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.     

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.     

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.     

Measurement of hydroxyl radical in rat blood vessel by microbore liquid chromatography and electrochemical detection: an on-line microdialysis study

Salicylic acid (0.5 mM) is used as a trapping reagent of hydroxyl radical, and the formed 2,3- and 2,5-dihydroxybenzoic acids were collected via an on-line microdialysis device from the blood vessels. This study revealed the use of a sensitive liquid chromatographic system with electrochemical detection for the determination of 2,3- and 2,5-dihydroxybenzoic acids. Mobile phase consisted of 0.1 M monochloroacetic acid, 10 mM EDTA, 0.5 mM sodium octylsulfate, 20% acetonitrile and 5% tetrahydrofuran in 1 l (pH 3.0 adjusted with 1 M NaOH), and the flow-rate of 0.05 ml/min were found to be optimum. Isocratic separation of these adducts on a microbore column (reversed-phase C₁₈, 150×1 mm I.D., 5 μm) was achieved within 10 min. The optimal applied potential of dihydroxybenzoic acids was set at 750 mV based on a hydrodynamic study. This method has the detection limits of 1.3 pmol/ml (or 0.2 ng/ml) for 2,3- and 2,5-dihydroxybenzoic acids in Ringer solution (at signal-to-noise ratio=3).     

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 blood and brain sampling of cephalexin in the rat by microdialysis and microbore liquid chromatography: application to pharmacokinetics studies

To circumvent the need for laborious sample clean-up and multiple blood sampling, a system was developed consisting of on-line microdialysis coupled to microbore liquid chromatography and ultraviolet detection. The system was designed for the simultaneous and continuous monitoring of unbound blood and brain cephalexin in the rat following single bolus intravenous administrations (10 mg/kg, n=6). Microdialysis probes were inserted into the jugular vein and brain striatum, respectively, for blood and brain sampling. Chromatographic conditions consisted of a mobile phase of methanol–100 mM monosodium phosphoric acid (20:80, 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 260 nm. The method was validated for response linearity as well as intra- and inter-day variabilities. Rapid appearance of cephalexin in the striatal dialysate suggested good blood–brain barrier penetration. This study provided pharmacokinetics information for cephalexin as well as demonstrated the applicability of this continuous sampling method for pharmacokinetics studies.     

On-line determination of fluconazole in blood and dermal rat microdialysates by microbore high-performance liquid chromatography

To study the distribution of fluconazole in the dermis of the rat, on-line microdialysis using double-site sampling coupled with a microbore HPLC system was developed. The chromatographic conditions consisted of a mobile phase of 20 mM diammonium phosphate-acetonitrile (75:25, v/v, pH 7.0) pumped through a microbore C(18) column at 40 microl/min. The eluent was monitored with UV detector with UZ flow cell (30 mm path length) at 210 nm. A microbore 10-port pneumatic valve fitted with two loops of 1 microl was used to collect and directly inject microdialysates from jugular and dermal probes. The retention time was 5.8 min for fluconazole and 10.1 min for its fluorinated analog, UK-54373 used as a retrodialysis marker. The assay was precise, with inter- and intra-assay relative standard deviation values of 0.64 and 0.71%, respectively, and with a good linearity (r=0.999) in the range of 0.15-20 microg/ml with only 1 microl injected onto the column. The LOD and LOQ values for fluconazole were 0.100 and 0.150 microg/ml, respectively. The applicability of the method was demonstrated by studying the disposition of fluconazole in blood and dermis following i.v. bolus at a dose of 10 mg/kg.     

Determination of SDZ ICM 567 in blood and muscle microdialysis samples by microbore liquid chromatography with ultraviolet and fluorescence detection

Fast, simple and accurate method for the determination of SDZ ICM 567, the 7-methoxy derivative of tropisetron, in microdialysates have been developed. Sampling by microdialysis from freely moving rats in the portal and jugular vein offers a new technology for pharmacokinetic studies by direct and continuous measurement of unbound drug concentrations with time. SDZ ICM 567 can be identified in small sample volumes of dialysates on a microbore high-performance liquid chromatography column-switching system with ultraviolet detection. In addition, determination of SDZ ICM 567 by fluorimetric detection has been developed for muscle microdialysates from rats. [¹⁴C]SDZ ICM 567 was used as reference substance for the estimation of the amount of substance transferred through the dialysis membrane. The radioactive measurement (RA) gave the recovery information, whereas the liquid chromatographic method detected the sum of [¹⁴C]SDZ ICM 567 and dialyzed SDZ ICM 567.     

