Liquid Chromatography With Electrochemical Detection Research Articles

Determination of phenols in sea water by liquid chromatography with electrochemical detection after enrichment by using solid-phase extraction cartridges and disks

The efficiency and the reproducibility of different solid-phase adsorbents such as C 18 cartridges, C 18 and poly(styrene-divinylbenzene) (PS-DVB) membrane extraction disks were evaluated for the extraction of phenols from water at μg 1 −1 concentration levels. Liquid chromatography with electrochemical detection (LC-ED) was used for the analysis of the organic extracts. A phosphate buffer-acetonitrile-methanol mixture as mobile phase and coulometric detection at + 750 mV were used. High recoveries (70–98%) and good reproducibility (relative standard deviation between 3 and 12%) were obtained using PS-DVB disks, which can be recommended for the enrichment of samples with low concentration of phenols (0.5 μg 1 −1). For more concentrated samples (10 μg 1 −1), the use of C 18 cartridges was preferred because smaller solvent and sample volumes were used and the analysis time was reduced.

Quantitative Analysis of 2-Oxoglutarate in Biological Samples Using Liquid Chromatography with Electrochemical Detection

This paper describes a technique for quantitative analysis of 2-oxoglutarate (α-ketoglutarate) in biological samples using liquid chromatography with electrochemical detection (LC-EC). This method utilizes a simplified, efficient sample preparation designed to select for 2-oxoglutarate and similar compounds by derivatization with phenylhydrazine. The response was linear over the range from 62.5 to 1000 ng/ml. The least quantifiable concentration was 62.5 ng/ml and the least detectable concentration was 25 ng/ml. To test the ability of the assay to measure 2-oxoglutarate in biological samples, this method was used to quantitate the 2-oxoglutarate content in chick osteoblast cultures and to determine the ability of the assay to accurately measure a standard addition of 500 ng/ml 2-oxoglutarate when added to a sample of the forementioned groups. The 2-oxoglutarate content of these cells was 6.67 ± 1.20 ng/μg DNA or 105 ± 18 ng/cell layer (mean ± 95% confidence interval) and the assay accurately measured the standard addition. This method was also used to quantitate 2-oxoglutarate content in whole embryonic chick calvariae containing 6.40 ± 0.95 ng/mg dry bone weight or 37.5 ± 5.5 ng/bone. This assay provides significantly lower detection limits than the currently available procedures and is suitable for determination of 2-oxoglutarate in biological samples where very low amounts of 2-oxoglutarate are found. This method is the first application of LC-EC for quantitating 2-oxoglutarate.

Determination of Reduced Glutathione in Human Plasma Using Liquid Chromatography with Electrochemical Detection with a Glassy Carbon Electrode Chemically Modified with Indium Ferricyanide

A stable indium ferricyanide-modified glassy carbon electrode (InHCF/GCE-2) was prepared and exhibited excellent electrocatalytic activity in the oxidation of reduced glutathione (GSH) in acidic media. Liquid chromatography with electrochemical detection (LCEC), incorporating the InHCF/GCE-2, has been employed for the determination of normal circulating levels of GSH in human plasma. The mobile phase consisted of 0.1 mol/liter phosphate butler (pH 3.0) containing 50 μmol/liter ethylenediaminetetraacetic acid (EDTA). A linear range was over two orders of magnitude. The limit of detection for GSH was 1.6 μmol/liter. The mean recovery of GSH added to the plasma was 98.7%. This was achieved following a simple sample pretreatment method, prior to LCEC, involving chelation of divalent cations with EDTA and subsequent acidification with orthophosphoric acid, using the LCEC method. The circulating level of GSH in plasma, found in three normal subjects, was 14-15 μmol/liter, which is in agreement with previously reported values.

