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Biomedical subjects

S M Lunte

Publications and source records attributed to S M Lunte.

At least 19 recordsLinked to original sources
PubMed2002

Comparison of in vitro BBMEC permeability and in vivo CNS uptake by microdialysis sampling.

The studies presented in this report were designed to assess the correlation of the bovine brain microvessel endothelial cell (BBMEC) apparent permeability coefficient (P(app)) and in vivo BBB penetration using microdialysis sampling. A mathematical model was developed to describe the relationship of brain extracellular fluid (ECF) concentration to free drug in plasma. The compounds studied have a broad range of physico-chemical characteristics and have widely varying in vitro and in vivo permeability across the blood-brain barrier (BBB). BBMEC permeability coefficients vary in magnitude from a low of 0.9 x 10(-5) cm/s to a high value of 7.5 x 10(-5) cm/s. Corresponding in vivo measurements of BBB permeability are represented by clearance (CL(in)) into the brain ECF and range from a low of 0.023 microl/min/g to a high of 12.9 microl/min/g. While it is apparent that in vitro data from the BBMEC model can be predictive of the in vivo permeability of a compound across the BBB, there are numerous factors both prior to and following entry into the brain which impact the ultimate uptake of a compound. Even in the presence of high BBB permeability, factors such as high plasma protein binding, active efflux across the BBB, and metabolism within the CNS can greatly limit the ultimate concentrations achieved. In addition, concentrations in the intracellular space may not be the same as concentrations in the extracellular space. While these data show that the BBMEC permeability is predictive of the in vivo BBB permeability, the complexity of the living system makes prediction of brain concentrations difficult, based solely on the in vitro measurement.

Animals↗
PubMed2001

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.

Animals↗
PubMed2001

Fabrication and evaluation of a carbon-based dual-electrode detector for poly(dimethylsiloxane) electrophoresis chips.

The first carbon-based dual-electrode detector for microchip capillary electrophoresis (CE) is described. The poly(dimethylsiloxane) (PDMS)-based microchip CE devices were constructed by reversibly sealing a PDMS layer containing separation and injection channels to another PDMS layer containing carbon fiber working electrodes. End-channel amperometric detection was employed and the performance of the chip was evaluated using catechol. The response was found to be linear between 1 and 600 microM with an experimentally determined limit of detection (LOD) of 500 nM and a sensitivity of 30 pA/microM. Collection efficiencies for catechol ranged from 36.0 to 43.7% at field strengths of 260-615 V/cm. The selectivity that can be gained with these devices is demonstrated by the first CE-based dual-electrode detection of a Cu(II) peptide complex. These devices illustrate the potential for a rugged and easily constructed microchip CE system with an integrated carbon-based detector of similar scale.

Carbon↗
PubMed2001

Microchip capillary electrophoresis/electrochemistry.

Microfabricated fluidic devices have generated considerable interest over the past ten years due to the fact that sample preparation, injection, separation, derivatization, and detection can be integrated into one miniaturized device. This review reports progress in the development of microfabricated analytical systems based on microchip capillary electrophoresis (CE) with electrochemical (EC) detection. Electrochemical detection has several advantages for use with microchip electrophoresis systems, for example, ease of miniaturization, sensitivity, and selectivity. In this review, the basic components necessary for microchip CEEC are described, including several examples of different detector configurations. Lastly, details of the application of this technique to the determination of catechols and phenols, amino acids, peptides, carbohydrates, nitroaromatics, polymerase chain reaction (PCR) products, organophosphates, and hydrazines are described.

Amino Acids↗
PubMed2001

Investigation of the metabolism of substance P at the blood-brain barrier using capillary electrophoresis with laser-induced fluorescence detection.

Substance P (SP) metabolism was investigated upon exposure to a monolayer of bovine brain microvessel endothelial cells (BBMECs), a cell culture model of the blood-brain barrier. SP was incubated with the BBMECs and its metabolism was followed as a function of time over a 5-h period. The resulting samples were derivatized with naphthalene-2,3-dicarboxaldehyde (NDA)/cyanide, separated, and detected using cyclodextrin-modified electrokinetic chromatography with laser-induced fluorescence detection (CDMEKC-LIF). Upon exposure to the BBMEC monolayer, SP rapidly degraded to produce the N-terminal (1-9), (1-4) and (1-7) and C-terminal (2-11) and (3-11) fragments. These results were compared with those in an earlier report from our laboratory, where SP metabolism was investigated in vivo by microdialysis sampling in rat striatum.

