Analysis of the Performance of a Flow Reactor for Use with Microcolumn HPLC

Beisler, Amy T.; Sahlin, Eskil; Schaefer, Kathleen; Weber, Stephen G.

doi:10.1021/ac034785d

Cited by 13 publications

(27 citation statements)

References 23 publications

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“…Biuret reagent was delivered by an Isco model DM syringe pump (Harvard Apparatus, Holliston, MA) at 0.3 μl/min. The post column derivatization was performed in a Capillary Taylor Reactor (CTR)41 with an inner diameter of 75 μm. The HPLC-EC system was depicted by Xu et al42.…”

Section: Methodsmentioning

confidence: 99%

Electroosmotic Sampling. Application to Determination of Ectopeptidase Activity in Organotypic Hippocampal Slice Cultures

Guy

Hamsher

et al. 2010

Anal. Chem.

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We hypothesize that peptide-containing solutions pulled through tissue should reveal the presence and activity of peptidases in the tissue. Using the natural ζ-potential in the organotypic hippocampal slice culture (OHSC), physiological fluids can be pulled through the tissue with an electric field. The hydrolysis of the peptides present in the fluid drawn through the tissue can be determined using capillary HPLC with electrochemical detection of the biuret complexes of the peptides following a postcolumn reaction. We have characterized this new sampling method by measuring the flow rate, examining the use of internal standards, and examining cell death caused by sampling. The sampling flow rate ranges from 60 to 150 nL/min with a 150 μm (ID) sampling capillary with an electric field (at the tip of the capillary) from 30 to 60 V/cm. Cell death can be negligible with controlled sampling conditions. Using this sampling approach, we have electroosmotically pulled Leu-enkephalin through OHSCs to identify ectopeptidase activity in the CA3 region. These studies show that a bestatin-sensitive aminopeptidase may be critical for the hydrolysis of exogenous Leu-enkephalin, a neuropeptide present in the CA3 region of OHSCs.Neuropeptides play a key role in brain and peripheral nervous system functions such as pain and learning 1 . They are mainly inactivated by ectopeptidases -outward-facing, membranebound peptidases that cleave the active peptides into inactive fragments 2-4 . Extracellular peptidases create active forms of BDNF 5 , substance P 6 , cholecystokinins 4 and alter the activity of dynorphins 7 . Recent work shows that peptidases are important in the degradation of amyloid 8,9 . Also, attenuated peptidase activity following stroke can contribute to neurotoxicity and an endogenous blocker of the ectopeptidase that cleaves enkephalin has powerful analgesic effects 10 . Thus, a deeper understanding of peptidase activity is necessary for understanding both normal and pathological brain function, as well as for the development of novel strategies for drug development.While the central focus of this paper is electroosmotic sampling, the determination of peptidase activity in tissue is an important direction. Peptidase activity has been a concern in microdialysis experiments. Microdialysis sampling of peptides is improved in the presence of peptidase inhibitors [11][12][13][14][15][16][17] . Recently, the Stenken group 18, 19 has developed microdialysis approaches to in vitro (enzyme solutions) determination of protease activity. In vitro (slice)

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Section: Methodsmentioning

confidence: 99%

Electroosmotic Sampling. Application to Determination of Ectopeptidase Activity in Organotypic Hippocampal Slice Cultures

Guy

Hamsher

et al. 2010

Anal. Chem.

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“…As long as the analyte can be oxidized by the homogeneous oxidant, which is true for many neurotransmitters and their metabolites, the PFET technique can be applied after separation with negligible band broadening. 15,16 In the present study, we report the successful combination of capillary HPLC with tris(2,2′-bipyridine)osmium(III) (1 3+ ) as the postcolumn PFET reagent. Capillary HPLC is especially suited for the analysis of biological samples due to its small sample load volume and high sensitivity.…”

