Malonne I. Davies

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.

A review of microdialysis sampling for pharmacokinetic applications

As an in vivo technique, microdialysis sampling reflects the composition of the extracellular fluid and can be used in virtually any tissue, organ or biological fluid. Microdialysis has several characteristics that make it a valuable addition to the classical techniques used in pharmacokinetic studies. Probe geometries are described and their suitability for particular target organs is discussed as is tissue response to probe implantation. The application of microdialysis sampling for pharmacokinetic and metabolism studies and its use in target tissues other than the brain have increased over the past decade. This review cites recent examples to illustrate the utility of microdialysis sampling for pharmacokinetic applications.

Detection of homocysteine by conventional and microchip capillary electrophoresis/electrochemistry

A method based on capillary electrophoresis (CE) with electrochemical (EC) detection for the determination of both total homocysteine (tHcy) and protein‐bound homocysteine (pbHcy) in plasma is described. Both end‐column and off‐column amperometric detection were investigated. Off‐column detection resulted in a more sensitive assay for the determination of homocysteine (Hcy). The detection limit for homocysteine was 500 nM using off‐column EC detection and the response was linear over the range 1–100 νM. Therefore, this assay is appropriate for the quantification of Hcy over the physiological concentration ranges found in all disease states. Methodologies for the determination of tHcy and pbHcy in human plasma were investigated and optimized and the concentrations of both pbHcy and tHcy in plasma obtained from a healthy individual were determined to be 2.79 ± 0.31 νM (n = 4) and 3.37 ± 0.15 νM (n = 3), respectively. The methodology was then transferred to a microchip CE‐EC format and Hcy and reduced glutathione (GSH) were detected. Future work will focus on the development of ancillary methodologies to identify the other forms of Hcy in vivo.

A review of membrane sampling from biological tissues with applications in pharmacokinetics, metabolism and pharmacodynamics

This review provides an overview of membrane sampling techniques, microdialysis and ultrafiltration, and cites illustrations of their applications in pharmacokinetics, metabolism and/or pharmacodynamics. The review organizes applications by target tissue and general type of information gleaned. It focuses on recently published microdialysis studies (1999 to this writing) and offers the first review of ultrafiltration sampling studies. The advantages and limitations of using microdialysis and ultrafiltration sampling as tools for obtaining pharmacokinetic and metabolism data are discussed. Numerous examples are described including studies in which several types of data are collected simultaneously. Reports that study local metabolism of drug delivered through the probe are also presented.

Investigation of the metabolism of substance P in rat striatum by microdialysis sampling and capillary electrophoresis with laser-induced fluorescence detection.

The metabolism of substance P (SP) was investigated in rat striatum using in vivo microdialysis. Substance P was perfused for 5 h at 0.2 microl/min, and its metabolism was followed for over 13 h. The resulting samples were derivatized precolumn with naphthalene-2,3-dicarboxaldehyde (NDA)/cyanide, separated and detected by cyclodextrin-modified electrokinetic chromatography with laser-induced fluorescence detection (CDMEKC-LIF). Substance P rapidly degraded to form the fragments (3-11), (1-9), (1-4) and, to a lesser extent, (1-7). The metabolites reached steady-state levels 2-3 h after addition of SP.

Simultaneous microdialysis sampling from multiple sites in the liver for the study of phenol metabolism

Microdialysis probes of the linear design were implanted in three regions of the liver of anesthetized rats. Probes planted 1 cm apart exhibited no cross communication as demonstrated by continuously perfusing one probe with phenol for over 2 hours while monitoring the other probes for phenol and its metabolites. Regional differences in metabolic profile were observed following intravenous administration of phenol. Significantly lower concentrations of the phase two metabolites, phenol-glucuronide and hydroquinone-glutathione conjugate, were found at the probe implanted at the anterior position relative to the probes at the median and posterior positions. The kinetics of elimination were not significantly different at any of these positions. Phenol was also directly delivered to the liver through the microdialysis probes. No differences in delivery of phenol or formation of metabolites were observed at the three regions.

