Determination and pharmacokinetic study of meropenem in rat bile using on-line microdialysis and liquid chromatography

Meropenem is a carbapenem antibiotic with a wide spectrum of activity against both Gram-positive and Gram-negative bacteria. Because of its clinical efficacy, meropenem is an excellent choice for the treatment of serious infections in both adults and children. The knowledge of tissue concentrations of antibiotic in an infection site is valuable for the prediction of treatment outcome. To investigate the biliary disposition of meropenem, we utilized a minimally invasive sampling technique with a shunt linear microdialysis probe for continuous sampling in the biliary excretion studies. Analysis of meropenem in the dialysates was achieved using a LiChrosorb RP-18 column (Merck, 250 x 4.6 mm I.D.; particle size 5 microm) maintained at ambient temperature. The mobile phase was 50 mM monosodium phosphoric acid-methanol (80:20, v/v, pH 3.0). The UV detector wavelength was set at 298 nm. The area under the concentration-time curve and elimination half-lives of meropenem were about 6144 +/- 1494 min microg/ml and 61 +/- 17 min, respectively. This study represents a successful application of the microdialysis technique, which is an effective method for pharmacokinetic and biliary drug excretion studies.     

Measurement and pharmacokinetic analysis of unbound cephaloridine in rat blood by on-line microdialysis and microbore liquid chromatography

A technique involving rapid sampling of cephaloridine in rat blood was achieved using a combination of microdialysis and sensitive microbore liquid chromatography. A microdialysis probe was inserted into the jugular vein/right atrium of a Sprague-Dawley rat. Then after a real-time collection of the analyte by microdialysis, the dialysate was automatically injected into a liquid chromatographic system via an on-line injector. Following a 2 h stabilization period after the surgical procedure, cephaloridine (20 mg/kg, i.v.) was then administered via the femoral vein. Isocratic elution of cephaloridine was carried out with a mobile phase containing methanol-20 mM monosodium phosphate (25:75, v/v, pH 5.5), and the flow rate of the mobile phase was 0.05 mL/min within 10 min. Intra- and inter-assay accuracy and precision of the assay were each less than 10%. The in vivo recovery of the cephaloridine from the microdialysate was 49.7 +/- 8.0% and 42.4 +/- 8.4% for 0.5 and 1 microg/mL standards (n = 6), respectively. Based on the pharmacokinetic analysis, the elimination half-life was 32.2 +/- 8.6 min by cephaloridine administration (20 mg/kg, i.v., n = 6).     

Determination of unbound cefamandole in rat blood by microdialysis and microbore liquid chromatography

To analyze unbound cefamandole in rat blood, a method combing microdialysis with microbore liquid chromatography has been developed. A microdialysis probe was inserted into the jugular vein/right atrium of male Sprague-Dawley rats to examine the unbound cefamandole level in the rat blood following cefamandole administration (50 mg/kg, i.v.). The dialysates were directly submitted to a liquid chromatographic system. Samples were eluted with a mobile phase containing acetonitrile-methanol-100 mM monosodium phosphate (pH 5.0; 15:20:65, v/v). The UV wavelength was set at 270 nm for monitoring the analyte. Using the retrograde method, at infusion concentrations of 1 microg/mL of cefamandole, the in vivo microdialysis recoveries were 55.44% for the rat blood (n = 6). Intra- and inter-assay accuracy and precision of the analyses were < or = 10% in the range of 0.1-10 microg/mL. Pharmacokinetic parameters were calculated from the recovery-corrected dialysate concentrations of cefamandole vs time data. The elimination half-life (t1/2,beta) was 21.6 +/- 1.6 min. The results suggest that the pharmacokinetics of unbound cefamandole in blood following cefamandole administration (50 mg/kg, i.v., n = 5) fit best to the two-compartmental model.     

