On-line microdialysis coupled with microbore liquid chromatography for the determination of unbound chloramphenicol and its glucuronide in rat blood

On-line microdialysis coupled with microbore liquid chromatography was used to investigate the pharmacokinetics of chloramphenicol and its glucuronide in rat blood. A microdialysis probe was inserted into a jugular vein of male Sprague–Dawley rats. Chloramphenicol succinate (20 mg/kg, intravenously) was then administered via a femoral vein. Dialysates were automatically injected onto a LC system, via an on-line injector. Samples were eluted with a mobile phase containing acetonitrile-10 mM monochloroacetic acid (30:70, v/v, pH 3.0). The UV detector wavelength was set at 278 nm. The limit of quantitation for chloramphenicol was 10 ng/ml. The in vitro recoveries of chloramphenicol and chloramphenicol glucuronide at 500 ng/ml were 32.2±0.3% and 11.4±0.7%, respectively (n=6). Intra- and inter-assay accuracy and precision of the analyses were ≤10% in the range of 0.01 to 5.0 μg/ml.     

Simultaneous measurement of blood and brain microdialysates of granisetron in rat by high-performance liquid chromatography with fluorescence detection

Simultaneous microdialysis probes in the blood and brain and sensitive high-performance liquid chromatography with fluorescence detection were used to examine the granisetron concentration in the jugular vein and frontal cortex of rats after drug administration. Two microdialysis probes were inserted into the right jugular vein and frontal cortex of male Sprague–Dawley rats to which granisetron (6 mg/kg, i.v.) had been administered. Dialysates were automatically collected using a microfraction collector. Samples were eluted with a mobile phase containing 25 mM acetate buffer (pH 4.8)–acetonitrile (72:28, v/v). Excitation and emission wavelengths were set at 305 and 360 nm, respectively, on a scanning fluorescence detector. The limit of quantification for granisetron was 0.5 ng/ml. The in vitro recovery of granisetron was 29.7±1.2% (n=6) for the jugular vein microdialysis probe and 6.1±0.5% (n=6) for the frontal cortex microdialysis probe. The increasing brain/blood concentration ratio of granisetron suggests that granisetron penetrates the blood–brain barrier.     

Determination of telithromycin in human plasma and microdialysates by high-performance liquid chromatography

A high-performance liquid chromatography method for the quantitative determination of telithromycin in biological fluids is described. The method is suitable for plasma and microdialysates from the interstitial space fluid of skeletal muscle and subcutaneous adipose tissue. Plasma samples were deproteinised with trichloroacetic acid and neutralised with sodium hydroxide. Microdialysates were analysed without further preparation step. Telithromycin was separated isocratically on a reverse-phase column using acetonitrile-0.03 M ammonium acetate, pH 5.2 (43:57, v/v) at a flow rate of 0.8 mlmin(-1), and fluorescence detection (excitation 263 nm, emission 460 nm). The calibration curve was linear from 0.01 to 5 microgml(-1). Within- and between-day imprecision and inaccuracy was < or =10%. The limits of quantification were 0.02 and 0.015 microgml(-1) for plasma and microdialysates, respectively. Since telithromycin is decomposed in aqueous solution at ambient temperature, it is strongly recommended to store samples frozen at -80 degrees C, to maintain the temperature at 4 degrees C during all preparation steps, and to analyse samples within 120 min after thawing.     

On-line solid-phase extraction of ceftazidime in serum and determination by high-performance liquid chromatography

A column-switching high-performance liquid chromatographic assay is described for the determination of ceftazidime (a third-generation cephalosporin) in human serum. The method does not require prior sample pretreatment. Serum is directly injected in a first chromatographic column for sample clean-up and extraction. Thereafter, using an on-line column-switching system, the drug is quantitatively transferred and separated on a second, analytical column followed by determination using ultraviolet absorption at 258 nm. The technique allows direct, rapid, precise, and simple determination of ceftazidime in serum over the range of 1–250 μg/ml using 12.5 μl of serum. This method was applied to study the pharmacokinetics of the drug in patients undergoing vascular surgery.     