Measurement and pharmacokinetic study of tetramethylpyrazine in rat blood and its regional brain tissue by high-performance liquid chromatography

We used a rapid, sensitive and reliable high-performance liquid chromatographic method for the determination of tetramethylpyrazine in rat brain tissue and plasma. The lower limit of quantification in plasma and brain tissue was 0.1 μg/ml and 0.1 μg/g, respectively, and only a small amount of plasma (100 μl) or brain tissue (100 μg) was required for analysis. The decline in the concentration of tetramethylpyrazine in plasma was generally two-exponential at a dose of 2, 5, or 10 mg/kg administered intravenously. Concentrations of tetramethylpyrazine in various regions of the brain (cerebral cortex, brainstem, striatum, hippocampus, cerebellum and midbrain) were not significantly different at 15 min following drug administration (10 mg/kg, i.v.). In additional analysis, mean concentration of the tetramethylpyrazine in rat plasma was approximately five-times greater than the drug in brain tissue.     

Determination of EETs using microbore liquid chromatography with fluorescence detection

Epoxyeicosatrienoic acids (EETs) are cytochrome P-450 metabolites of arachidonic acid involved in the regulation of vascular tone. The method of microbore column high-performance liquid chromatography with fluorescence detection was developed to determine 14,15-EET, 11, 12-EET, and the mixture of 8,9-EET and 5,6-EET. Tridecanoic acid (TA) was used as an internal standard. EETs were reacted with 2-(2, 3-naphthalimino)ethyl trifluoromethanesulfonate (NT) to form highly fluorescent derivatives. A C(18) microbore column and a water-acetonitrile mobile phase were used for separation. Samples were excited at 259 nm, and the fluorescence was detected at 395 nm. The overall recoveries were 88% for EETs and 40% for TA. EETs were detected in concentrations as low as 2 pg (signal-to-noise ratio = 3). The method was used to determine the EET production from endothelial cells (ECs). Bradykinin and methacholine (10(-6) M) stimulated an increase in the production of EETs by ECs two- and fivefold, respectively. This sensitive method may be used for determination of EETs at low concentrations normally detected in complex biological samples.     

High-sensitivity phenylthiohydantoin amino acid analysis using conventional and microbore chromatography

Reversed-phase microbore high-performance liquid chromatography was investigated for high-sensitivity analysis of phenylthiohydantoin (PTH) amino acids. A mixed nitrile alkylsilane bonded phase was developed and ternary gradient elution conditions were devised for resolution 150 × 4.6 mm I.D. column and transferred to a 150 × 1 mmI.D. microbore column. The performance of these columns was evaluated in terms of PTH amino acid resolution, enhanced sample detectability, and retention time precision. For this work a general purpose high-performance liquid chromatograph was modified to reduce extra column band broadening and a preformed gradient elution technique was developed to achieve rapid analysis times at microbore flow-rates. The microbore high-performance liquid chromatographic system is useful for high-sensitivity analysis of PTH amino acids in micro-sequencing applications.     

Determination of extracellular hesperidin in blood and bile of anaesthetized rats by microdialysis with high-performance liquid chromatography: a pharmacokinetic application

A method coupled with microdialysis technique and liquid chromatography was applied in the continuous and concurrent in vivo monitoring of extracellular hesperidin in the blood and bile of anaesthetized rats. Hesperidin was intravenously administered via the femoral vein. Sampling was achieved using two microdialysis probes, which were implanted into the jugular vein and into the bile duct. Dialysates of blood and bile were both directly injected onto the liquid chromatographic system, so no further clean-up procedures were required. Separation was performed using a reversed phase ODS-2 microbore column 150 mm x 1 mm i.d., particle size 5 microm with mobile phase of acetonitrile-0.1M ammonium acetate (30:70, v/v) at flow-rate of 0.05 ml/min. The UV detection for hesperidin was set at a wavelength of 283 nm. This method was used to determine the pharmacokinetics of hesperidin and its interaction in the presence of cyclosporin A, which is a P-glycoprotein modulator. The results indicate that the curve of area under the concentration versus time (AUC) for hesperidin in bile was significantly greater than that for hesperidin in blood at the dose of 30 mg/kg. The blood-to-bile distribution ratio (k = AUC(bile)/AUC(blood)) was 8.9 +/- 2.5 for hesperidin at 30 mg/kg. Following cyclosporin A treatment, the distribution ratio was reduced to 3.2 +/- 0.6. In conclusion, hesperidin goes through hepatobiliary elimination against the concentration gradient from blood to bile, and this hepatobiliary excretion of hesperidin may be regulated by the P-glycoprotein.     