Determination and pharmacokinetic analysis of salvianolic acid B in rat blood and bile by microdialysis and liquid chromatography

Salvianolic acid B is an herbal ingredient isolated from Salvia miltiorrhiza. An in vivo microdialysis sampling method coupled to high-performance liquid chromatography has been developed for continuous monitoring of protein-unbound salvianolic acid B in rat blood and bile. Microdialysis probes were inserted into the jugular vein/right atrium and bile duct of Sprague-Dawley rats, and a dose of 100 mg/kg salvianolic acid B was then administered via the femoral vein. Dialysates were collected and directly injected into a liquid chromatographic system. Salvianolic acid B was eluted using a microbore reversed-phase ODS 5 microm (150 mm x 1 mm I.D.) column. Isocratic elution of salvianolic acid B was achieved within 10 min using the liquid chromatographic system. The chromatographic mobile phase consisted of acetonitrile-methanol-20 mM monosodium phosphoric acid (pH 3.5) (10:30:60, v/v/v) containing 0.1 mM 1-octanesulfonic acid with 0.05 ml/min. The wavelength of the UV detector was set at 290 nm. Salvianolic acid B in both blood and bile dialysates was adequately determined using the liquid chromatographic conditions described, although the blank bile pattern was more complex. The retention times of salvianolic acid B in rat blood and bile dialysates were found to be 7.2 min. Peak-areas of salvianolic acid B were linear (r2 > 0.995) over a concentration range of 0.1-50 microg/ml. In vivo recoveries of microdialysis probes of salvianolic acid B in rat blood and bile averaged 22 +/- 2% and 41 +/- 1%, respectively. This study indicates that salvianolic acid B undergoes hepatobiliary excretion.     

Determination and pharmacokinetic profile of omeprazole in rat blood, brain and bile by microdialysis and high-performance liquid chromatography

The disposition and biliary excretion of omeprazole was investigated following i.v. administration to rats at 10 mg/kg. We used a microdialysis technique coupled to a validated microbore HPLC system to monitor the levels of protein-unbound omeprazole in rat blood, brain and bile, constructing the relationship of the time course of the presence of omeprazole. Microdialysis probes were simultaneously inserted into the jugular vein toward right atrium, the brain striatum and the bile duct of the male Sprague-Dawley rats for biological fluid sampling after the administration of omeprazole (10 mg/kg) through the femoral vein. The concentration-response relationship from the present method indicated linearity (r2>0.995) over a concentration range of 0.01-50 microg/ml for omeprazole. Intra-assay and inter-assay precision and accuracy of omeprazole fell well within the predefined limits of acceptability. Following omeprazole administration, the blood-to-brain coefficient of distribution was 0.15, which was calculated as the area under the concentration versus time curve (AUC) in the brain divided by the AUC in blood (k=AUCbrain/AUCblood). The blood-to-bile coefficient of distribution (k=AUCbile/AUCblood) was 0.58. The decline of unbound omeprazole in the brain striatum, blood and bile fluid suggests that there was rapid exchange and equilibration between the compartments of the peripheral and central nervous systems. In addition, the results indicated that omeprazole was able to penetrate the blood-brain barrier and undergo hepatobiliary excretion.     

Measurement of unbound ranitidine in blood and bile of anesthetized rats using microdialysis coupled to liquid chromatography and its pharmacokinetic application

