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.     

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 μg/g, respectively. The glucuronide conjugate of naringenin was evaluated by the deconjugated enzyme β-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.     

Measurement of unbound caffeic acid in rat blood by on-line microdialysis coupled with liquid chromatography and its application to pharmacokinetic study

To monitor the levels of caffeic acid in rat blood, an on-line microdialysis system coupled with liquid chromatography was developed. The microdialysis probe was inserted into the jugular vein/right atrium of male Sprague-Dawley rats. Caffeic acid (100 mg/kg, i.v.) was then administered via the femoral vein. Dialysates were automatically injected onto a liquid chromatographic system via an on-line injector. Samples were eluted with a mobile phase containing methanol–100 mM monosodium phosphoric acid (35:65, v/v, pH 2.5). The UV detector wavelength was set at 320 nm. The detection limit of caffeic acid was 20 ng/ml. The in vivo recoveries of the microdialysis probe for caffeic acid at 0.5 and 1 μg/ml were 48.34±2.68 and 47.64±3.43%, respectively (n=6). Intra- and inter-assay accuracy and precision of the analyses were ≤10% in the range of 0.05 to 10 μg/ml. Pharmacokinetics analysis of results obtained using such a microdialysis–chromatographic method indicated that unbound caffeic acid in the rat fitted best to a biexponential decay model.     

Quantitative determination of olanzapine in rat brain tissue by high-performance liquid chromatography with electrochemical detection

A sensitive assay was developed for the measurement of olanzapine in rat brain tissue using HPLC with electrochemical detection. The assay has a lower limit of quantitation of 0.5 ng/ml in tissue homogenate and utilizes a liquid–liquid extraction followed by reversed-phase HPLC for the quantitative analysis of olanzapine. The method provided a linear response for olanzapine over a concentration range of 0.5–100 ng/ml with a coefficient of determination (r²) greater than 0.9995. The extraction efficiencies of olanzapine and internal standard (LY170158) were greater than 82% in brain tissue. The intra-assay and inter-assay relative errors ranged from −5.38 to 17.60% and −3.25 to 10.53%, respectively. The intra-assay and inter-assay RSD values were in the range of 1.12 to 6.96% and 3.78 to 6.68%. Long-term stability studies showed that brain tissue homogenate samples spiked with olanzapine and internal standard are stable at −70°C for at least 110 days. However, a room temperature stability study showed that olanazapine was not stable in brain homogenate if the sample was exposed at 25°C longer than 2 h. This method has been used for the study of the disposition and pharmacokinetics of olanzapine in male Sprague–Dawley rats.     

Determination and pharmacokinetic study of taxifolin in rabbit plasma by high-performance liquid chromatography

Taxifolin has been widely used in the treatment of cerebral infarction and sequelae, cerebral thrombus, coronary heart disease and angina pectoris. A reliable sensitive reversed-phase high-performance liquid chromatography (RP-HPLC) method with UV detection for the pharmacokinetic study of taxifolin in rabbit plasma after enzymatic hydrolysis was developed and validated for the first time. Taxifolin, with biochanin A as the internal standard, was extracted from plasma samples by liquid/liquid extraction after hydrolysis with β-glucuronidase and sulfatase. Chromatographic separation was conducted on a Luna C18 column (4.6 mm×150 mm, 5 μm particle size) and pre-column (2.0 mm, the same sorbent). Two-step linear gradient elution with acetonitrile and 0.03% water solution of trifluoroacetic acid as mobile phase at a flow rate of 1.0 ml/min was used. The UV detector is set at 290 nm. The elution time for taxifolin and biochanin A was approximately 7.9 and 18.3 min, respectively. The calibration curve of taxifolin was linear (r>0.9997) over the range of 0.03–5.0 μg/ml in rabbit plasma. The limit of detection (LOD) and limit of quantification (LOQ) for taxifolin were 0.03 and 0.11 μg/ml, respectively. The present method was successfully applied for the estimation of the pharmacokinetic parameters of taxifolin following intravenous and oral administration of lipid solution to rabbits. The absolute bioavailability of taxifolin after oral administration of lipid solution was 36%.     

