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Biomedical subjects

S M Lunte

Publications and source records attributed to S M Lunte.

At least 37 records · Page 2Linked to original sources
PubMed1997

Determination of kynurenic acid by capillary electrophoresis with laser-induced fluorescence detection.

Kynurenic acid (KA) is an excitatory amino acid receptor antagonist that is believed to play an important role in a host of diseases of the neuropsychiatric and central nervous system. A method for the determination of KA in microdialysate samples using capillary electrophoresis (CE) separation with laser-induced fluorescence (LIF) detection is described. CE is advantageous for the analysis of microdialysis samples due to its short analysis times and small sample volume requirements. Three complexation approaches were evaluated in an attempt to achieve the best limit of detection. The best approach was found to be pre-column complexation with inclusion of Zn(II) in the background electrolyte. After optimization of the zinc acetate concentration and pH, a limit of detection of 1 nM KA was achieved. However, when KA was present in the dialysate, the limit of detection increased 50-fold. Even though the endogenous levels of KA in rat brain are below this limit of detection, this methodology could be used to monitor the increase of KA levels in rat brain following dosing with its precursors, tryptophan and kynurenine.

Animals↗
PubMed1997

Capillary zone electrophoresis separation of tryptophan and its metabolites, including quinolinic acid.

Capillary zone electrophoresis with UV absorbance detection was used to separate tryptophan and ten of its metabolites. Run buffers of pH 4.0-10.0 were evaluated for their effect on resolution; a pH 9.6 buffer was found to give optimum separation of all components. Ethylenediaminetetraacetic acid (EDTA), which prevents complexation of some analytes with polyvalent cations, was included in the run buffer to insure good peak shape and reproducible mobilities. The resulting method was used to detect the presence of quinolinic acid in a urine sample.

Buffers↗
PubMed1997

Evaluation of capillary electrophoresis with post-column derivatization and laser-induced fluorescence detection for the determination of substance P and its metabolites.

A method for the detection of substance P and its metabolites using capillary electrophoresis with post-capillary derivatization and laser-induced fluorescence detection is described. The post-capillary chemical derivatization system employs naphthalene-2,3-dicarboxaldehyde and beta-mercaptoethanol. Two reactor designs were evaluated for the determination of substance P and its metabolites. The fluorescent spectroscopic properties of the derivatives under optimal separation conditions were also examined. The final system was evaluated for the investigation of substance P metabolism in brain following perfusion of the striatum with substance P using microdialysis sampling.

Animals↗
PubMed1997

Capillary electrophoresis in pharmaceutical analysis.

Capillary electrophoresis (CE) is a separation technique particularly suited to the analysis of pharmaceutical compounds. This review offers a detailed discussion of the four common modes of detection coupled to CE-UV absorption, fluorescence, electrochemical, and mass spectrometry-and gives examples of the use of these methods in pharmaceutical analyses. Sample preparation and pretreatment techniques used for CE separations are described, as well as methods of preconcentration including hydrophobic retention, affinity concentration, sample stacking, and isotachophoresis. The use of affinity CE, chiral CE, and capillary gel electrophoresis for analysis of pharmaceuticals is covered in detail, and recent advances in capillary electrochromatography and CE on a chip are also discussed.

Electrophoresis, Capillary↗
PubMed1996

Complete capillary electrophoretic separation of substance P and its metabolites at neutral pH using ionic run buffer additives.

Substance P (SP), a bioactive peptide that is present in the extracellular fluid of the brain at picomolar concentrations, has been proposed to be a neurotransmitter and a neuromodulator in the central and peripheral nervous systems. This paper describes the development of analytical methodology for the separation of SP and its metabolites by capillary electrophoresis at neutral pH. Since SP is a cationic peptide, it tends to adsorb to silica surfaces. In this work, phytic acid was employed as a run buffer additive to eliminate the interaction of SP and its cationic N-terminus metabolites with ionized silanol groups. The separation of the C-terminus metabolites was then accomplished through the use of sulfobutyl ether beta-cyclodextrin, which imparted a negative charge to these otherwise neutral metabolites through complexation of the phenylalanine residue. The final separation buffer consisted of 150 mM boric acid, 15 mM phytic acid and 5 mM SBE(IV)beta-cyclodextrin at pH 7. Under these conditions, it was possible to separate SP from all eight of its reported metabolites.

