[Norepinephrine of the rat myocardium during repeated ischemia].

Character of tissue changes as well as their reversibility could vary depending on the duration of myocardial ischemia. Long (over 30 min) ischemia leads to a massive release of myocardial interstitial norepinephrine. We tried to investigate changes in the myocardial sympathetic system produced by a relatively long episode of ischemia-repeperfusion. Myocardial norepinephrine has been collected by means of microdialysis probe during repeated occlusions of the left descending coronary artery. It was shown that long episode of occulusion-reperfusion resulted in suppression of massive norepinephrine release in response to second (test) occlusion. The features of norepinephrine release during successive occlusions make it possible to associate this process with the reversibility of the ischemic tissue damages.

Correlation of the capacity factor in vesicular electrokinetic chromatography with the octanol:water partition coefficient for charged and neutral analytes.

PURPOSE: The aim of this study was to develop a method based upon electrokinetic chromatography (EKC) using oppositely charged surfactant vesicles as a buffer modifier to estimate hydrophobicity (log P) for a range of neutral and charged compounds. METHODS: Vesicles were formed from cetyltrimethylammonium bromide (CTAB) and sodium n-octyl sulfate (SOS). The size and polydispersity of the vesicles were characterized by electron microscopy, dynamic light scattering, and pulsed-field gradient NMR (PFG-NMR). PFG-NMR was also used to determine if ion-pairing between cationic analytes and free SOS monomer occurred. The CTAB/SOS vesicles were used as a buffer modifier in capillary electrophoresis (CE). The capacity factor (log k') was calculated by determining the mobility of the analytes both in the presence and absence of vesicles. Log k' was determined for 29 neutral and charged analytes. RESULTS; There was a linear relationship between the log of capacity factor (log k') and octanol/water partition coefficient (log P) for both neutral and basic species at pH 6.0, 7.3, and 10.2. This indicated that interaction between the cation and vesicle was dominated by hydrophobic forces. At pH 4.3, the log k' values for the least hydrophobic basic analytes were higher than expected, indicating that electrostatic attraction as well as hydrophobic forces contributed to the overall interaction between the cation and vesicle. Anionic compounds could not be evaluated using this system. CONCLUSION: Vesicular electrokinetic chromatography (VEKC) using surfactant vesicles as buffer modifiers is a promising method for the estimation of hydrophobicity.

Determination of myocardial norepinephrine in freely moving rats using in vivo microdialysis sampling and liquid chromatography with dual-electrode amperometric detection.

Myocardial norepinephrine (NE) is considered a meaningful parameter for estimation of cardiac function. Long lasting changes in myocardial NE appear to be not only a consequence of pathologic processes in the myocardium, but may be a factor responsible for some diseases (e.g. increased propensity for arrhythmias or negative effect on left ventricular contractility in congestive heart failure). In this respect monitoring of myocardial NE is of great importance. A microdialysis sampling technique coupled with liquid chromatography with electrochemical detection (LCEC) was developed to measure the in vivo NE concentration in the myocardial interstitium of conscious, freely moving rats. LCEC using a dual-electrode amperometric detection in the series configuration provided detection limits for NE of 10 pg/ml in 20 microl microdialysis samples. Microdialysis probes of the linear design were implanted in the myocardial tissue in the periphery of the left descending coronary artery. The basal steady-state concentration of NE in myocardial dialysate of awake, freely moving rats was found to be 0.17+/-0.026 ng/ml. Delivery through the microdialysis probe of the NE reuptake inhibitor desipramine (DMI) at a concentration of 0.1 mM increased NE release to 153+/-13% of control. If the concentration of DMI in the perfusate was increased to 1.0 mM, NE release increased to only 166+/-21% of control.

pH-mediated field-amplified sample stacking of pharmaceutical cations in high-ionic strength samples.