To investigate the pharmacokinetics of unbound ranitidine in rat blood and bile, multiple microdialysis probes coupled to a liquid chromatographic system were developed. This study design was parallel in the following groups: the control-group of six rats received ranitidine alone (10 and 30 mg/kg, i.v.), the treated-group rats were co-administered with ranitidine and cyclosporine (P-glycoprotein (P-gp) inhibitor) or quinidine (both organic cation transport (OCT) and P-gp inhibitors) in six individual rats. Microdialysis probes were inserted into the jugular vein and the bile duct for blood and bile fluids sampling, respectively. Ranitidine in the dialysate was separated by a reversed-phase C18 column (Zorbax, 150 mm x 4.6 mm i.d.; 5 microm) maintained at ambient temperature. Samples were eluted with a mobile phase containing acetonitrile-methanol-tetrahydrofuran-20 mM K2HPO4 (pH 7.0) (24:20:10:946, v/v), and the flow rate of the mobile phase was 1 ml/min. The optimal UV detection for ranitidine was set at wavelength 315 nm. Between 20 and 30 min after drug administration (10 or 30mg/kg), the ranitidine reached the maximum concentration in the bile. The bile-to-blood distribution ratio (AUC(bile)/AUC(blood)) was 9.8 +/- 1.9 and 13.9 +/- 3.8 at the dosages of 10 and 30 mg/kg, respectively. These studies indicate that ranitidine undergoes hepatobiliary excretion which against concentration gradient from bile-to-blood. In addition, the AUC of ranitidine in bile decreased in the treatment of cyclosporine or quinidine, which suggests that the hepatobiliary excretion of ranitidine was partially regulated by P-glycoprotein or organic cation transporter.     

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.     

Determination of phthalate esters from food-contacted materials by on-line microdialysis and liquid chromatography

Phthalates (endocrine disrupters) released from food-contacted plastics into aqueous solution during microwave conditioning were measured with microdialysis enrichment on-lined high-performance liquid chromatography. The released phthalates in aqueous solution were diffused through a cellular dialysis membrane into the perfusion stream and thus enriched prior to HPLC analysis. Conditions for obtaining optimum enrichment such as the hollow dialysis fiber, flow-rate and polarity modifier in perfusion stream, pH, added-salt and stirring rate in sample solution, as well as chromatographic conditions were investigated. Experimental results indicated that microdialysis enrichment with a 20-cm polysulfone hollow dialysis fiber and heptane as the perfusate at 0.10-microL/min flow-rate to collect phthalates from aqueous sample in 0.5M KCl matrix (optional pH) at 250 rpm stirring offered the optimum enriched efficiency. The dimethyl phthalate (DMP), diethyl phthalate (DEP) and dibutyl phthalate (DBP) were well separated within 16 min by a C-18 column and eluted gradient from 40 to 90% aqueous acetonitrile (at pH 6.0) and 1.0 to 1.5 mL/min flow-rate. Detection was carried out with an UV detector at 225 nm. The enrichment factors were 14, 140 and 201 (at 0.10-microL/min perfusate flow-rate) for DMP, DEP and DBP, respectively, with less than 4% RSD. The proposed method provided a very simple, fast and eco-friendly enriched procedure to determine the extent of phthalates migration from disposable plastic materials into drinking soup.     

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 μg/ml and the limit of quantification was 0.05 μg/ml. The in vivo recovery of the chlorogenic acid of the microdialysis probe, based on a 5 μg/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 of unbound cocaine in blood, brain and bile of anesthetized rats using microdialysis coupled with liquid chromatography and verified by tandem mass spectrometry

To investigate the disposition of unbound cocaine in the rat blood, brain and bile, we demonstrate an in vivo multiple sampling microdialysis system coupled with liquid chromatography for cocaine assay and verified by tandem mass spectrometry. Three microdialysis probes were concurrently inserted into the jugular vein, bile duct and brain striatum of each anesthetized rat. After a period of 2 h post-surgical stabilization, cocaine (10 mg kg(-1)) was administered through the femoral vein. Separation of unbound cocaine from various biological fluids was applied to a reversed-phase C(18) column (250 x 4.6 mm I.D., 5 microm). The mobile phase consisted of acetonitrile--10 mm potassium dihydrogen phosphate buffer (25:75, v/v, pH 4.0) and 0.8% diethylamine at a flow rate of 1 mL min(-1). The UV detector wavelength was set at 235 nm. The results indicate that cocaine penetrates the blood--brain barrier with a rapid distribution. However, unbound cocaine in the bile dialysate was not detectable in the UV detection. We therefore use LC--tandem mass spectrometry to detect the bile fluid after cocaine administration (3 mg kg(-1), i.v.). The results indicate that cocaine goes through hepatobiliary excretion.     

