Craig E. Lunte

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.

AAPS-FDA workshop white paper: Microdialysis principles, application, and regulatory perspectives

Many decisions in drug development and medical practice are based on measuring blood concentrations of endogenous and exogenous molecules. Yet most biochemical and pharmacological events take place in the tissues. Also, most drugs with few notable exceptions exert their effects not within the bloodstream, but in defined target tissues into which drugs have to distribute from the central compartment. Assessing tissue drug chemistry has, thus, for long been viewed as a more rational way to provide clinically meaningful data rather than gaining information from blood samples. More specifically, it is often the extracellular (interstitial) tissue space that is most closely related to the site of action (biophase) of the drug. Currently microdialysis (μD) is the only tool available that explicitly provides data on the extracellular space. Although μD as a preclinical and clinical tool has been available for two decades, there is still uncertainty about the use of μD in drug research and development, both from a methodological and a regulatory point of view. In an attempt to reduce this uncertainty and to provide an overview of the principles and applications of μD in preclinical and clinical settings, an AAPS-FDA workshop took place in November 2005 in Nashville, TN, USA. Stakeholders from academia, industry and regulatory agencies presented their views on μD as a tool in drug research and development.

Electrochemistry of catechol-containing flavonoids

The electrochemical properties of four structurally related flavonoids, quercetin, quercetin-3-O-rhamnose (quercitrin), quercetin-3-O-rutinose (rutin) and luteolin were investigated. These flavonoids were shown to undergo homogenous chemical reactions following oxidation at a glassy carbon electrode. These reactions were studied using cyclic voltammetry and rotating ring-disk voltammetry. Both first-order and zero-order processes were observed. The rate of the zero-order process was strongly dependent on the substituent at the C-3 position of the flavonoid. The rate of the first-order process was independent of substitution. Two products were observed using liquid chromatography. These products did not correspond to previously reported products of enzymatic oxidation. The products were not stable under conditions for isolation.

Capillary electrophoresis with electrochemical detection employing an on-column Nafion joint

Abstract The construction and evaluation of an on-column joint utilizing Nafion tubing for the isolation of the electrical circuit from the detection end of a capillary zone electrophoresis system is described. The joint enables electrochemical detection to be performed without adverse effects from the applied high voltage. The joint is both simple to construct and durable. The electrochemical detector employing a carbon fiber working electrode exhibited high coulometric efficiencies and a detection limit of 6 · 10−9M or 34.8 amol for hydroquinone. A high efficiency, of the order of 185 000 theoretical plates, was achieved for this compound. This system was evaluated for the detection of phenolic acids in apple juice and for the determination of naphthalene-2,3-dicarboxaldehyde derivatized amino acids in a brain homogenate. The use of voltammetry as a method of compound verification was also demonstrated.

p-aminophenyl phosphate: an improved substrate for electrochemical enzyme immnoassay

An alternative substrate is described for enzyme immunosaasay with electrochemical detection. Alkaline phosphatase (EC.3.1.3.1) activity is determined by using p-aminophenyl phosphate as the enzyme substrate. Enzyme-generated p-aminophenol is detected amperometrically at a glassy carbon electrody by liquid chromatography with electrochemical detection. The oxidation potential obtained for the detectionof p-aminophenol is lower than that for phenol, the previously used substrate product. The detection limit for p-aminophenol is 0.20pmol. A detection limit of 30 pg ml-1 for digoxin and a 5-min incubationtime for the enzyme reaction were obtained with the new system.

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.

In Vivo Microdialysis Sampling in the Bile, Blood, and Liver of Rats to Study the Disposition of Phenol

Methods for continuous in vivo sampling in the bile, blood, and liver extracellular fluid are described. These methods are based on microdialysis sampling in anesthetized rats. A new flow-through microdialysis probe is described for sampling bile while maintaining normal bile flow. All three sites are simultaneously and continuously sampled to provide concentration–time profiles at multiple sites in a single experimental animal. This technique is demonstrated by studying the hepatic metabolism and biliary excretion of phenol in rats. Following an i.v. infusion of phenol, the major hepatic metabolite was found to be phenyl-glucuronide. Hydroquinone and 2-glutathionyl–hydroquinone were also detected but at lower concentrations. A similar pattern of metabolites was found in the bile and blood. For all of the metabolites, bile concentrations are higher than liver concentrations, indicating that the metabolites are actively excreted into the bile.

Monitoring excitatory amino acid release in vivo by microdialysis with capillary electrophoresis- electrochemistry

Capillary electrophoresis (CE) with electrochemical detection (ED) was used to determine extracellular levels of aspartate, glutamate and alanine in samples from the frontoparietal cortex of the rat which were obtained by microdialysis. The method was used to monitor the effect on the overflow of the excitatory amino acids aspartate and glutamate of an influx of high concentrations of potassium ion. Samples were derivatized with naphthalenedialdehyde-cyanide prior to analysis. Detection limits for aspartate and glutamate were 80 and 100 nM, respectively. CE-ED is extremely useful for the analysis of microdialysis samples because of the very small sample volumes required by this analytical technique. The use of ED provides the requisite sensitivity and allows verification of peak purity by voltammetry.

Microdialysis Sampling for Determination of Plasma Protein Binding of Drugs

The use of microdialysis sampling to study the binding of drugs to plasma proteins was evaluated. Microdialysis sampling is accomplished by placing a short length of dialysis fiber in the sample and perfusing the fiber with a vehicle. Small molecules in the sample, such as drugs, diffuse into the fiber and are transported to collection vials for analysis. Larger molecules, such as proteins and protein-bound drugs are excluded by the dialysis membrane. Microdialysis was found to give values for in vitro protein binding in plasma equivalent to those determined by ultrafiltration. Microdialysis offers advantages in terms of maintaining equilibria and experimental versatility. Microdialysis sampling also provides potential use for in vivo determinations of protein binding.

Dermal Microdialysis Sampling in Vivo

Microdialysis sampling of the dermis in vivo was accomplished using a linear microdialysis probe. In contrast to previous studies using a commercial cannula-style microdialysis probe, the linear probe had no effect on the flux of drug through the skin in vitro. The extent of tissue damage in vivo due to probe implantation was evaluated by histological examination and microdialysis delivery studies. Tissue damage due to implantation of the linear probe was minimal with no bleeding or edema observed. Infiltration of lymphocytes into the tissue was observed beginning 6 hours after probe implantation with scar tissue beginning to form after approximately 32 hours. The infiltration of lymphocytes had no effect on the behavior of implanted microdialysis probes. Delivery of 5-fluorouracil was between 20 and 25% for six different probes implanted in six different animals demonstrating good probe-to-probe and implantation-to-implantation reproducibility. Constant delivery was maintained for at least 24 hours in all cases indicating that experiments of at least 24 hour duration are feasible. The dermal concentration of topically applied 5-FU cream, Efudex®, was continuously monitored by an implanted microdialysis probe demonstrating the feasibility of this technique as for monitoring skin drug levels in vivo. The dermal concentration of 5-FU following topical application was approximately 40-fold higher for in vitro excised skin than for in vivo intact skin.