New dual electrochemical detector for microbore liquid chromatography determination of dopamine and serotonin in rat striatum dialysates

A new type of liquid chromatographic (LC) dual thin-layer amperometric detector for the simultaneous measurement of trace levels of dopamine and serotonin in microdialysates is described. The concentrations of these analytes in rat dialysates are usually in the sub-nanomolar concentration range (typically, 0.10–5.00 pg in 5-μl dialysates). With this dual electrode, a glass-lined microbore column provides excellent sensitivity, selectivity, and separation. In addition, a three- to five-fold improvement in anodic current or cathodic responses over conventional dual electrodes in microbore LC can be achieved. Due to the irreversible electrochemical properties of some interference peaks, this dual electrode provides reliable measurement of dopamine based on the cathodic signal. The detection limit (signal-to-noise RATIO=3) of this assay is 0.02 pg per injection for dopamine or serotonin. This new dual electrode allows the simultaneous measurements of basal dopamine and serotonin in rat striatum dialysates without the use of re-uptake inhibitors in perfusion medium.     

Quantitative analysis of PD 0332991 in mouse plasma using automated micro-sample processing and microbore liquid chromatography coupled with tandem mass spectrometry

In the oncology therapeutic area, the mouse is the primary animal model used for efficacy studies. Often with mouse pharmacokinetic (PK) and pharmacokinetic/pharmacodynamic (PK/PD) studies, less than 20 μL of total plasma sample volume is available for bioanalysis due to the small size of the animal and the need to split samples for other measurements such as biomarker analyses. The need to conduct automated "small volume" sample processing for quantitative bioanalysis has therefore increased. An automated fit for purpose protein precipitation (PPT) method using a Hamilton MicroLab Star (Reno, NV, USA) to support mouse PK and PK/PD studies for an oncology drug candidate PD 0332991, (a specific inhibitor of cyclin-dependent kinase 4 (CDK-4) currently in development) for processing "small volumes" was developed. The automated PPT method was achieved by extracting and processing 10 μL out of a minimum sample volume of 15 μL plasma utilizing the Hamilton MicroLab Star. A 96-conical shallow well plate by Agilent Technologies, Inc (Wilmington, DE, USA) was the labware of choice used in the automated Hamilton "small volume" method platform. Analyses of a 10 μL plasma aliquot from 15 μL of plasma study samples were conducted by both automated and manual PPT method. All plasma samples were quantitated using a Sciex API 4000 triple quadrupole mass spectrometer coupled with an Eksigent Express HT Ultra HPLC system. The chromatography was achieved using an Agilent microbore C(18) Extend, 1.0 × 50 mm, 3.5 μm column at a flow rate of 0.150 mL/min with a total run time of 1.8 min. Accuracy and precision of standard and QC concentration levels were within 90-107% and <14%, respectively. Calibration curves were linear over the dynamic range of 1.0-1000 ng/mL. PK studies for PD 0332991 were conducted in female C3H mice following intravenous administration at 1mg/kg and oral administration at 2mg/kg. PK values such as area under curve (AUC), volume of distribution (Vd), clearance (Cl), half life (T(1/2)) and bioavailability (F%) demonstrated less than 11% difference between the automated Hamilton and manual PPT methods. The results demonstrate that the automated Hamilton PPT method can accurately and precisely aliquot 10 μL of plasma from 15 μL or larger volume plasma samples. The fit for purpose Hamilton PPT method is suitable for routine analyses of plasma samples from micro-sampling PK and PK/PD samples to support discovery studies.     

Determination of baicalin in rat cerebrospinal fluid and blood using microdialysis coupled with ultra-performance liquid chromatography-tandem mass spectrometry

An in vivo microdialysis sampling method coupled with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was employed for continuous simultaneous monitoring of unbound baicalin in rat blood and brain. Microdialysis probes were inserted into the jugular vein and brain cerebrospinal fluid (CSF) of Sprague-Dawley rats then, following administration of baicalin at doses of 24mg/kg via the candal vein, samples were collected every 20min and injected directly into the UPLC-MS/MS system. In vitro recoveries of the probes were 19.26% and 18.38%, while in vivo recoveries of the probes were 15.0% and 17.52% for blood and brain, respectively. This improved method offers a rapid quantitative procedure for the determination of baicalin with a retention time of only 1.6min. The lower limit of quantification (LLOQ) and the lower limit of detection (LLOD) based on a signal-to-noise ratio of 5 were 2.37 and 0.1ng/ml for anticoagulant citrate dextrose (ACD) solution, and 1.185 and 0.3ng/ml for artificial cerebrospinal fluid (aCSF), respectively. The pharmacokinetics results indicated that baicalin could pass through the blood-brain barrier (BBB) and was detectable in brain dialysate. These in vivo microdialysis-based measurements provide a technique for simple sampling and rapid sensitive analysis of unbound baicalin in rat blood and CSF and for further application in pharmacokinetic studies.     