Rapid assay of the monoamine content in small volumes of rat plasma

A method for the simultaneous measurement of serotonin catecholamines, and their metabolites in rat plasma by ultrafiltration and microbore liquid chromatography with electrochemical detection (LC-ED) in small volumes is established. Prior to the LC assay, sixteen plasma ultrafiltrates are readily prepared within 30 min in the present study. The present method, applying a dual-electrode detection technique, provides an additional reliable assignment or measurement of peaks by identifying the peaks on the basis of their redox ratios. In addition, the important early-eluting peaks and interfering peaks are eliminated in the cathodic chromatogram resulting in a reliable measurement of norepinephrine, epinephrine, and 3,4-dihydroxyphenylacetic acid. Isocratic separation of serotonin, catecholamines, and their metabolites by a microbore column is achieved within 15 min. Hence, theoretically, over 90 analyses can be performed in a working day. The limit of detection (signal-to-noise ratio = 3) of this method is ca. 0.2–0.5 pg per injection for all analytes. The required volume of the plasma samples can be less than 100 μl. Hence, the remainder of the plasma sample can be analysed for other substances. This rapid, simple, and sensitive method can thus be used as a research tool in the simultaneous measurement of rat plasma serotonin, catecholamines, and their metabolites.

Rapid and Simultaneous Determination of Monoamine Oxidase A and Monoamine Oxidase B Activities in Mouse Brain Homogenates by Liquid Chromatography with Electrochemical Detection

Sensitive and rapid enzymatic assays have been developed and optimized to measure the separate and combined activities of monoamine oxidase A (MAO-A) and monoamine oxidase B (MAO-B) in mouse brain tissue homogenates using liquid chromatography with electrochemical detection (LCEC). The selectivity for the two isozymes is primarily afforded by use of selective substrates, 5-hydroxytryptamine (5-HT) for MAO-A and 3-methoxy-4-hydroxybenzylamine (MHBA) for MAO-B. The selectivity of the separate assays is further enhanced by the use of inhibitors, deprenyl to block MAO-B and clorgyline to block MAO-A. The dual assay procedure, which employs no inhibitors, shows remarkably enhanced selectivity for each of the isozymes through the use of the two substrates; the preferred substrate for one isozyme acts as an effective competitive inhibitor of the nontargeted substrate for that isozyme, leading to substantially decreased activity for the latter substrate. Using the dual assay, kinetic constants determined for MAO-A (mean ± SD) were: K m,5-HT = 39 ± 7 μM, V max,5-HT = 37.6 ± 2.1 pmol/mg wet tissue/min, K mMHBA = 341 ± 75μM, and V max,MHBA = 27.7 ± 2.3 pmol/mg wet tissue/min; those for MAO-B were: K mMHBA = 108 ± 11μM, V max,MHBA = 44.3 ± 1.2 pmol/mg wet tissue/min, K m,5-HT = 1704 ± 122 μM, and V max,5-HT 12.0 ± 0.3 pmol/mg wet tissue/min. The separate isozyme procedures, when used without selective inhibitors, reflect only 88.3% of the MAO-A activity using the 5-HT velocity and only 66.0% of the MAO-B activity using the MHBA velocity. On the other hand, when the dual assay is employed, 98% of the observed 5-HT velocity can bedirectly attributed to MAO-A, and 94% of the observed MHBA velocity can be directly attributed to MAO-B. The dual assay was employed to demonstrate the relative change in the activity of these two enzymes in whole mouse brain between 27 and 74 days of age. During this time, the MAO-B activity increased from ∼ 40 to ∼ 60% of the total MAO activity. Under typical conditions, results can be easily obtained from any of the three procedures outlined for 100 samples in less than 2 working days, including only 3.5 h for the LCEC portion.

Ferrocene tagging of amines, amino acids and peptides for liquid chromatography with electrochemical detection

Ferrocenecarboxylic acid chloride (FAC) and ferrocenesulfonyl chloride (FSC) were investigated as ferrocene tagging reagents for amine and amino acid determination by liquid chromatography with electrochemical detection (LC-EC). Whereas FSC gives low sulfonamide yields, FAC reacts readily with both primary and secondary amines and amino acids to yield stable amides with are reversibly oxidized in mixed acetonitrile—aqueous solutions at potentials 220 mV positive of ferrocene oxidation. A derivatization procedure is presented which yields quantitative derivatization of micromolar concentrations of amines and amino acids and the resulting derivatives are amenable to reversed-phase chromatographic separation coupled with dual-electrode detection schemes. A mixture of fourteen amino acids was derivatized and separated by gradient elution LC-EC within 45 min. FAC derivatization was applied for monitoring the enzymatic hydrolysis of 200 mmol of the tripeptide Gly—Phe—Leu by leucine aminopeptidase M. FAC derivatization of peptides is also demonstrated for the substance P fragment 4–11 ( N-terminus-proline) and subsequent LC-EC determination of the FAC derivative yielded a detection limit of 500 fmol.