Capillary electrophoretic separation of samples of physiological origin typically have both poor resolution and efficiency due to destacking. We have previously reported a stacking method for concentration of catecholamines in artificial dialysate, or Ringer's solution. However, pH-mediated sample stacking of other cations has not been investigated. In this report, pH-mediated stacking has been extended to eletripan, dofetilide, doxazosin, sildenafil, UK-103,320, UK-202,581, and CP-122,288. These compounds were chosen without prior structural screening except that they were cationic at the pH of our background electrolyte (BGE). Capillary electrophoretic behavior of samples in BGE is compared with those of samples in Ringer's solution with and without pH-mediated acid stacking. Results indicate that the peak heights and efficiencies for acid-stacked samples are increased compared to the unstacked samples in Ringer's solution or BGE. For example, the peak efficiencies for 5 s injections of eletriptan in BGE and Ringer's solution are 138,000 and 72,000 plates, respectively. In contrast, a 10 s injection of eletriptan followed by acid injection for 16 s produces a peak with 246,000 plates. Evaluation of the stacking effect was performed by comparison of the peak height at similar peak efficiencies for samples in Ringer's solution with and without stacking. Using this method, pH-mediated acid stacking provides a 10- to 27-fold sensitivity enhancement for the seven cations.

Cellulose acetate decoupler for on-column electrochemical detection in capillary electrophoresis.

A new decoupler for on-column electrochemical detection in capillary electrophoresis is presented. The decoupler is constructed by etching a series of holes through the side of the separation capillary with a CO2 laser and then coating the holes with cellulose acetate. The decoupler shows isolation of the detection circuit for separation currents up to 30 microA. Detection limits below 1 nM were achieved for four model compounds, including anions, neutrals, and cations, using the laser-etched decoupler. This decoupler design combines excellent mechanical stability, effective shunting of high separation currents, and ease of manufacture.

Microdialysis sampling of the isothiazolone, PD-161374, and its thiol and disulfide metabolites.

A method based on microdialysis sampling combined with high-performance liquid chromatography (HPLC) has been developed for monitoring the anti-HIV agent PD-161374 (isothiazolone) and its thiol and disulfide metabolites in blood. It was demonstrated that unlike blood withdraw and extraction, microdialysis sampling can preserve the distribution among the isothiazolone and its metabolites in blood. The use of a narrow-bore HPLC system, combined with the relatively high probe extraction efficiency (approximately 50%) from the flexible probe design in this work, allows the direct and quantitative determination of the drug and its major metabolites at submicromolar level.

Analytical considerations for microdialysis sampling.

Adaptations in microdialysis probe designs have made it possible to obtain samples from the extracellular fluid of a variety of tissues with high temporal resolution. The resulting small volume samples, often with low concentration of the analyte(s) of interest, present a particular challenge to the analytical system. Rapid separations can be coupled on-line with microdialysis to provide near real-time data. By combining microdialysis sampling with a liquid chromatographic or capillary electrophoretic separation and a highly sensitive detection method, a separation-based sensor can be developed. Such sensors have been applied to the investigation of drug entities as well as to study endogenous analytes.

On-line preconcentration methods for capillary electrophoresis.

The limits of detection (LOD) for capillary electrophoresis (CE) are constrained by the dimensions of the capillary. For example, the small volume of the capillary limits the total volume of sample that can be injected into the capillary. In addition, the reduced pathlength hinders common optical detection methods such as UV detection. Many different techniques have been developed to improve the LOD for CE. In general these techniques are designed to compress analyte bands within the capillary, thereby increasing the volume of sample that can be injected without loss of CE efficiency. This on-line sample preconcentration, generally referred to as stacking, is based on either the manipulation of differences in the electrophoretic mobility of analytes at the boundary of two buffers with differing resistivities or the partitioning of analytes into a stationary or pseudostationary phase. This article will discuss a number of different techniques, including field-amplified sample stacking, large-volume sample stacking, pH-mediated sample stacking, on-column isotachophoresis, chromatographic preconcentration, sample stacking for micellar electrokinetic chromatography, and sweeping.