Amino Acid Sequence↗
PubMed2001

Microchip electrophoretic separation systems for biomedical and pharmaceutical analysis.

The application of microchip capillary electrophoresis (CE) systems to biomedical and pharmaceutical analysis is described and reviewed. Fabrication, instrumentation, and operation of the systems are discussed. An overview of applications is presented, covering four main areas: DNA sequencing, genetic analysis, immunoassays, and protein and peptide analysis. These systems have the potential to dramatically change the way that biochemical analyses are performed.

Animals↗
PubMed2001

Carbon paste-based electrochemical detectors for microchip capillary electrophoresis/electrochemistry.

The first reported use of a carbon paste electrochemical detector for microchip capillary electrophoresis (CE) is described. Poly(dimethylsiloxane) (PDMS)-based microchip CE devices were constructed by reversibly sealing a PDMS layer containing separation and injection channels to a separate PDMS layer that contained carbon paste working electrodes. End-channel amperometric detection with a single electrode was used to detect amino acids derivatized with naphthalene dicarboxaldehyde. Two electrodes were placed in series for dual electrode detection. This approach was demonstrated for the detection of copper(II) peptide complexes. A major advantage of carbon paste is that catalysts can be easily incorporated into the electrode. Carbon paste that was chemically modified with cobalt phthalocyanine was used for the detection of thiols following a CE separation. These devices illustrate the potential for an easily constructed microchip CE system with a carbon-based detector that exhibits adjustable selectivity.

Carbon↗
PubMed2000

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.

Animals↗
PubMed2000

Dual-electrode electrochemical detection for poly(dimethylsiloxane)-fabricated capillary electrophoresis microchips.

The development of a poly(dimethylsiloxane)-based (PDMS-based) microchip electrophoresis system employing dual-electrode electrochemical detection is described. This is the first report of dual-electrode electrochemical detection in a microchip format and of electrochemical detection on chips fabricated from PDMS. The device described in this paper consists of a top layer of PDMS containing the separation and injection channels and a bottom glass layer onto which gold detection electrodes have been deposited. The two layers form a tight reversible seal, eliminating the need for high-temperature bonding, which can be detrimental to electrode stability. The channels can also be temporarily removed for cleaning, significantly extending the lifetime of the chip. The performance of the chip was evaluated using catechol as a test compound. The response was linear from 10 to 500 microM with an LOD (S/N = 3) of 4 microM and a sensitivity of 45.9 pA/microM. Collection efficiencies for catechol ranged from 28.7 to 25.9% at field strengths between 200 and 400 V/cm. Dual-electrode detection in the series configuration was shown to be useful for the selective monitoring of species undergoing chemically reversible redox reactions and for peak identification in the electropherogram of an unresolved mixture.

Dimethylpolysiloxanes↗
PubMed2000

Separation of naphthalene-2,3-dicarboxaldehyde-derivatized-substance P and its metabolites by micellar electrokinetic chromatography.

This paper describes the development of analytical methodology for the separation of naphthalene-2,3-dicarboxaldehyde (NDA)-derivatized substance P (CBI-SP) and five lysine-containing metabolites by micellar electrokinetic chromatography (MEKC). The effect of surfactant composition and organic modifiers on the separation was investigated. The final separation buffer consisted of 80 mM sodium cholate in 50 mM N-tris (hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), pH 7. All six lysine-containing peptides were separated under these conditions.

Amino Acid Sequence↗
PubMed2000

Optimization of the conditions for biuret complex formation for the determination of peptides by capillary electrophoresis with ultraviolet detection.

Capillary electrophoresis with UV detection was utilized to optimize copper complexation conditions for the analysis of neuropeptides. Complexation was confirmed by monitoring the response at a visible wavelength. Four complexation strategies were used to compare the UV response of native peptides and their respective copper complexes. All four strategies resulted in complete complexation, but on-capillary complexation provided significant advantages over precapillary and pre-/on-capillary. An increase in UV absorbance along with peak stacking resulted in a significantly greater response using the on-capillary technique. Also, on-capillary complexation does not require dilution of the sample. The effects of temperature and copper concentration were also investigated. The utility of this method for the separation of an enkephalin peptide mixture is presented.