Determination of (-)-epigallocatechin gallate in rat blood by microdialysis coupled with liquid chromatography

A liquid chromatographic method coupled with microdialysis was used to determine the protein-unbound (-)-epigallocatechin-3-gallate (EGCG) in rat blood. EGCG and dialysates were separated using a Merck RP-18e column maintained at ambient temperature, and a mobile phase comprised of acetonitrile-10 mM monopotassium phosphate (pH 3.82) (20:80, v/v) with a flow rate of 1.0 ml/min. The UV detector wavelength was set at 206 nm. The detection limit for EGCG was 10 ng/ml. The concentration-response relationship was linear (r2 > 0.995) over a concentration range of 0.05-10 microg/ml; intra- and inter-assay precision and accuracy of EGCG fell within predefined limits. Pharmacokinetic parameters of EGCG were assessed using compartmental models. The disposition of EGCG in the rat blood suggests that EGCG was fitted by two-compartmental model. The distribution and elimination half-lives were 6 and 72 min respectively, after the dosage of 30 mg/kg.     

Determination of thiobarbituric acid adduct of malondialdehyde using on-line microdialysis coupled with high-performance liquid chromatography.

An on-line analytical system for the continuous monitoring of malondialdehyde (MDA) was developed. This method involves the use of microdialysis perfusion, on-line derivatization and on-line HPLC analysis. This method gave a linear response for MDA concentrations and HPLC peak areas in the range from 0.051 microM to 2.43 microM. The intra-day (RSD = 1.6-10.5%) and inter-day (RSD = 1.1-9.3%) precisions were acceptable. The average in vitro probe recovery of MDA standard was 18.4 +/- 1.0%. The detection limit was 0.03 microM, corresponding to 0.6 pmol for an injection volume of 20 microl. This method was used for in vitro peroxidation investigations, which provided evidence for elevated MDA levels following the incubation of metal ions to a linoleic acid solution.     

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.     

Determination of bisphenol A in rat brain by microdialysis and column switching high-performance liquid chromatography with fluorescence detection

A sensitive column switching HPLC-fluorescence detection for determination of bisphenol A (BPA) in rat brain by coupling with microdialysis was developed. A microdialysis probe was inserted into the hypothalamus of rat brain and an artificial cerebrospinal fluid was used for perfusion. BPA in brain dialysate was subjected to a fluorescent derivatization with 4-(4,5-diphenyl-1H-imidazol-2-yl)benzoyl chloride (DIB-Cl), and the excess reagent was removed by a column-switching technique. Separation was carried out on two ODS semimicro-columns with the mobile phase of acetonitrile-H(2)O-methanol-tetrahydrofuran (55:10:35:2.5, v/v) and acetonitrile-0.1 M acetate buffer (pH 3.0)-methanol (35:10:55, v/v) at a flow rate of 0.10 and 0.15 mL/min for a precolumn and a separation column, respectively. Fluorescence intensity was monitored at 475 nm with excitation of 350 nm. BPA could be sensitively detected at 0.3 ppb in 60 micro L brain microdialysate at a signal-to-noise ratio of 3. By the proposed method, concentrations of BPA in rat brain and plasma were monitored for 8 h after single i.v. or oral administration. It is proved that BPA is capable of penetrating the blood-brain barrier. The ratio of the area under the concentration-time curve of BPA in rat brain to that in blood was estimated to be about 3.0-3.8%.     