Microdialysis sampling with on-line microbore HPLC for the determination of tirapazamine and its reduced metabolites in rats.

An on-line microdialysis microbore HPLC method is described for the determination of the bioreductive anti-tumor agent, tirapazamine (3-amino-1,2,4-benzotriazine-1,4-di-N-oxide, SR4233, WIN59075, Tirazone, TPZ) and its two major reduced metabolites, 3-amino-1,2,4-benzotriazine-1-N-oxide (SR4317) and 3-amino-1,2,4-benzotriazine (SR4330). Detection limits of 0.003 microM, 0.005 microM and 0.007 microM were obtained for tirapazamine, SR4317 and SR4330, respectively. Linear ranges of 0.011-20 microM, 0.017-20 microM and 0.025-20 microM for tirapazamine, SR4317 and SR4330 permitted quantitative analysis of all three compounds in microdialysis samples. Typical intra-day reproducibilities (n = 7) of 4.1% (tirapazamine), 6.6% (SR4317), 9.9% (SR4317), and 1.8% (tirapazamine), 2.4% (SR4317) and 2.6% (SR4330) were obtained at the 0.12 microM and 1.2 microM levels, respectively. Inter-day reproducibilities (n = 5) of 3.4% (tirapazamine), 1.8% (SR4317), 4.5% (SR4330) and 2.5% (tirapazamine), 2.5% (SR4317) and 1.7% (SR4330) were obtained at the 0.12 microM and 1.2 microM levels, respectively. The use of an on-line microdialysis HPLC system, permitted the determination of tirapazamine, SR4317 and SR4330 in blood and muscle tissue of rats with a high temporal resolution of sampling. The pharmacokinetics of tirapazamine and its metabolites were studied in the muscle and blood of rats previously administered an intraperitoneal dose of tirapazamine.

Detection of a urinary biomaker for oxidative DNA damage 8-hydroxydeoxyguanosine by capillary electrophoresis with electrochemical detection.

8-Hydroxydeoxyguanosine (8-OHdG) is present in urine as a result of oxidative DNA damage associated with age-related diseases such as cancer. In this report a method is presented for the detection of 8-OHdG in human morning urine utilizing capillary electrophoresis with electrochemical detection (CEEC). The limit of detection for a aqueous standard of 8-OHdG is 50 nM (signal to noise ratio S/N = 3). A single solid-phase extraction (SPE) step with a C18 column is used for sample cleanup and 20-fold preconcentration of the urine before analysis by CEEC. Optimized conditions for analysis of extracted urine are E(app) = 0.5 V vs. Ag/AgCl with 20 mM sodium borate/20% MeOH v/v, pH 9, as the background electrolyte, and a separation voltage of 22 kV. The concentration of 8-OHdG varied from 6 to 86 nM with an average value of 42 +/- 26.9 nM for four healthy female and four healthy male subjects between the ages of 23 and 43.

Analysis of illicit drugs by nonaqueous capillary electrophoresis and electrochemical detection.

Nonaqueous capillary electrophoresis (NACE) was applied to the determination of illicit drugs. The complete separation of amphetamine, methamphetamine, 3,4-methylene dioxy amphetamine (MDA), 3,4-methylene dioxy methamphetamine (MDMA), mescaline, cocaine and benzoylecgonine was obtained using an acetonitrile based buffer solution containing 10 mM sodium acetate and 1 M acetic acid. Electrochemical detection using a Pt microdisk electrode set to a potential of +1.8 V was found to be selective for MDA, MDMA and mescaline. The detection limits for these compounds were in the low ng/mL range which is between 2 and 3 orders of magnitude lower compared to UV-detection.

pH-mediated field amplification on-column preconcentration of anions in physiological samples for capillary electrophoresis.