Angiotensin II↗
PubMed2000

Detection of neuropeptides using on-capillary copper complexation and capillary electrophoresis with electrochemical detection.

Capillary electrophoresis with electrochemical detection using a carbon fiber electrode in conjunction with on-capillary copper complexation was evaluated for the determination of peptides in standard and biological matrices. Peptides composed of 2-10 amino acids were investigated. A comparison was made between the responses obtained for peptides containing the oxidizable residue tyrosine and those obtained for their respective copper complexes. Electrochemical detection of non-tyrosine-containing peptides and a cyclic peptide was also demonstrated. A separation of leucine (Leu)-enkephalin and five metabolites was developed and then used for the investigation of Leu-enkephalin metabolism in plasma. The appearance of the des-tyrosine (des-Tyr) Leu-enkephalin, which cannot be detected directly at a carbon electrode, was monitored using the on-capillary complexation technique. Direct injection of the plasma sample was possible using this methodology.

Angiotensins↗
PubMed1999

Hydroquinone-based derivatization reagents for the quantitation of amines using electrochemical detection.

Two new reagents, NDTE (2,5-dihydroxyphenylacetic acid, 2,5-bis-tetrahydropyranyl ether p-nitrophenyl ester) and HLTE (homogentisic gamma-lactone tetrahydropyranyl ether), are described for the chemical derivatization of primary and/or secondary amines to form an electrochemically active product. These reagents undergo reaction with the aforementioned analytes to form a product possessing the hydroquinone moiety, thus allowing for reversible electrochemical detection at mild oxidation potentials. The reactivity of each reagent was demonstrated by using N-ethylbenzylamine (EBzA) and the dipeptide isoleucine leucine methyl ester as model analytes. The investigation included the isolation and identification of the intermediates and final products from derivatization of EBzA. These isolated standards were subsequently characterized with respect to electrochemical properties by means of cyclic voltammetry. In LC-EC experiments, the concentration limit of detection (CLOD) of the purified EBzA product was determined to be 5 nM (100 fmol) at a detection potential of +200 mV vs Ag/AgCl ([Cl-] = 3 M). The CLOD values obtained by LC-EC after derivatization of aqueous solutions of EBzA and Ile-Leu-OMe with NDTE were 25 nM (250 fmol) and 250 nM (2.5 pmol), respectively.

Amines↗
PubMed1999

Postcolumn reaction detection with dual-electrode capillary electrophoresis-electrochemistry and electrogenerated bromine.

This is the first report of postcolumn amperometric reaction detection for capillary electrophoresis and dual-electrode detection. Bromide present in the run buffer is oxidized to bromine at the first electrode and subsequently detected at a second electrode downstream. Analytes that react with bromine cause a decrease in signal at the downstream electrode that is proportional to analyte concentration. Bromine is known to react with a variety of compounds, including thiols, thioethers, disulfides, amines, and unsaturated organic compounds. In this paper, the development of a new wire--wire on-capillary dual electrode that is well suited to bromine-based post-column reaction detection is described. System performance was evaluated using glutathione, cysteine, and methionine as test analytes. The final optimized system could be operated continuously for 24 h and was stable for day-to-day use for at least two weeks. The response for cysteine was linear from 0.5 to 20 microM with a limit of detection of approximately 80 nM.

Acetylcysteine↗
PubMed1999

Tubular-wire dual electrode for detection of thiols and disulfides by capillary electrophoresis/electrochemistry.

A new dual-electrode detector for capillary electrophoresis is described. The detector consists of an integrated gold tubular electrode as the generator electrode and a gold wire electrode for detection. The detector configuration, including electrode size and position, has been optimized in terms of detection sensitivity and separation efficiency. After amalgamation of the dual electrode with mercury, the capillary electrophoresis/electrochemistry system was employed for simultaneous detection of thiols and disulfides. The response of cystine was found to be linear from 1 microM to 1 mM with a LOD of 0.5 microM (S/N = 3) and sensitivity of 60 pA/microM. The detection limits represent 200-fold improvement over previously reported dual-electrode designs for the detection of disulfides. The use of this detector for identification of thiol- and disulfide-containing peptides was demonstrated with a tryptic digest of ribonuclease A.

Disulfides↗