Measurement of free and esterified carnitine in tissue extracts by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) method for the determination of L-carnitine in clamped and frozen rat livers is described. L-carnitine + acetyl-CoA in equilibrium with acetyl-L-carnitine + CoASH Using the above enzymatic reaction, release of CoASH is stoichiometric with the L-carnitine added. The present method has made possible the determination of carnitine in liver tissues, which is difficult by the conventional enzymatic spectrophotometric method using 5,5'-dithiobis(2-nitrobenzoic acid), owing to acetyl-CoA hydrolysis during prolonged incubations at pH 7.8.     

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.     

Tryptophan hydroxylase system in brain tissue slices assayed by high-performance liquid chromatography

A new method was developed to study the unsupplemented tryptophan hydroxylase system in brain tissue slices from the raphe nuclei of the rat by high-performance liquid chromatography (HPLC) with fluorescence detection. Tryptophan hydroxylase activity was measured by determining 5-hydroxytryptophan (5-HTP) accumulation in raphe nuclei slices containing all of the enzyme system (the hydroxylase, tetrahydrobiopterin, and dihydropteridine reductase) in the presence of NSD-1055 (an inhibitor of aromatic l-amino acid decarboxylase). An optimum temperature was observed at 25°C and the reaction progressed linearly for 60 min. The hydroxylation of tryptophan was maximal by the addition of 0.2 mM tryptophan in the medium. A maximum 1.5-fold activation was shown at 0.2 mM 6-methyltetrahydropterin in the presence of 10 mM dithiothreitol. Dithiothreitol alone did not affect the activity. A 1.5-fold activation was observed when incubation was carried out under gas phase of 95% oxygen and 5% CO₂ instead of air. The activity was inhibited by 75% at 10^?4 M p-chlorophenylalanine. Both A-23187, a calcium ionophore, and dibutyryl cyclic AMP (DBc-AMP) stimulated the hydroxylation of tryptophan. The activation by A-23187 plus DBc-AMP was more than additive, suggesting the two activating mechanisms by Ca²⁺ and cyclic AMP may be operating synergistically.     

Measurement and pharmacokinetic analysis of unbound ceftazidime in rat blood using microdialysis and microbore liquid chromatography

To evaluate the biodisposition of ceftazidime in rat blood, a rapid and simple microbore liquid chromatographic technique together with a microdialysis sampling technique were developed. This method involves an on-line design for blood dialysate directly injected into a microbore liquid chromatographic system. The chromatographic conditions consisted of a mobile phase of methanol–acetonitrile–100 mM monosodium phosphoric acid (pH 3.0) (10:10:80, v/v/v) pumped through a microbore reversed-phase column at a flow-rate of 0.05 ml/min. With the detection wavelength set at 254 nm, a good linear correlation was observed between the peak area and the ceftazidime concentration at 0.1 to 50 μg/ml (r=0.999). Microdialysis probes, being custom-made, were screened for acceptable in vivo recovery while chromatographic resolution and detection were validated for response linearity, as well as intra-day and inter-day variabilities. This method was then applied to the pharmacokinetic profiling of ceftazidime in blood following intravenous 50 mg/kg administration to rats. The pharmacokinetics was calculated from the corrected data for dialysate concentrations of ceftazidime versus time. This method has been used to study ceftazidime pharmacokinetics in rats and has proven to be rapid and reproducible.     

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 propofol in rat whole blood and plasma by high-performance liquid chromatography

A simple, accurate and sensitive high-performance liquid chromatographic method was developed for the determination of propofol, an intravenous anaesthetic agent, in rat whole blood or plasma samples. The method is based on precipitation of the protein in the biological fluid sample and direct injection of the supernatant into an HPLC system involving a C₁₈ reversed-phase column using a methanol-water (70:30) mobile phase delivered at 1 ml/min. Propofol and the internal standard (4-tert.-octylphenol) were quantified using a fluorescence detector set at 276 nm (excitation) and 310 nm (emission). The analyte and internal standard had retention times of 6.3 and 10.5 min, respectively. The limit of quantification for propofol was 50 ng/ml using 100 μl of whole blood or plasma sample. Calibration curves were linear (r²=0.99) over a 1–10 μg/ml concentration range and intra- and inter-day precision were between 4–11%. The assay was applied to the determination of propofol whole blood pharmacokinetics and propofol whole blood to plasma distribution ratios in rats.     