Two limitations of capillary electrophoresis (CE) are the low sample loadability of the capillary and an incompatibility with high ionic strength samples. Several strategies have been described to preconcentrate and lower the ionic strength of physiological samples prior to CE analysis. These have included both off-capillary and on-capillary approaches. We have previously described a version of on-column field-amplification stacking termed pH-mediated stacking. pH-mediated stacking was initially developed for the separation of cations. In this report, we describe the application of pH-mediated sample stacking to anions. In this method, an electrokinetic injection is used to introduce analyte anions into the CE system and simultaneously replace the sample matrix cations with ammonia from the background electrolyte. Base is then electrokinetically injected to neutralize the sample zone and create a low conductivity region across which the analyte anions will stack. Using this method, a sensitivity enhancement of more than 66-fold was achieved without loss in separation efficiency relative to normal electrokinetic injection. Detection limits of 0.3 microM for four phenolic acids in a physiological sample were achieved using simple UV absorbance detection. The limit to the amount of sample that could be loaded using this technique was the length of the separation capillary. To further increase the amount of sample that could be loaded, a double-capillary system was developed. Using the double-capillary system the sensitivity was increased more than 300-fold and detection limits of 0.06 microM were achieved.

End-column amperometric detection in capillary electrophoresis: influence of separation-related parameters on the observed half-wave potential for dopamine and catechol.

Capillary electrophoresis (CE) was coupled to a micro-electrode-based end-column amperometric detector. The influences of separation voltage, CE buffer concentration, and capillary-to-electrode distance on the observed hydrodynamic voltammetry of dopamine and catechol were studied using a separation capillary with an i.d. of 25 microns. It was found that an increased CE voltage, increased buffer concentration, or decreased capillary-to-electrode distance resulted in a positive shift of the observed half-wave potentials for both dopamine and catechol. At a constant separation current of 1.6 microA, the observed half-wave potential was found to increase with applied separation voltage. Furthermore, when experiments were carried out with a platinum quasi-reference electrode instead of a Ag/AgCl reference electrode, similar shifts in half-wave potential were observed. These results indicate that the observed shifts are an effect of the separation voltage rather than the separation current or a change in the reference potential. The characteristics of end-column detection with and without a fracture decoupler were compared. It was found that the effects of separation voltage, CE buffer concentration, and capillary-to-electrode distance were minimized by the use of a decoupling device. The observed half-wave potentials for dopamine and catechol were more positive when a CE capillary without a decoupler was employed compared to when a decoupler was used. Additionally, using the fracture decoupler, the observed half-wave potentials for both dopamine and catechol were approximately the same as when no CE voltage was applied (i.e., when the hydrodynamic voltammograms were recorded under flow injection conditions).

Peer reviewed: instrumental analysis at the University of Kansas: an experiment in problem-based learning.

Students work in groups to solve real-world problems.

[An improved method for microdialysis study of noradrenaline level in rat myocardium]. / Usovershenstvovannaia metodika mikrodializnogo issledovaniia soderzhaniia noradrenalina v miokarde krysy.

A level of myocardial norepinephrine (NE) is considered as one of the meaningful parameters in the estimation of myocardium functioning. Long lasting changes of myocardial NE appear to be not only a sequence of pathologic processes in myocardium, but could also be a factor responsible for some diseases. An improved microdialysis sampling technique with HPLC-ED analysis was developed to measure in vivo NE content in a rat myocardial interstitium. Linear polyacrylonitryl 5 mm probes were flushed with methanol while being immersed in water. In vitro NE recovery with the flow rate of perfusate was found at the level 67.9 + 0.4%. Probes were implanted into the rat myocardium under the general anaesthesia. Myocardial dialysate was collected during 20 min into plastic vials, containing 10 microliters of 0.1 M perchloric acid. Low noise, high sensitivity dual electrochemical detection was used for the determination of NE amount in the samples. Overall sensitivity threshold was found about 0.3 pg of NE in 20 microliters. Steady state concentration of NE in myocardial dialysate was found 54 + 7 pg/ml. 20 min before cardiac arrest the concentration of NE in myocardial dialysate was found to be increased 4-5 times. 40 min after cardiac arrest further increase in NE content was registered up to 1-4 ng per ml of perfusate. It is suggested that the method will be useful for routine study of myocardial NE.