Amino Acid Sequence↗
PubMed1996

Dual-electrode detection for capillary electrophoresis/electrochemistry.

The extremely low sample volumes required for capillary electrophoresis and the high sensitivity and selectivity of electrochemical detection make capillary electrophoresis/electrochemistry (CEEC) a very useful method for bioanalysis. In this paper, two types of dual-electrode detectors for CEEC are described. The first employs a ring-disk microelectrode placed in a wall-jet configuration and is used for the selective detection of substances undergoing chemically reversible oxidations. Collection efficiencies obtained for catecholamines with this configuration were between 25 and 35%. The second electrode design consists of two adjacent carbon fibers embedded in an epoxy matrix and is analogous to the parallel dual-electrode configuration used in liquid chromatography/electrochemistry. This configuration can be used to confirm peak identity and purity by operating the electrodes at two different potentials. Alternatively, it is possible to perform simultaneous oxidative and reductive electrochemical detection.

Catecholamines↗
PubMed1996

Capillary electrophoresis with pulsed amperometric detection of carbohydrates and glycopeptides.

The utility of capillary electrophoresis with pulsed amperometric detection (CE-PAD) for the analysis of carbohydrate-containing samples from a variety of biological sources is described. CE-PAD was used to separate a mixture of oligosaccharides obtained from bovine fetuin and to monitor the desialylation process used in the characterization of the oligosaccharides. Additionally, the high resolving power of the system was demonstrated using a series of glycopeptides obtained from recombinant coagulation factor VIIa, which possess the same decapeptide core but differ in the extent of sialylation. Deglycosylation of these glycopeptides for characterization purposes resulted in a mixture of carbohydrates and peptides. Unlike CE with UV detection, this system gave good responses for all analytes, demonstrating the unique ability of PAD to respond to the electrochemical features of diverse classes of biomolecules such as carbohydrates and peptides. Finally, CE-PAD was applied to the analysis of a tryptic digest of recombinant tissue plasminogen activator. The use of different detection potentials in sequential runs on a sample gave structural information about the peptides, such as glycosylation. A brief review of prior applications of CE-PAD to the analysis of standard mixtures of simple saccharides is also presented.

Electrophoresis, Capillary↗
PubMed1995

Determination of alpha-difluoromethylornithine in blood by microdialysis sampling and capillary electrophoresis with UV detection.

A procedure is described for the analysis of alpha-difluoromethylornithine (DFMO), an anti-cancer agent, in plasma microdialysis (MD) samples. DFMO has been shown to be effective alone or in combination with other agents in the treatment of several cancers. Precolumn derivatization of DFMO with naphthalene-2,3-dicarboxaldehyde-cyanide (NDA-CN) in pH 10.0 borate buffer results in the rapid formation of a stable mono-derivatized product (N-substituted 1-cyanobenz[f]isoindole, CBI), which is UV active. An analytical method has been developed to separate CBI-DFMO from NDA-CN derivatization products of 20 standard amino acids using capillary electrophoresis (CE). This method is then employed for the determination of DFMO in plasma microdialysis samples. Separation of DFMO from other components in the dialysate was achieved within 20 min. The response for DFMO in Ringer's solution was linear over the range of 1.2 x 10(-6) to 1.6 x 10(-4) M after derivatization. The detection limit of DFMO in the plasma dialysate is 5 microM using UV detection at 254 nm. This method has been proven to have adequate sensitivity for quantitation of DFMO in i.v. microdialysate samples and has been successfully applied to monitoring the pharmacokinetics of DFMO by CE-UV.

Animals↗
PubMed1995

Characterization of glycopeptides from recombinant coagulation factor VIIa by high-performance liquid chromatography and capillary zone electrophoresis using ultraviolet and pulsed electrochemical detection.

Four glycopeptide (GP) fractions containing glycosylated Asn 322 were isolated from a tryptic digest of recombinant coagulation factor VII by reversed-phase-HPLC (RP-HPLC). Characterization of the GPs by enzymatic desialylation and RP-HPLC as well as by enzymatic deglycosylation, RP-HPLC, and high-pH anion-exchange chromatography indicated that the four GPs consisted of the same decapeptide but with 0, 1, 2, or 4 residues of sialic acid. In comparison to HPLC, capillary zone electrophoresis (CZE) using uv and pulsed electrochemical detection (PED) afforded improved separation of GPs from each other and from contaminants. CZE-uv and CZE-PED of the desialylated GPs and deglycosylated GPs corroborated the results obtained with the chromatographic methods.