Determination of the dopamine agonist rotigotine in microdialysates from the rat brain by microbore column liquid chromatography with electrochemical detection

Rotigotine, an investigational dopamine agonist formulated as a patch, is being studied in Parkinson's disease. A microdialysis technique, in combination with microbore column liquid chromatography and electrochemical detection, was developed to monitor rotigotine levels in the brain. Microdialysis probes were inserted into the striata of anesthetized rats, and samples were collected during perfusion with Ringer's solution. Rotigotine was separated using a C18 reversed-phase column. The mobile phase consisted of 50mM Na(2)HPO(4) x 2H(2)O, 2.5 mM sodium octyl sulfonate, and pH 4.5; 35% volume to volume acetonitrile. The flow rate was 30 microl/min, and the potential of the glassy carbon electrode was set to +850 mV. The method allowed monitoring of the time course of brain extracellular rotigotine levels with a detection limit of 1 nM following either intravenous (0.5 mg/kg) or subcutaneous (5.0 mg/kg) rotigotine injection.     

Determination of lauroyl-indapamide in rat whole blood by high-performance liquid chromatography

A method based on a liquid-liquid extraction procedure followed by high-performance liquid chromatography (HPLC) coupled with UV-visible detection is described and validated for the determination of lauroyl-indapamide in rat whole blood. The blood sample was extracted with diethyl ether after the addition of 10% trifluoroacetic acid (aq.). The chromatographic separation was performed on a Chromasil ODS column, using methanol-acetonitrile-tetrahydrofuran-0.2% trifluoroacetic acid (170:20:15:38, v/v/v/v) as the mobile phase. The UV detection wavelength was set at 240 nm. The extraction recovery of lauroyl-indapamide was ranged from 76.5 to 82.6%, and the calibration curve had a good linearity in the range of 0.048-200 microg/ml (r = 0.9976). The method presents appropriate intra-day and inter-days repeatabilities, showing values below 7.4% in terms of the percentage of relative standard deviation (R.S.D.). The method proposed is simple, rapid and sensitive, being useful for pharmacokinetic studies in rats.     

Simple determination of cyclosporine in human whole blood by high-performance liquid chromatography

A simple and reproducible high-performance liquid chromatography (HPLC) method was developed for determination of cyclosporine (CyA, also known as cyclosporin A) in human whole blood. The method entailed direct injection of the blood samples after deproteination using acetonitrile. Chromatography was carried out using an ODS column under isocratic elution with acetonitrile-5mM disodium hydrogen phosphate (75:25, v/v), pH 5.1 at 70 degrees C and a detector set at 210 nm. The mean absolute recovery of cyclosporine from blood was 97%, and the linearity was assessed in the range of 100-3000 ng/ml blood, with a correlation coefficient of greater than 0.999. The limit of quantification and detection of the present method were 100 and 50 ng/ml, respectively. This method has been used to analyze several hundred human blood samples for bioavailability studies.     

Chiral determination of mirtazapine in human blood plasma by high-performance liquid chromatography

A method is described for the determination of the two enantiomers of mirtazapine in human blood plasma by high-performance liquid chromatography. Measurements were performed on drug free plasma spiked with mirtazapine and used to prepare and validate standard curves. Levels of enantiomers of mirtazapine were also measured in patients being treated for depression with racemic mirtazapine. Mirtazapine was separated from plasma by solid-phase extraction using CERTIFY columns. Chromatographic separation was achieved using a Chiralpak AD column and pre-column and compounds were detected by their absorption at 290 nm. Imipramine was used as an internal standard. The assay was validated for each analyte in the concentration range 10–100 ng/ml. The coefficient of variance was 16% and 5.5% for(+)-mirtazapine for 10 and 100 ng/ml control specimens respectively and 15% and 7.3% for mirtazapine for 10 and 100 ng/ml control specimens respectively. This assay is appropriate for use in the clinical range. The range of plasma mirtazapine concentrations from eleven patients taking daily doses of 30–45 mg of racemate was <5 to 69 ng/ml for (+)-mirtazapine and 13–88 ng/ml for (−)-mirtazapine for blood specimens collected 10–17.5 h after taking the dose.     

