Yen-Fei Chen

Pharmacokinetics of gastrodin and its metabolite p-hydroxybenzyl alcohol in rat blood, brain and bile by microdialysis coupled to LC-MS/MS

Gastrodin is a pharmacologically active substance isolated from Gastrodia elata Blume with sedation, anti-convulsion and anti-epilepsy activities. A rapid and sensitive liquid chromatography technique coupled to tandem mass spectrometry (LC-MS/MS) system was developed to determine gastrodin and its metabolite p-hydroxybenzyl alcohol (HBA) in rat blood, brain and bile collected using microdialysis technique. The analytes were separated using a reversed phase column (4.6 mm x 150 mm, 5 microm). The mobile phase for column separation was 30% methanol with a flow rate of 0.6 mL/min. As a post-column addition, 1% ammonium hydroxide solution (in methanol) was additionally pumped via a T-connection using a chromatographic pump (BAS PM-80, USA) at a flow rate of 0.2 mL/min after the column separation. A LC-MS/MS system equipped with a negative electrospray ionization (ESI) source in multiple reaction monitoring (MRM) mode was used to monitor m/z 285.0-->122.9 and m/z 123.0-->105.0 transitions for gastrodin and HBA, respectively. The lower limit of quantification (LLoQ) for gastrodin and HBA were 0.5 and 2 ng/mL, respectively. The calibration curves were linear over the range of 0.5-5,000 ng/mL and 2-1,000 ng/mL for gastrodin and HBA with a coefficient of determination >0.995, respectively. This selective and sensitive method is useful for the determination of gastrodin and HBA and in the pharmacokinetic studies of these compounds.

Pharmacokinetic study of levofloxacin in rat blood and bile by microdialysis and high-performance liquid chromatography

The aim of this study was to develop a rapid and sensitive method for the simultaneous determination of unbound levofloxacin in rat blood and bile using high-performance liquid chromatography coupled with microdialysis for further pharmacokinetic study. Microdialysis probes were simultaneously inserted into the jugular vein toward the right atrium and the bile duct of male Sprague-Dawley rats for biological fluid sampling after administration of levofloxacin 3 mg/kg through the femoral vein. Levofloxacin and dialysates were separated using a Merck LiChrospher reversed-phase C18 column maintained at ambient temperature. The mobile phase was comprised of acetonitrile-1 mM 1-octanesulfonic acid (40:60, v/v, pH 3.0 adjusted with orthophosphoric acid). The fluorescence response for levofloxacin was observed at excitation and emission wavelengths of 292 and 494 nm, respectively. The detection limit of levofloxacin was 50 ng/ml. Intra-day and inter-day precision and accuracy of levofloxacin measurements fell well within the predefined limits of acceptability. The disposition of levofloxacin in the blood and bile fluid suggests that there was rapid exchange and equilibration between the blood and hepatobiliary systems, and the plasma level of levofloxacin was greater than that of the bile. Thus, levofloxacin undergoes hepatobiliary excretion but might not be related to the P-glycoprotein transport system.

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 microg/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 < or = 10% in the range of 0.05 to 10 microg/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.

Determination of chlorogenic acid in rat blood by microdialysis coupled with microbore liquid chromatography and its application to pharmacokinetic studies.

To investigate the pharmacokinetics of unbound chlorogenic acid, a sensitive microbore liquid chromatographic method for the determination of chlorogenic acid in rat blood by microdialysis has been developed. A microdialysis probe was inserted into the jugular vein of male Sprague-Dawley rats, to which chlorogenic acid (20, 40, 60 or 80 mg/kg, i.v.) had been administered. On-line microdialysate was directly injected into a microbore column using a methanol-100 mM sodium dihydrogenphosphate (30:70, v/v, pH 2.5 adjusted with orthophosphoric acid) as the mobile phase and ultraviolet detection at 325 nm. The method is rapid, easily reproduced, selective and sensitive. The limit of detection for chlorogenic acid was 0.01 microg/ml and the limit of quantification was 0.05 microg/ml. The in vivo recovery of the chlorogenic acid of the microdialysis probe, based on a 5 microg/ml standard, was approximately 49-65% (n=6). The disposition of chlorogenic acid at each dose was best fitted to a two-compartment pharmacokinetic model. The area under the concentration curve increased greater than in direct proportion with the dose and terminal disposition become much slower as the dose was increased. The results indicated that the pharmacokinetics of unbound chlorogenic acid in rat blood is non-linear.

Measurement and pharmacokinetics of unbound 20(S)-camptothecin in rat blood and brain by microdialysis coupled to microbore liquid chromatography with fluorescence detection.

To characterize the pharmacokinetics of protein-free camptothecin in blood and brain we implanted microdialysis probes into the jugular vein and striatum of rats for unbound drug sampling and determination. Camptothecin (2 or 5 mg/kg, i.v., n=6) was then administered from the femoral vein, and microdialysates were collected from blood and brain of both sites and assayed by a validated microbore scale high-performance liquid chromatographic method. The mobile phase consisted of methanol-100 mM monosodium phosphoric acid (35:65, v/v, pH 2.5) with a flow-rate 0.05 ml/min. The fluorescence response for camptothecin was observed at excitation and emission wavelengths of 360 and 440 nm, respectively. Pharmacokinetic parameters were calculated from the corrected data for dialysate concentrations of camptothecin versus time. The results suggest that the pharmacokinetics of unbound camptothecin in blood and brain can be fitted best to a two- and one-compartment model, respectively. Camptothecin rapidly entered the extracellular fluid of brain striatum at 10 min following camptothecin administration.