Determination of amphetamine in rat brain by in vivo microdialysis and ion-pairing liquid chromatography with electrospray tandem mass spectrometry

An ion-pairing liquid chromatography/electrospray tandem mass spectrometry (LC/ES-MS/MS) method with in vivo microdialysis for the determination of amphetamine in rat brain has been developed. A microdialysis probe was surgically implanted into the striatum of the rat and artificial cerebrospinal fluid (aCSF) was used as the perfusion medium. Samples were collected and then analyzed off-line by LC/ES-MS/MS. A reversed-phase C18 column was employed for LC separation and MS/MS was utilized for detection. Trifluoroacetic acid (TFA) was added to the mobile phase (acetonitrile/water) as an ion-pairing reagent. Detection was by ES-MS/MS directly, and no post-column addition of organic modifier was needed. Dual linear ranges were determined from 0.1-0.5 microg/mL and 0.005-0.1 microg/mL, respectively. The detection limit, based on a signal-to-noise ratio of 3, was 0.001 microg/mL (5 nM). Good precision and accuracy were obtained. The applicability of this newly developed method was demonstrated by continuous monitoring of amphetamine concentrations in rat brain. Amphetamine reached a maximum concentration of 0.086 +/- 0.017 microg/mL over 20-40 min after a single 3.0 mg/kg intraperitoneal administration.     

Determination of drug-protein interactions by combined microdialysis and high-performance liquid chromatography

Summary A simple and rapid method for determination of the parameters of the interaction between drugs and protein, including the association constant and the number of binding sites, has been developed by use of a microdialysis sampling technique combined with high-performance liquid chromatography. The drug and protein (carbamazepine (5H-dibenz[b,f]flazepine-5-carboxamide, CBZ) and human serum albumin (HSA) were used as examples) were mixed in different molar ratios in 0.067 M potassium phosphate buffer, pH 7.4, and incubated at 37°C in a water-bath. The microdialysis probe was the used to sample the mixed CBZ-HSA solution at a perfusion rate of 1 μL min⁻¹. The concentration of CBZ in the microdialysate was determined by reversed-phase high-performance liquid chromatography. Relative recovery (R), determined in vitro under similar conditions, was approximately 42.7%; theRSD ofR was approximately 1.85%. The estimated association constant (K) and the number of the binding sites,n, on one molecule of HSA were 1.06×10⁴ M⁻¹ and 0.880, respectively, which is in good agreement with the literature values determined by high-performance frontal analysis. The potential use of microdialysis is also discussed.     

Continuous monitoring of unbound flomoxef levels in rat blood using microdialysis and its new pharmacokinetic analysis

Microdialysis sampling method coupled to high-performance liquid chromatography with UV detection was applied to continuous monitoring of in vivo unbound flomoxef concentration in rat blood. By comparison with ultrafiltration method, it was demonstrated that it gave reliable results for the unbound drug monitoring in blood. Furthermore, a new method was presented for the calculation of pharmacokinetic parameters from the data obtained by the microdialysis method.     

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 matrine in rat plasma by high-performance liquid chromatography and its application to pharmacokinetic studies

A simple high-performance liquid chromatography (HPLC) method is described for the determination of matrine in rat plasma. The plasma was deproteinized with acetonitrile that contained an internal standard (phenacetin) and was separated from the aqueous layer by adding sodium chloride. Matrine was extracted into the acetonitrile layer with high yield, and determined by reversed-phase HPLC (column: YMC-pack ODS-A, 5 μm, 150×4.6 mm, I.D.; eluent: acetonitrile-0.02 mol ammonium acetate buffer-triethylamine (35:65:0.035, v/v/v) and ultraviolet detection (220 nm). The limit of quantitation for matrine was 200 ng ml⁻¹ in plasma, and the recovery was greater than 89%. The assay was linear from 0.5 to 50.0 μg ml⁻¹. Variation over the range of the standard curve was less than 6%. The method was used to determine the concentration-time profiles of matrine in the plasma following oral administration of matrine aqueous solution or bolus injection from which the fractions of matrine reaching the systemic circulation were estimated by a deconvolution method for the first time.