Simultaneous determination of catecholamines in rat brain tissue by high-performance liquid chromatography

A novel and highly sensitive method has been developed for the determination of catecholamines [noradrenaline (NA), dopamine (DA), serotonin (5-HT) and their metabolites 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA)] in brain tissue. The method uses isocratic reversed-phase HPLC with amperometric end-point detection. The calibration curve was linear over the range 10–150 pg on-column. The assay limits of detection for NA, DA, 5-HT, 5-HIAA and HVA were 3.8, 3.8, 6.8, 5 and 7.5 pg on-column, respectively. The mean inter- and intra-assay relative standard deviations (RSDs) over the range of the standard curve were less than 5%. The absolute recoveries averaged 99.1%, 99.5%, 97.7%, 99.5% and 98.8% for NA, DA, 5-HT, 5-HIAA and HVA, respectively.     

Quantitative determination of Aconitum alkaloids in blood and urine samples by high-performance liquid chromatography

An HPLC method has been developed for the simultaneous determination of the toxic Aconitum alkaloids, aconitine, mesaconitine and hypaconitine in blood and urine samples. The samples were initially subjected to solid phase extraction using Oasis MCX cartridges, and the alkaloids were separated on an XTerra RP18 column, gradient-eluted with acetonitrile: ammonium hydrogen carbonate buffer. Calibration curves were linear in the range 2.75-550 ng for aconitine and hypaconitine, and 3-600 ng for mesaconitine: the limit of detection was 0.1 ng (signal-to-noise ratio of 3) for each alkaloid. The described analysis proved to be sensitive, rapid and economical, and will be applied in the identification and determination of these alkaloids in forensic and therapeutic drug monitoring.     

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.     

Sensitive detection of human globin chains by microbore high-performance liquid chromatography and its forensic application

This paper describes a simple and sensitive high-performance liquid chromatographic (HPLC) method for the detection of human globin chains in blood and bloodstains. The method involves direct injection of the filtered samples of dilute hemolysates or bloodstain extracts onto a microbore C₄ reversed-phase column (2.1 mm I.D.) with UV detection at 220 nm. Microbore HPLC offers a significant improvement in sensitivity with little loss of the resolution of globin chains and only small variations in the determination of γ chain composition. The detection limit of hemoglobin (Hb) was 0.1 μg, which is equivalent to about 1 nl of fresh whole blood. Umbilical cord blood could be differentiated from adult blood in stains that were up to twenty weeks old, by the presence of γ globin chains. The present method will be useful for detection of abnormal Hbs and for the determination of γ chain composition in clinical laboratories, as well as in the practice of forensic science for the analysis of minute amounts of blood and bloodstains.     

Free malonaldehyde determination in tissues by high-performance liquid chromatography

A high-performance liquid chromatographic method was developed for the quantification of free malonaldehyde (MA) in tissues. HPLC separation was performed using a TSK G1000 PW column (7.5-mm i.d. X 30 cm) with a mobile phase of 0.1 M Na3PO4 buffer, pH 8.0, at a flow rate of 0.6 ml/min. The eluant was monitored at 267 nm. Free MA in the tissue sample was separated and quantified in approximately 50 min. The lowest amount of MA that can be determined by this HPLC technique is approximately 1 ng per injection. This method was successfully applied to rat liver and beef, pork, and chicken muscle and was compared to the thiobarbituric acid (TBA) test. It was found to be more sensitive, accurate, and specific for the determination of free MA than the TBA method.     