Determination of unbound 20(S)-camptothecin in rat bile by on-line microdialysis coupled to microbore liquid chromatography with fluorescence detection

To evaluate the biliary excretion of unbound camptothecin, a flow-through microdialysis probe was constructed for bile sampling. The shunt linear probe was connected from the bile duct, between the liver side to the duodenum to avoid obstruction of the bile duct or bile salt waste. For automatic analysis of microdialysate, an on-line injector was connected to a microbore high-performance liquid chromatographic column with fluorescence detection. Samples were eluted with a mobile phase containing methanol-100 mM monosodium phosphoric acid (35:65, v/v, pH 2.5, adjusted with orthophosphoric acid). The limit of quantification was 1 ng/ml for camptothecin. Following camptothecin administration (5 mg/kg, i.v.), it was found in the bile microdialysate. It was concluded that the in vivo microdialysis technique yields useful data on the biliary excretion of camptothecin. This method is suitable for additional pharmacokinetic studies in rat bile.

Simultaneous measurement of cefuroxime in rat blood and brain by microdialysis and microbore liquid chromatography: Application to pharmacokinetics

To characterize the pharmacokinetics of cefuroxime in rat blood and brain, microdialysis probes were inserted into the jugular vein and brain striatum, respectively. Cefuroxime (20 mg/kg, i.v.) was administered via the femoral vein. Blood microdialysates were automatic injected onto microbore liquid chromatography via an on-line injectors. The mobile phase consisted of methanol-100 mM monosodium phosphoric acid (25:75, v/v, pH 5.0) with a flow-rate of 0.05 ml/min. Ultraviolet detector was set at a wavelength of 280 nm for cefuroxime. The present assay enhanced the detection sensitivity and enabled the determination of cefuroxime down to 5 ng/ml. The pharmacokinetic data demonstrated that the area under the concentration curve (AUC) ratio of unbound cefuroxime in rat brain and blood was about 4.2% after cefuroxime (20 mg/kg, i.v.) administration. These results provided further evidence that cefuroxime could penetrate the blood-brain barrier.

Concurrent quantification and pharmacokinetic analysis of cefotaxime in rat blood and brain by microdialysis and microbore liquid chromatography.

A simple but effective coupling of microdialysis and microbore liquid chromatograph with UV detection technique was applied to the simultaneous studying of the pharmacokinetics of cefotaxime in both the peripheral compartment and central nervous system. The mobile phase consisted of methanol-100 mM monosodium phosphoric acid (25:75, v/v, pH 5.5) pumped through a C18 microbore column at a flow-rate of 0.05 ml/min. Detection of cefotaxime was set at a UV wavelength of 254 nm. Microdialysis probes were inserted into the jugular vein and striatum of the rat. Following stabilization of microdialysate levels, rats received cefotaxime (20 mg/kg, i.v., n=6) via the femoral vein, and complete concentration versus time profiles for blood and striatum were constructed. The results indicated that cefotaxime rapidly (within 10 min) entered the extracellular fluid of brain striatum following intravenous administration. Noncompartmental pharmacokinetics analysis indicated that the area under the concentration versus time ratio of cefotaxime in rat brain and blood was 6.9%, suggesting appreciable blood-brain barrier penetration. The method was relatively simple, imposed minimal physiological perturbance as it involved no body fluid consumption and sampled in particular protein-unbound drugs, generally believed to be the active fraction.

Pharmacokinetics of metronidazole in rat blood, brain and bile studied by microdialysis coupled to microbore liquid chromatography

Metronidazole is a synthetic nitroimidazole-derived antibacterial and antiprotozoal agent used for the treatment of infections involving gram-negative anaerobes. The aim of this study is to develop an in vivo microdialysis with microbore high-performance liquid chromatographic system for the pharmacokinetic study of metronidazole in rat blood, brain and bile. In addition, to investigate the disposition mechanism of metronidazole, the P-glycoprotein modulator and cytochrome P450 inhibitor were concomitantly administered. Separation of metronidazole from various biological fluids was applied to a microbore reversed-phase ODS 5 microm (150 x 1 mm I.D.) column. Its mobile phase consists of an acetonitrile-50 mM monosodium phosphate buffer (pH 3.0) containing 0.1% triethylamine (10:90, v/v) with a flow-rate of 0.05 ml/min. The UV detector wavelength was set at 317 nm. The results suggest that metronidazole penetrates the blood-brain barrier (BBB) and goes through hepatobiliary excretion. However, these pathways of BBB penetration and hepatobiliary excretion of metronidazole may not be related to the P-glycoprotein.

On-line microdialysis coupled with microbore liquid chromatography with ultraviolet detection for continuous monitoring of free cefsulodin in rat blood

A microdialysis method followed by a microbore liquid chromatographic ultraviolet detection procedure has been performed for the assay of unbound cefsulodin in rat blood. A microdialysis probe was inserted into the jugular vein for blood sampling. This method involves an on-line design for submitting dialysate into the liquid chromatographic system. The chromatographic conditions consisted of a mobile phase of methanol-100 mM monosodium phosphoric acid (10:90, v/v, pH 5.0) pumped through a microbore reversed-phase column at a flow-rate of 0.05 ml/min. Detection wavelength was set at 265 nm. Microdialysis probes, being laboratory-made, were screened for acceptable in vivo recovery while chromatographic resolution and detection were validated for response linearity as well as intra- and inter-day variabilities. The method was then applied to pharmacokinetics profiling of cefsulodin in the blood following intravenous administration of cefsulodin (20 mg/kg) in rats. Pharmacokinetics were calculated from the corrected data for dialysate concentrations of cefsulodin versus time. Based on pharmacokinetic calculation, cefsulodin best fitted to a two-exponential disposition. This study provided specific pharmacokinetic information for protein-unbound cefsulodin and demonstrated the applicability of this continuous sampling method for pharmacokinetic study.