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confidence: 96%

Simultaneous Determination of Biogenic Monoamines in Rat Brain Dialysates Using Capillary High-Performance Liquid Chromatography with Photoluminescence Following Electron Transfer

et al. 2006

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Simultaneous determination of biogenic monoamines such as dopamine, serotonin, and 3-methoxytyramine in brain is important in understanding neurotransmitter activity. This study presents a sensitive determination of biogenic monoamines in rat brain striatum microdialysates using capillary high-performance liquid chromatography with the photoluminescence following electrontransfer detection technique. Separation conditions were optimized by changing the concentration of an ion-interaction agent and the percentage of an organic modifier. The high concentration of ioninteraction agent enabled the amines as a class to be separated from interfering acids, but also made the separation very long. To shorten the separation time, 10% (v/v) acetonitrile was used as the organic modifier. Eight chromatographic runs during a 3-h period were analyzed in terms of retention times, peak heights, and peak widths. Chromatograms are very reproducible, with less than 1% changes in peak height over 3 h. Typical concentration detection limits at the optimum separation conditions were less than 100 pM for metabolic acids and ~200 pM for monoamines. The injection volume of the sample was 500 nL. Thus, the mass detection limits were less than 50 amol for metabolic acids and ~100 amol for monoamines. Typical separation time was less than 10 min. To validate the technique, the separation method was applied to the observation of drug-induced changes of monoamine concentrations in rat brain microdialysis samples. Local perfusion of tetrodotoxin, a sodium channel blocker, into the striatum of an anesthetized rat decreased dopamine, 3-methoxytyramine, and serotonin concentrations in dialysates. Successive monitoring of striatal dialysates at a temporal resolution of 7.7 min showed that the injection of nomifensine transiently increased dopamine and 3-methoxytyramine concentrations in rat brain dialysate.Biogenic monoamines, such as dopamine (DA) and serotonin (5HT), play important roles as major neurotransmitters in the mammalian central nervous system. 1 Reliable measurement of extracellular neurotransmitter concentration is thus important in understanding the central nervous system and its underlying physiology.Several methods have been used to monitor the extracellular level of neurotransmitters and their metabolites without separation. 2-5 However, some neurons do not contain only one neurotransmitter; thus, changes in the neurotransmitter concentrations are often interrelated. Moreover, the real biological response is more likely due to the interplay of all the released neurotransmitters. Therefore, it is highly desirable to monitor the level of several neurotransmitters simultaneously. Measuring extracellular concentrations of neurotransmitter metabolites, including 3, 4-dihydroxyphenylacetic acid (DOPAC), 3-methoxytyramine * To whom correspondence should be addressed. E-mail: sweber@pitt.edu.. (3MT), 5-hydroxyindole-3-acetic acid (5HIAA), and homovanillic acid (HVA), is also important in understanding the metabolic pathways and ...

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“…The capillary HPLC system was described before[35, 36], so the process is briefly summarized as follows.…”

Section: Methodsmentioning

confidence: 99%

Carbon fiber/epoxy composite ring–disk electrode: Fabrication, characterization and application to electrochemical detection in capillary high performance liquid chromatography

Weber

2009

Journal of Electroanalytical Chemistry

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Carbon fiber/epoxy composite materials, which are manufactured using the pultrusion process, are commercially available in various shapes and sizes at very low cost. Here we demonstrate the application of such a material as an electrochemical detector in a flow system. Cyclic voltammetry shows that the material's electrochemical behavior resembles that of glassy carbon. Using tube and rod composites, we successfully fabricated a ring-disk electrode with a 20 μm gap between the ring and the disk. The narrow gap is favorable for mass transfer in the generator-collector experiment. This composite ring-disk electrode is assembled in a thin-layer radial-flow cell and used as an electrochemical detector. The disk electrode, placed directly opposite to the flow inlet, is operated as a generator electrode with the ring electrode being a collector. The high collection efficiency on the ring electrode (0.8 for a chemically reversible species) enhances the detection selectivity.

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Analysis of the Performance of a Flow Reactor for Use with Microcolumn HPLC

Cited by 13 publications

References 23 publications

Electroosmotic Sampling. Application to Determination of Ectopeptidase Activity in Organotypic Hippocampal Slice Cultures

Electroosmotic Sampling. Application to Determination of Ectopeptidase Activity in Organotypic Hippocampal Slice Cultures

Simultaneous Determination of Biogenic Monoamines in Rat Brain Dialysates Using Capillary High-Performance Liquid Chromatography with Photoluminescence Following Electron Transfer

Carbon fiber/epoxy composite ring–disk electrode: Fabrication, characterization and application to electrochemical detection in capillary high performance liquid chromatography

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