Microdialysis sampling coupled on-line to microseparation techniques

Microdialysis sampling is a powerful tool for continuously monitoring the extracellular concentration of compounds in tissues in vivo. In order to fully utilize the high temporal resolution but small sample volume of the microdialysis technique, several approaches to coupling microdialysis sampling on-line to microseparation techniques have been developed. This article will describe the analytical challenges of microdialysis sampling. Direct coupling of the microdialysis system to the analytical system can provide many benefits. The application of on-line microseparation techniques will be reviewed.

Evaluation of an osmotic pump for microdialysis sampling in an awake and untethered rat

The feasibility of using an osmotic pump in place of a syringe pump for microdialysis sampling in rat brain was investigated. The use of an osmotic pump permits the rat to be free from the constraints of the standard tethered system. The in vitro flow rates of a microdialysis syringe pump (set at 10.80 microl/h) and the osmotic pump (pump specifications were 11.35 microl/h) with no probe attached were compared, yielding results of 10.87 microl/h+/-1.7% and 10.95 microl/h+/-8.0%, respectively. The average of four flow rate experiments in vivo yielded R.S.D.s less than 10% and an average flow rate of 11.1 microl/h. Following the flow rate studies, in vivo sampling of neurotransmitters was accomplished with the osmotic pump coupled to a microdialysis probe implanted in the brain. Finally, after determination of basal levels of 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), and 5-hydroxyindole-3-acetic acid (5-HIAA) in the rats, the rats were dosed with benserazide followed by l-3,4-dihydroxyphenylalanine (l-DOPA). The results from the dosing study showed at least a 10-fold increase in compounds in the l-DOPA metabolic pathway (DOPAC and HVA) and a slight or no increase in 5-HIAA (serotonin metabolic pathway.) These results indicate that the osmotic pump is a viable alternative to the syringe pump for use in microdialysis sampling.

Using a microdialysis shunt probe to monitor phenolphthalein glucuronide in rats with intact and diverted bile flow

Abstract This study is undertaken to demonstrate that the shunt microdialysis probe is a valuable tool for profiling analytes in the rat bile duct while preserving enterohepatic circulation (EHC). Phenolphthalein (PT) is well known to be efficiently converted to its glucuronide adduct and enterohepatically cycled. Phenolphthalein glucuronide (PTG) has been used as marker to study both liver function and EHC. Using a microdialysis shunt probe, the concentration of PTG in the bile can be monitored. In this study, the PTG profiles were obtained in anesthetized rats with the bile flow either diverted or intact. Characterization of the PTG extraction efficiency (EE) using microdialysis showed that the in vitro EE of PTG was affected by the presence of bile salts, whereas the bile salts have no effect on the EE of caffeine. In addition, it was shown that the bile salt concentration must be balanced on both sides of the membrane in order to obtain the expected flow rate through the perfusion channel of the probe. A 2% solution of bile salts in Ringers (BSR) was sufficient as the perfusate against rat bile in the shunt. With BSR as the perfusate, consistent in vitro EE results (by both recovery and delivery experiments) for PTG were obtained. These in vitro results compared favorably with EE values determined in vivo.

Preliminary evaluation of several disinfection/sterilization techniques for use with microdialysis probes.

This study evaluated the suitability of some disinfection and sterilization methods for use with microdialysis probes. Disinfection or sterilization should minimize the tissue inflammatory reaction and improve the long-term health of rats on study and ensure the quality of data obtained by microdialysis sampling. Furthermore, the treatment should not negatively impact probe integrity or sampling performance. The techniques chosen for evaluation included two disinfection methods (70% ethanol and a commercial contact lens solution) and two sterilization methods (hydrogen peroxide plasma, and e-beam radiation). Linear microdialysis probes treated by these processes were compared to untreated probes removed from the manufacturers packaging as if sterile (the control group). The probes were aseptically implanted in the livers of rats and monitored for 72 hours. The parameters chosen to evaluate probe performance were relative sample mass recovery and the relative in vivo extraction efficiency of the probe for caffeine. Post mortem bacterial counts and histopathology examination of liver tissue were also conducted. The probes remained intact and functional for the entire study period. The methods tested did not acutely alter the probes although hydrogen peroxide plasma and contact lens solution groups showed reduced extraction efficiencies. Minimal tissue damage was observed surrounding the probes and acute inflammatory reaction was mild to moderate. Low numbers of bacterial colonies from the implantation sites indicates that the health of animals in this study was not impaired. This was also true for the control group (untreated probe).