile-water (20:80, v/v) containing 0.1 M acetic acid pH 5.0, running through a microBondapak CN column. The method was capable of separating the two isomeric forms of DHA (alpha, beta). The retention times of alpha-DHA, beta-DHA, ARN and ART were 4.6, 5.9, 7.9 and 9.6 min, respectively. Validation of the assay method was performed using both extraction systems. The two extraction systems produced comparable recoveries of the various analytes. The average recoveries of ART, DHA and ARN over the concentration range 80-640 ng/ml were 86-93%. The coefficients of variation were below 10% for all three drugs (ART, alpha-DHA, ARN). The minimum detectable concentrations for ART and alpha-DHA in spiked plasma samples were 5 and 3 ng/ml, respectively. The method was found to be suitable for use in clinical pharmacokinetic study.     

Percutaneous nephrostolithotomy. What is its role in 1997?

A fully automated isocratic high-performance liquid chromatographic method for the determination of 9-cis-retinoic acid, 13-cis-retinoic acid, all-trans-retinoic acid, 4-oxo-13-cis-retinoic acid and 4-oxo-all-trans-retinoic acid, has been developed using on-line solid-phase extraction and a column switching technique allowing clean-up and pre-concentration in a single step. A 500-microliter sample of serum was diluted with 750 microliters of a solution containing 20% acetonitrile and the internal standard 9,10-dimethylanthracene. About 1000 microliters of this mixture was injected on a 20 x 4.6 mm I.D. poly ether ether ketone (PEEK) pre-column with titanium frits packed with Bondapak C18, 37-53 microns, 300 A particles. Proteins and very polar compounds were washed out to waste, from the pre-column, with 0.05% trifluoroacetic acid (TFA)-acetonitrile (8.5:1.5, v/v). More than 200 aliquots of diluted serum could be injected on this pre-column before elevated back-pressure enforces replacement. Components retained on the pre-column were backflushed to the analytical column for separation and detection at 360 nm. Baseline separation was achieved using a single 250 x 4.6 mm I.D. Suplex pKb-100 column and a mobile phase containing 69:10:2:16:3 (v/v) of acetonitrile-methanol-n-butanol-2% ammonium acetate-glacial acetic acid. A total time of analysis of less than 30 min, including sample preparation, was achieved. Recoveries were in the range of 79-86%. The limit of detection was 1-7 ng/ml serum and the precision, in the concentration range 20-1000 ng/ml, was between 1.3 and 4.5% for all five compounds. The method was applied for the analysis of human serum after oral administration of 60 mg Roaccutan. The method is well suited for pharmacological studies, while the endogenous levels of some retinoic acid isomers are below the limit of quantitation.     

The correction of the immune and microcirculatory disorders in patients with chronic hepatitis of alcoholic and viral etiologies

A simple, rapid and sensitive method for the clean-up and analysis of cefoxitin in serum and tissue is described. Serum (0.5 ml) and tissue (100 mg) samples after homogenization underwent high speed centrifugation. Chromatography was performed on a muBondapak C18 cartridge using a mobile phase of 0.005 M potassium dihydrogen phosphate-acetonitrile-glacial acetic acid (77.5:22:0.5, v/v/v) with a flow-rate of 2.0 ml/min. Ultraviolet detection occurred at 235 nm. The procedure produced a linear curve for the concentration range 100-5000 ng/ml. The assay produced accurate, repeatable and rapid results for both tissue and serum samples without the need for chemical extraction.     

The interaction of antitumor-active anthraquinones with biologically important redox couples: I. Spectrophotometric investigation of the interaction of carminic acid and mitoxantrone with the iron (II, III) and copper (I, II) redox couples

Studying the interaction of antitumor-active anthraquinones with biologically important redox couples is important in understanding the possible reductive or oxidative mode of metabolism of these antineoplastic agents coupled with the formation of free radicals. The interactions of such anthraquinones, i.e., carminic acid (CA) and mitoxantrone (Mx) with iron(II, III) and copper(I, II) redox couples in oxygenated and deaerated solutions, were investigated by UV-Visible and IR-spectroscopy. The superoxide radical reagent, nitroblue tetrazolium (NBT), was added to the metal and anthraquinone solutions and their binary mixtures at varying pH. Formazan, the reduction product of NBT, was produced mainly as a result of Fe(II)-NBT and Fe(II)-Mx-NBT interactions. The ternary mixtures of the lower valencies of iron and copper with CA and NBT exhibited intensive charge-transfer bands in the visible region, while metal-Mx-NBT combinations did not produce such bands, possibly due to the blockage of the redox-active aminoethanolamine side-chains of Mx through coordination with the metals. Copper-Mx combinations showed an oxygen sensitivity as spectral evidence was obtained for the oxidative transformation of Mx to the cyclic primary metabolite. The results were evaluated in regard to the possible oxidative activation of the studied anthracenediones with iron and copper systems.     

Anti-tumor gene therapy

A high-performance liquid chromatographic method with ultraviolet detection is described for the simultaneous measurement of pyrimethamine and sulphadoxine in human plasma. After an automated liquid-solid extraction on a C8 cartridge, the compounds are separated on a C18 column by isocratic elution; the mobile phase is methanol-acetonitrile-water (10:25:65, v/v/v) with triethylamine (1%) and adjusted to pH 5.6 with phosphoric acid. The eluent is monitored with an ultraviolet detector at 240 nm. The limit of quantification was 10 ng/ml for pyrimethamine and 22 microg/ml for sulphadoxine. No chromatographic interferences can be detected from endogenous compounds, other anti-malarial drugs or major drugs used for the treatment of children. Sulphadimethoxine is used as an internal standard. The method is accurate and precision is good with relative standard deviations lower than 6%. The chromatographic procedure takes 11 min. The method is comparatively rapid, simple, sensitive and can be used for therapeutic drug monitoring, clinical and pharmacokinetic studies.     

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.