Amino Acid Sequence↗
PubMed1995

Continuous in vivo monitoring of amino acid neurotransmitters by microdialysis sampling with on-line derivatization and capillary electrophoresis separation.

A separation-based biosensor has been developed that is capable of near-real-time analysis of aspartate and glutamate with a temporal resolution of less than 2 min in anesthetized or awake, freely moving animals. The instrument consists of a microdialysis sampling system, an on-line reactor, an injection interface, and a CE-LIF system. Primary amine analytes are derivatized with NDA/CN following microdialysis sampling using an on-line reactor to produce fluorescent CBI derivatives. The reaction takes approximately 1 min. The derivatized sample then travels to a microinjection valve which alternately sends CE running buffer and reacted microdialysis sample to the CE column via an injection interface. The interface allows a controllable volume of 10-20 nL to be injected onto the CE separation capillary. Separation of aspartate and glutamate from the other amino acids present in the microdialysis sample was achieved within 70 s. Detection limits for glutamate and aspartate using laser-induced fluorescence detection were 0.1 microM. The linear dynamic range was acceptable for the determination of aspartate and glutamate in dialysate samples where the levels are between 1 and 10 microM. Full automation of the system was achieved by computer control of the valve, the interface, and the data collection system. The performance of this system was demonstrated in an anesthetized rat by monitoring ECF levels of aspartate and glutamate released in brain after stimulation with high concentrations of K+.

Animals↗
PubMed1995

Direct determination of amino acids by capillary electrophoresis/electrochemistry using a copper microelectrode and zwitterionic buffers.

Sixteen free amino acids were separated by capillary electrophoresis (CE) using zwitterionic buffers of pH 9.8 and detected amperometrically using a copper microelectrode. Low conductivity buffers make possible the use of higher buffer concentrations. This depresses the electroosmotic flow, leading to a better separation of the amino acids compared to that obtained using strong electrolytes. The use of zwitterionic buffers also minimizes the amount of Joule heating in the capillary leading to more efficient separations. Perhaps most importantly for electrochemical (EC) detection, these buffers reduce the noise due to high separation current at the EC detector. The increased signal-to-noise ratio (S/N) results in much better detection limits for amino acids than those reported previously for capillary electrophoresis/electrochemistry (CEEC) with a copper electrode. Detection limits ranged from 10 to 400 nM, and the response was linear over three orders of magnitude for most of the amino acids. The system demonstrated good long-term stability and reproducibility with a relative standard deviation (RSD) of less than 5% for both the migration time and peak current (n = 20). This method was employed for the determination of amino acids in urine and in brain microdialysate samples.

Amino Acids↗
PubMed1994

Determination of the number and distribution of oligosaccharide linkage positions in O-linked glycoproteins by capillary electrophoresis.

O-Linked oligosaccharide chains are found in a wide variety of glycoproteins. Determination of the number of O-linked serine and threonine residues is an important step in glycoprotein characterization. Alkaline release of the oligosaccharide chains from the peptide chain followed by bisulfite addition to the unsaturated residues generates sulfonates. Hydrolysis of treated samples followed by derivatization with naphthalene 2,3-dicarboxyaldehyde and cyanide results in analytes which are separated and quantitated by capillary electrophoresis with UV detection. The utility of the method was demonstrated using bovine submaxillary mucin yielding results that agreed with previously published values.

Animals↗
PubMed1994

Determination of metal ions by capillary electrophoresis using on-column complexation with 4-(2-pyridylazo)resorcinol following trace enrichment by peak stacking.

Co2+, Fe2+, Cu2+ and Zn2+ were separated and detected following derivatization with 4-(2-pyridylazo)resorcinol by capillary electrophoresis with UV-Vis detection. Parameters which were examined include both on-column and pre-column complexation, limit of detection, capillary loadability, linear dynamic range and reproducibility. A sample stacking technique was investigated in order to obtain better detection limits and greater sensitivity. Detection limits of 1 x 10(-8) M were achieved for Co2+, Fe2+ and Zn2+ and 4 x 10(-7) M for Cu2+. Mass detection limits were 0.2 fmol for Co2+, Fe2+ and Zn2+ and 7.0 fmol for Cu2+. The use of this method for the determination of metals in vitamin tablets and a pond water sample is presented.