Determination of bupivacaine and three of its metabolites in rat urine by capillary electrophoresis.

A capillary electrophoretic (CE) method for the analysis of urinary extracts of the local anesthetic, bupivacaine, and its three main metabolites, desbutylbupivacaine, 3'-hydroxybupivacaine, and 4'-hydroxybupivacaine, in rat urine has been developed. The limits of detection were 0.22 microM for desbutylbupivacaine and bupivacaine, 0.15 microM for 3'-hydroxybupivacaine, and 0.16 microM for 4'-hydroxybupivacaine. The linear range was from 0.7 microM to 16.8 microM for all four compounds. Migration time and peak height reproducibilities, and extraction efficiencies were determined for all four compounds. Peak height reproducibilities (n = 5) for the overall method were improved through the use of prilocaine as an internal standard. Peak height reproducibilities were 5.6% RSD for desbutylbupivacaine and bupivacaine, and 9.9% RSD for 3'-hydroxybupivacaine and 4'-hydroxybupivacaine. Migration time reproducibilities (n = 5) were 2.4% for all compounds. Urine samples were collected from rats administered therapeutic doses of bupivacaine and extracted using a solid-phase extraction method (SPE). Separation of bupivacaine and its metabolites was achieved in 15 min.

On-column sample preconcentration using sample matrix switching and field amplification for increased sensitivity of capillary electrophoretic analysis of physiological samples.

An on-line sample concentration method using sample matrix switching and field amplification peak stacking has been developed. A microbore LC guard column is used to slightly retain the analytes in order to switch from a high ionic strength sample matrix (the physiological fluid) to a low ionic strength matrix (the LC mobile phase). The eluted LC peak is then trapped in a CE system and preconcentrated by field amplification peak stacking. The concentrated sample peak is then analyzed by CE. Compared to normal hydrodynamic injection, the sensitivity was increased by more than 500-fold without loss in resolution. A limit of detection of less than 10 nM for a physiological sample was achieved using UV adsorption detection. This method can be used for negatively or positively charged analytes.

Development of a capillary electrophoretic separation of an N-(substituted)-glycine-peptoid combinatorial mixture.

Capillary electrophoresis was used for the separation of a combinatorially synthesized N-(substituted)-glycine (NSG) peptoid mixture. This mixture consisted of 24 trimeric compounds sharing a common backbone structure but differing in the side chain attached at the N-terminal residue. Standards of the individual components were unavailable so that development of the separation was based on the mixture. A variety of buffer additives were investigated to enhance the CE resolution of this diverse mixture. Ion-pairing agents, cyclodextrins and organic modifiers were all evaluated as buffer additives. The best separations were achieved using a combination of buffer additives, each serving a different purpose in the separation. Heptane sulphonic acid (HSA) was used to reduce hydrophobic intramolecular interactions. Methyl-beta-cyclodextrin was used to provide host-guest interactions in order to resolve the very hydrophobic components of the NSG-peptoid mixture. The optimized run buffer consisted of 250 mM sodium phosphate buffer, pH 2.0, with 25 mM HSA and 40 mg/ml BCD and resulted in the resolution of 21 peaks for the 24 peptoids in the combinatorial mixture.

Monitoring oxygen partial pressure in tissue using microdialysis sampling and membrane coated oxygen sensors.

The use of microdialysis sampling coupled to oxygen sensors for monitoring oxygen tension in the tissue of animals was examined. The response time of the microdialysis sampling system was not different than oxygen sensors implanted directly in the tissue. The microdialysis system could be calibrated in vivo using the no-net-flux calibration method while directly implanted oxygen sensors had to rely on in vitro calibration. No difference was found in the oxygen tensions determined in vivo between microdialysis sampling and direct sensor implantations.