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.     

Concurrent measurement of unbound genistein in the blood, brain and bile of anesthetized rats using microdialysis and its pharmacokinetic application

Genistein, the major isoflavone in soybeans, has been shown to have a wide range of effects. We used an HPLC-UV combined with microdialysis method to detect unbound genistein in rat blood, brain and bile. Genistein dialysates were eluted with a mobile phase containing acetonitrile-water (40:60, v/v, pH 3.5 adjusted by 0.1% acetic acid). Samples were separated using a phenyl (5 microm) column maintained at ambient temperature. The UV detector wavelength was set at 259 nm. The flow rate was 1.0 m/min. The limit of quantitation for genistein was 50 ng/ml. The in vitro recoveries of genistein were 31 +/- 1, 13 +/- 1 and 59 +/- 4% in microdialysis probes of blood, brain and bile, respectively (n = 4). Inter- and intra-assay accuracy and precision of the analysis were less than 10% in the concentration ranges of 0.05-5.0 microg/ml. A small ratio of genistein penetrates the blood-brain barrier (BBB) and goes through hepatobiliary excretion after genistein administration (10 or 30 mg/kg, i.v.). The brain-to-blood (AUC(brain)/AUC(blood)) and bile-to-blood (AUC(bile)/AUC(blood)) distribution ratios were 0.04 +/- 0.01 and 1.85 +/- 0.42, respectively for the dosage of genistein 30 mg/kg. After co-administration of cyclosporine, a P-glycoprotein (P-gp) inhibitor, the distribution ratios of genistein in brain and bile were not significantly altered. These results suggest that the BBB penetration and hepatobiliary excretion of genistein may not regulated by P-gp.     

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 and pharmacokinetic study of unbound cefepime in rat bile by liquid chromatography with on-line microdialysis

Biliary excretion and intestinal reabsorption in enterohepatic circulation play major dispositional roles for some drugs. To investigate biliary excretion of drug, we inserted a microdialysis probe into the bile common duct of rat between the liver and the duodenum. In order to avoid the obstruction of bile fluid or bile salt waste, a shunt linear microdialysis probe was used for simultaneous and continuous sampling following intravenous administration of cefepime (50 mg/kg, i.v.). Separation and quantitation of cefepime in the dialysates were achieved using a LiChrosorb RP-18 column (Merck; 250x4.6 mm I.D., particle size 5 microm) maintained at ambient temperature. Samples were eluted with a mobile phase containing 100 mM monosodium phosphoric acid (pH 3.0)-methanol (87:13, v/v). The UV detector wavelength was set at 270 nm. The result indicates that the elimination half-life of cefepime in bile was 64.01+/-9.32 min. This study also served as an example for the microdialysis application in the biliary excretion study of drug.     

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.     

Concurrent determination of thalidomide in rat blood, brain and bile using multiple microdialysis coupled to liquid chromatography

A rapid and sensitive system of liquid chromatography coupled with microdialysis was developed for the simultaneous determination of unbound thalidomide in rat blood, brain and bile for pharmacokinetic study. Microdialysis probes were concurrently inserted into the jugular vein toward the right atrium, the brain striatum and the bile duct of the anesthetized Sprague-Dawley rats for biological fluid sampling after the administration of thalidomide (5 mg kg(-1)) through the femoral vein. Thalidomide and dialysates were separated using a Zorbax ODS C(18) column and a mobile phase comprising acetonitrile-methanol-0.1 mm 1-octanesulufonic acid (32:3:65, v/v/v, pH 5.3) at flow rate of 1 mL min(-1). The UV wavelength was set at 220 nm. The concentration-response relationship was linear (r(2)>0.995) over a concentration range of 0.025--25 microg mL(-1). The intra-assay and inter-assay precision and accuracy of thalidomide fell within 7%. The average in vivo recoveries were 0.31+/- 0.02,0.046+/- 0.004 and 0.57+/- 0.02 (n=6), respective to the dialysates of blood, brain and bile, with thalidomide at concentrations 2, 5 and 10 microg mL(-1). The disposition of thalidomide in the blood, brain and bile fluid suggests that there is a rapid thalidomide exchange and equilibration between the blood and brain systems. In addition, thalidomide undergoes hepatobiliary excretion.     

Simultaneous determination of berberine in rat blood,liver and bile using microdialysis coupled to high-performance liquid chromatography

High-performance liquid chromatography coupled to microdialysis was used for the simultaneous determination of unbound berberine in rat blood, liver and bile for a pharmacokinetic study. Microdialysis probes were simultaneously inserted into the jugular vein toward the right atrium, the median lobe of the liver, and the bile duct of male Sprague-Dawley rats for biological fluid sampling after administration of berberine (10 mg/kg) through the femoral vein. Berberine and dialysates were separated using a Zorbax SB-phenyl column and a mobile phase comprised of acetonitrile-methanol-20 mM monosodium phosphate (pH 3.0) (35:20:45, v/v) together with 0.1 mM 1-octanesulfonic acid. The detection limit for berberine was 10 ng/ml. The concentration-response relationship was linear (r2 > 0.995) over the concentration range 0.05-50 microg/ml; intra-assay and inter-assay precision and accuracy for berberine fell within predefined limits. The disposition of berberine in the blood, liver and bile fluid suggests that berberine might be metabolized in the liver and undergo hepatobiliary excretion.     