Buffers↗
PubMed1994

On-line coupling of in vivo microdialysis sampling with capillary electrophoresis.

Microdialysis sampling has become an important means of continuously monitoring reactions in vivo. This sampling technique places a constraint on the analysis method because of the very small sample volume provided. On the other hand, microdialysis provides the advantage of clean samples that do not require cleanup prior to analysis. An on-line coupling of microdialysis sampling to capillary electrophoretic (CE) analysis is described that uses the advantages of microcolumn separations to overcome the small volume limitation. An interface was designed which converts the continuous microdialysis sample stream into discrete 60-nL sample plugs and then injects a portion of this plug into the CE system. The on-line interface provided precision of 2.6% with minimal band broadening or peak height loss relative to off-line sampling. Using a high-speed micellar electrokinetic chromatography (MEKC) separation, resolution of the investigational antineoplastic SR 4233 from its main metabolite SR 4317 was achieved in less than 60 s. This allowed the on-line system to achieve a 90-s temporal resolution for determining the pharmacokinetics of SR 4233 in vivo.

Animals↗
PubMed1994

Pharmaceutical and biomedical applications of capillary electrophoresis/electrochemistry.

The use of capillary electrophoresis/electrochemistry (CEEC) for the analysis of microdialysis samples obtained for pharmacokinetic and neurochemical studies is described, as well as the development of new types of electrodes and waveforms which increase the selectivity of this technique for specific classes of analytes. CEEC with a carbon fiber electrode was employed for the analysis of microdialysis samples. Microdialysis is an in vivo sampling technique that yields very small samples for analysis (less than 1 microL). Therefore, capillary electrophoresis, with its small volume requirements, is an excellent choice for the analytical method. CEEC was used to study the pharmacokinetics of L-dopa and the release of aspartate and glutamate following a high K+ infusion in the brain. Several modified electrodes which increase the applicability of CEEC in pharmaceutical and biomedical analysis are described. One of these is a gold/mercury electrode which is highly selective for thiols and was used for the determination of glutathione in a rat brain. An alternative method for the detection of thiols employed a chemically modified electrode containing cobalt phthalocyanine. In this case, an electrocatalyst reduces the overpotential of thiols at the carbon electrode and makes it possible to detect them at a much lower and more selective oxidation potential. This electrode was used for the detection of cysteine in urine. The development of pulsed amperometric detection for capillary electrophoresis is also described and is demonstrated by the detection of glucose in blood. Lastly, a method for the detection of peptides based on the formation of a copper complex and detection at a carbon fiber electrode is discussed.

Animals↗
PubMed1994

Separation and quantitation of the amino acid neurotransmitters in rat brain by capillary electrophoresis.

Capillary electrophoresis (CE) has been used to separate the CBI derivatives of the neurotransmitters gamma-aminobutyric acid, glycine, glutamate, aspartate, norepinephrine and dopamine from 18 other amino acids present in the rat brain. The procedure, which requires an injection volume of < 10 nl, gave detection limits of 24-40 fmol using UV detection at 420 nm. Efficiencies for the derivatized amino acids varied from 344,000 to 444,000 theoretical plates. The method was applied successfully to the quantitation of the amino acid neurotransmitters and several other amino acids in a rat brain homogenate.

Animals↗
PubMed1993

Determination of peptides by capillary electrophoresis-electrochemical detection using on-column Cu(II) complexation.

A Cu(II)-coated capillary has been developed for the determination of peptides by capillary electrophoresis with electrochemical detection. Capillaries were prepared by forcing a solution 48 microM in CuSO4, 120 microM in tartaric acid, 2.4 mM in NaOH and 120 microM in KI through them for 25 min; the resulting capillaries are stable for at least 12 h. Under alkaline conditions, peptides complex with Cu(II) present on the walls of the capillary to form Cu(II)-peptide complexes which can be detected oxidatively at a carbon fiber electrode. Di-, tri-, tetra- and pentaglycine were determined with a detection limit of 7 x 10(-7) M for triglycine. N-Terminal-blocked peptides can also be determined via this method. This system is more sensitive than direct detection of peptides by UV at 210 nm and exhibits higher selectivity than commonly employed derivatization procedures based on reactions with a primary amine functionality.

Amino Acid Sequence↗