Measurement of nitric oxide metabolites in brain microdialysates by a sensitive fluorometric high-performance liquid chromatography assay

Nitric oxide (NO), formed from arginine by a specific neuronal NO synthase, is an important neurotransmitter in various regions of the central nervous system. While intracerebral microdialysis is an elegant technique to study local extracellular neurotransmitter concentrations in vivo, NO metabolites (nitrate, nitrite (NO(x))) are difficult to study at high temporal resolution because of low tissue concentrations and small sample volumes. We developed a sensitive fluorometric high-performance liquid chromatography (HPLC)-coupled NO(x) assay adapted for the use in brain microdialysate samples. The assay includes an initial enzymatic step in which nitrate is reduced to nitrite. Nitrite is acidified to N2O3, which reacts with 2,3-diaminonaphthalene to form 1-(H)-naphthotriazole. This reaction product can be readily isolated and quantitated by HPLC with fluorometric detection. The theoretical assay sensitivity is less than 1 nM, but numerous sources of contamination must be eliminated in the sampling and assaying process to reliably monitor brain NO(x) outflow by microdialysis.     

Determination of unbound cefmetazole in rat blood by on-line microdialysis and microbore liquid chromatography: a pharmacokinetic study

A specific and sensitive microbore liquid chromatographic method for the determination of unbound cefmetazole in rat blood was developed. A microdialysis probe was inserted into the jugular vein/right atrium of a Sprague–Dawley rat. Cefmetazole (10 mg/kg, i.v.) was then administered via the femoral vein. Dialysates were automatically injected into a liquid chromatographic system via an on-line injector. Isocratic elution of cefmetazole was achieved by LC–UV within 10 min. Intra- and inter-assay accuracy and precision of the assay were 10%. The detection limit of cefmetazole was 20 ng/ml. Pharmacokinetic analysis of results indicated that unbound cefmetazole levels in rats best fit a biexponential decay model.     

Analysis of barbiturates in blood by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) assay for the identification and quantification of barbiturates in blood at therapeutic levels has been developed. An ODS-silica column is used with an eluent of 40% methanol at pH 8.5. The barbiturates are detected at 240 nm. The sample preparation procedure involves extraction of unfractionated blood (100 μl) with hexane—diethyl ether (50:50, v/v) and is very rapid. Talbutal is used as an internal standard.The method has been applied to the determination of five barbiturates (amylobarbitone, butobarbitone, cyclobarbitone, pentobarbitone and quinalbarbitone) in blood after therapeutic doses of the drugs. An application of the HPLC assay to forensic casework is demonstrated.     

Quantitative determination of domperidone in rat plasma by high-performance liquid chromatography with fluorescence detection

A sensitive and selective analytical method for the determination of domperidone in rat plasma is described. The procedure involves liquid–liquid extraction followed by reversed-phase high-performance chromatographic analysis with fluorometric detection at 282 nm for excitation and 328 nm for emission. The detection limit was 1 ng ml⁻¹ using 1 ml of plasma. This assay procedure should be useful for the pharmacokinetic study of domperidone in small animals such as rats.     

Simultaneous determination of felbamate and four metabolites in rat cerebrospinal fluid by high-performance liquid chromatography

An isocratic liquid chromatographic method for direct sample injection has been developed for the quantitation of felbamate and four metabolites in rat cerebrospinal fluid. The method uses 0.050- or 0.025-ml aliquots of cerebrospinal fluid diluted with equal volumes of internal standard. Chromatography is performed on a 150 mm × 4.6 mm I.D. Spherisorb ODS2, 3-μm HPLC column eluted with a phosphate buffer—acetonitrile—methanol (820:120:60, v/v/v) mobile phase and ultraviolet absorbance detection at 210 nm. The linear quantitation ranges are: felbamate and the 2-hydroxy metabolite 0.195–200 μg/ml, the propionic acid metabolite 0.195–50.0 μg/ml, the p-hydroxy metabolite 0.781 to 50.0 μg/ml, and the monocarbamate metabolite 0.098–50.0 μg/ml.