Applications of tandem microbore liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis/electroblotting in microsequence analysis

Protein isolation by microbore HPLC is compared with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/electroblotting methods for several major proteins from rabbit muscle. Although single-mode HPLC or SDS-PAGE/electroblotting provides excellent speed and sensitivity for submicrogram-level protein purification, neither one alone has adequate resolution for separating such a complex protein mixture. Tandem procedures, utilizing two different modes of HPLC in separate steps or a combination of single HPLC separation and SDS-PAGE/electroblotting, offer the necessary versatility. One of the major concerns in this investigation was to evaluate electroblotting techniques for microsequencing. The Aebersold et al. procedure (R.H. Aebersold, D.B. Teplow, L.E. Hood, and S.B.H. Kent (1986) J. Biol. Chem. 261, 4229-4238) was substantially modified and improved; the details of this work will be published elsewhere. These changes significantly improve repetitive yields at the low microgram level without producing high backgrounds. At lower levels the recovery of sequenceable protein currently limits our ability to obtain useful results. Starting with 250-750 micrograms of rabbit muscle crude extract, several proteins (15-70 kDa) were isolated by tandem microbore LC and PAGE/electroblotting for amino-terminal sequence analysis. It appears that the combination of electroblotting and microbore LC represents a powerful approach for microsample preparation.     

Determination of an arylacetamide antiarrhythmic in rat blood and tissues using reversed-phase high-performance liquid chromatography

A method was developed for quantification of (+)-trans-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzo[b- ]thiophene-4-acetamide (compound I), an antiarrhythmic drug, in rat whole blood, heart, brain, liver and skeletal muscle. Blood and tissue samples were homogenized and purified by chemical extraction. Chromatographic separations were achieved using reversed-phase high-performance liquid chromatography (HPLC) coupled with UV detection (215 nm). Drug recoveries from the extraction procedure ranged from 77 to 90%. Within- and between-day reproducibility of peak area (coefficient of variation) ranged from 1.1 to 15.7%. The detection limit was 80–200 ng/ml (in a 500-μl extracted solution) depending on the type of biological sample. This method was used in a pharmacokinetic study of compound I disposition in rats after a bolus intravenous dose of 3.1 mg/kg.     

Determination of celecoxib in human plasma and rat microdialysis samples by liquid chromatography tandem mass spectrometry

Methods for the determination of celecoxib in human plasma and rat microdialysis samples using liquid chromatography tandem mass spectrometry are described. Celecoxib and an internal standard were extracted from plasma by solid-phase extraction with C₁₈ cartridges. Thereafter compounds were separated on a short narrow bore RP C₁₈ column (30×2 mm). Microdialysis samples did not require extraction and were injected directly using a narrow bore RP C₁₈ column (70×2 mm). The detection was by a PE Sciex API 3000 mass spectrometer equipped with a turbo ion spray interface. The compounds were detected in the negative ion mode using the mass transitions m/z 380→316 and m/z 366→302 for celecoxib and internal standard, respectively. The assay was validated for human plasma over a concentration range of 0.25–250 ng/ml using 0.2 ml of sample. The assay for microdialysis samples (50 μl) was validated over a concentration range of 0.5–20 ng/ml. The method was utilised to determine pharmacokinetics of celecoxib in human plasma and in rat spinal cord perfusate.     

High-performance liquid chromatographic analysis with electrochemical detection of biogenic amines using microbore columns

High-performance liquid chromatography with electrochemical detection (HPLC-ED) is a popular method for measuring biogenic amines, owing to its simplicity, versatility, sensitivity, and specificity. Recent developments in microbore column HPLC-ED have been facilitated by miniaturization of solvent delivery, column packing, sample injection and micro-flow cell construction. The aim of this paper is to present an overview of recent developments in microbore column HPLC-ED, in terms of advantages and limitations. This paper covers the recent advancements and important factors of HPLC-ED analysis of biogenic amines using microbore columns. Particular emphasis is placed on applying this technique to microdialysis, for which great sensitive is required. Its potential in future biomedical applications is also discussed.