Simultaneous determination of nicotine and its metabolite, cotinine, in rat blood and brain tissue using microdialysis coupled with liquid chromatography: pharmacokinetic application

To elucidate the disposition of nicotine in the brain is important because the neuropharmacological effects from nicotine exposure are centrally predominated. The aim of the present study was to develop a rapid and simple method for the simultaneous determination of unbound nicotine and its main metabolite, cotinine, in rat blood and brain tissue. We coupled a multiple sites microdialysis sampling technique with HPLC-UV system to characterize the pharmacokinetics of both nicotine and cotinine. Microdialysis probes were inserted into the jugular vein/right atrium and brain striatum of Sprague-Dawley rats, and nicotine (2 mg/kg, i.v.) was administered via the femoral vein. Dialysates were collected every 10 min and injected directly into a HPLC system. Both nicotine and cotinine were separated by a phenyl-hexyl column (150 mm x 4.6 mm) from dialysates within 12 min. The mobile phase consisted of an acetonitrile-methanol-20 mM monosodium phosphate buffer (55:45:900, v/v/v, pH adjusted to 5.1) with a flow-rate of 1 ml/min. The wavelength of the UV detector was set at 260 nm. The limit of quantification for nicotine and cotinine were 0.25 microg/ml and 0.05 microg/ml, respectively. Intra- and inter-day precision and accuracy of both measurements fell well within the predefined limits of acceptability. The blood and brain concentration-time profile of nicotine and cotinine suggests that nicotine is easily to get into the central nervous system and cotinine exhibits a long retention time and accumulates in blood.     

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.     

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 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.     

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 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.     

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 salbutamol in human plasma and urine using liquid chromatography coupled to tandem mass spectrometry and its pharmacokinetic study

A sensitive and selective liquid chromatography coupled to tandem mass spectrometry (LC‐MS/MS) was developed and validated for the determination of salbutamol in human plasma and urine, and successfully applied to the pharmacokinetic study of salbutamol in Chinese healthy volunteers after inhalation of salbutamol sulfate aerosol. Salbutamol and the internal standard (IS) acetaminophen in plasma and urine were extracted with ethyl acetate, separated on a C₁₈ reversed‐phase column, eluted with mobile phase of acetonitrile–ammonium acetate (5 m m ; 30:70, v/v), ionized by positive ion pneumatically assisted electrospray and detected in the multi‐reaction monitoring mode using precursor → product ions of m/z 240.2 → 148.1 for salbutamol and 152 → 110 for the IS. The lower limits of quantitation of salbutamol in human plasma and urine by this method were 0.02 and 1 ng/mL, respectively. The specificity, matrix effect, recovery, sensitivity, linearity, accuracy, precision and several stabilities were validated for salbutamol in human plasma and urine. In conclusion, the validation results showed that this method is robust, specific and sensitive, and can successfully fulfill the requirement of clinical pharmacokinetic study of salbutamol in healthy Chinese volunteers. Copyright © 2011 John Wiley & Sons, Ltd.     

Measurement of free hydroxytyrosol in microdialysates from blood and brain of anesthetized rats by liquid chromatography with fluorescence detection

Hydroxytyrosol [4-(2-hydroxyethyl)-1,2-benzenediol] is a well known natural polyphenolic component with antioxidative effects from olive oil and an aglycone of acteoside. In order to examine the in vivo metabolism of acteoside to hydroxytyrosol and the distribution of hydroxytyrosol in the blood and brain, microdialysis coupled to a liquid chromatographic system was developed to evaluate the pharmacokinetics of free-form hydroxytyrosol in rat blood and brain. Probes were implanted in the jugular vein and the brain hippocampus for blood and brain sampling purposes. Hydroxytyrosol in the microdialysis samples was separated by a reversed-phase C18 column and eluted with a mobile phase containing acetonitrile – 2% acetic acid (pH 2.6) (12:88, v/v), using a flow rate for the mobile phase of 1 mL/min. Fluorescence detection for hydroxytyrosol was set at 281 nm and 316 nm for excitation and emission wavelengths, respectively. Hydroxytyrosol and endogenous interference could be resolved within 10 min by the developed chromatographic method. The results indicated that acteoside was metabolized immediately to hydroxytyrosol in vivo and eliminated rapidly from the blood, and hydroxytyrosol could enter the brain. The blood-to-brain distribution ratio was defined by dividing the area under concentration versus time (AUC) ratio of AUC_brain/AUC_blood, which represents the AUC for brain and blood. The results suggested that the P-glycoprotein was not involved in the brain efflux transport of hydroxytyrosol.     

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.