Determination of tryptophan and metabolites in rat brain and pineal tissue by reversed-phase high-performance liquid chromatography with electrochemical detection

Tryptophan and many of its indole metabolites were separated using reversed-phase high-performance liquid chromatography (HPLC) and determined using electrochemical detection. This was accomplished isocratically using an acetate—citric acid eluent with various amounts of methanol. Brain and pineal tissue was analyzed for several tryptophan metabolites. Tissue preparation required only homogenization in acidic solution and centrifugation prior to application to the HPLC column. Detection limits in the low picogram range were found for those indoles separated.     

Simultaneous determination of bile acids in rat liver tissue by high-performance liquid chromatography

A method for the simultaneous determination of bile acids in rat liver tissue by high-performance liquid chromatography was developed. Without prior fractionation and alkaline hydrolysis, 30 unconjugated, glycine- and taurine-conjugated bile acids were detected by post-column enzymatic reaction and fluorescence detection. They were separated on a reversed-phase column using a linear gradient solvent system of 10 mM tribasic ammonium phosphate–acetonitrile–methanol (44:12:5, v/v/v) and 20 mM dibasic ammonium phosphate–acetonitrile–methanol (2:1:2, v/v/v). The limits of detection were 1–5 pmol, and calibration curves were linear for concentrations ranging between 10 and 4000 pmol per 10 μl injection. This rapid and reliable method is effective for measuring bile acid levels in liver tissue not only of rats but also of patients with hepatobiliary and other diseases.     

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.     

Determination of azosemide and its metabolite in plasma, blood, urine and tissue homogenates by high-performance liquid chromatography

High-performance liquid chromatographic methods were developed for the determination of azosemide and its metabolite, M1, in human plasma and urine and rabbit blood and tissue homogenates. The methods involved deproteinization of the biological samples: 2.5 volumes of acetonitrile were used for the determination of azosemide and 1 volume of saturated Ba(OH)₂ and ZnSO₄ for that of M1. A 50-μl aliquot of the supernatant was injected onto a C₁₈ reversed-phase column in each instance. The mobile phases employed were 0.03 M phosphoric acid—acetonitrile (50:40, v/v) for azosemide and 0.03 M phosphoric acid/0.2 M acetic acid—acetonitrile (83:17, v/v) for M1. The flow-rate was 1.5 ml/min in both instances. The column effluent was monitored by ultraviolet detection at 240 and 236 nm for azosemide and M1, respectively. The retention times for azosemide and M1 were 6.0 and 8.3 min, respectively. The detection limits for both azosemide and M1 in both human plasma and urine were 50 ng/ml. The coefficients of variation of the assay were generally low (below 11.0%) for plasma, urine, blood and tissue homogenates. No interferences from endogenous substances or other diuretics tested were observed.     

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.     

Determination of 5-fluorouracil and its main metabolites in plasma by high-performance liquid chromatography: Application to a pharmacokinetic study

This paper describes a relatively simple and sensitive high-performance liquid chromatographic assay (HPLC) with ultraviolet absorbance detection for 5-fluorouracil (5-FUra) and its two main metabolites, 5-fluorouridine (5-FUrd) and 5-fluoro-2′-deoxyuridine (5-FdUrd), in plasma. In this study, two plasma clean-up procedures involving addition of internal standard, solid-phase and liquid-liquid extractions have been developed. A reversed-phase Kromasil C₁₈ column was used. The detection was performed at 268 nm for 5-FUra and at 275 nm for the two metabolites. Linear detection responses were obtained for concentrations ranging from 25 to 1000 ng/ml. The average recovery from plasma was 35, 42 and 48% for 5-FUra, 5-FUrd and 5-FdUrd, respectively. Precision, expressed as C.V., ranged from 2.7 to 13% and the mean recovery from 94 to 105%. The limits of quantitation and detection of the three analytes were 20 and 10 ng/ml, respectively. The method was used to monitor the pharmacokinetic profile of 5-FUra and its two metabolites in patients with metastatic colorectal cancer.