The nigrostriatal dopaminergic system in MPTP-treated mice shows more prominent recovery by syngeneic adrenal medullary graft than by allogeneic or xenogeneic graft

Following systemic injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), young (2-month-old) C57BL/6 mice show decreased dopaminergic (DA) nigrostriatal fibers and DA concentration in the striatum. We transplanted syngeneic, allogeneic and xenogeneic adrenal medullary grafts into the striatum of the MPTP-treated young mice and compared the survivability of grafted chromaffin cells and the recovery of intrinsic host DA fibers using computerized image analysis of tyrosine hydroxylase (TH)-immunoreactive (IR) fibers and high performance liquid chromatography with electrochemical detection (LCEC). The grafted syngeneic adrenal chromaffin cells survived better than allogeneic or xenogeneic chromaffin cells, and host DA nigrostriatal fiber recovery was more prominent in mice with a syngeneic graft than in mice with an allogeneic or xenogeneic graft. However, the degree of host fiber recovery in mice with allogeneic or xenogeneic mice was greater than in mice with a sham operation alone, even though the allografts and xenografts had no surviving chromaffin cells. Allografts and xenografts showed prominent rejection responses, with T lymphocyte infiltration in addition to macrophages. We conclude that a syngeneic adrenal graft survives better than an adrenal allograft or xenograft and promotes recovery of the intrinsic host nigrostriatal DA fibers. We also conclude that grafted chromaffin cell survivability influences the degree of host DA fiber recovery following MPTP depletion. Adrenal medullary grafts to Parkinsonian patients are currently under way in a large number of hospitals; we suggest that greater attention be paid to methods which lead to enhanced survival of the grafted chromaffin cells, since survivability might be closely related to the functional recovery of these patients.

Identification of metabolites of 3,4-methylenedioxymethamphetamine in rats

Liquid chromatography with electrochemical detection (LC/ECD) and gas chromatography/mass spectrometry (GC/MS) were used to identify metabolites of N-methyl-3,4-methylenedioxyamphetamine (MDMA) in samples of rat plasma and urine. Several potential metabolites, based on what is known about the metabolism of the desmethyl analog (i.e., MDA), were synthesized as standards to aid in the identification of the MDMA metabolites. MDA and N-methyl-1-(4-hydroxy-3-methoxy-phenyl) -2-aminopropane (3 b) were identified in urine by HPLC and confirmed by GC/MS. 1-(4-Hydroxy-3-methyoxyphenyl)2-aminopropane, (3 a), N-methyl-1-(3-hydroxy-4-methoxyphenyl)-2-aminopropane (2 b) and 1-(3,4-dihydroxyphenyl)-2-aminopropane (4 a) were tentatively identified by LC/ECD but insufficient sample size precluded confirmation by mass spectrometry. MDA was also identified in brain and plasma extracts. Because MDA is a metabolite of MDMA in humans, and because it has been speculated that the neurotoxic effects of MDA and MDMA may be due to a metabolite, the results of the present study may ultimately aid our understanding of the neurotoxic mechanism of these drugs of abuse.

Simultaneous measurement of tetrahydrobiopterin (THBP) and biogenic amines by liquid chromatography with electrochemical detection

This report describes a rapid and sensitive method for measuring tetrahydrobiopterin (THBP) and biogenic amines simultaneously by liquid chromatography with electro-chemical detection (LC-ECD). The coefficient of variation for THBP was 4.87 % and the minimum detectable amount of THBP was approximately 20 pg. These results indicate that this simple reverse-phase ion-pair chromatography system can be used for the simultaneous analysis of endogenous THBP and biogenic amines without long sample preparation time.

Electrochemical investigations of immunologically reactive procainamide metabolites

As a result of the implication of N-oxidized procainamide metabolites in drug-related lupus (DRL), the electrochemical behaviour of these compounds was investigated and a coulometric synthesis of the nitroso derivative developed using a previously described carbon packed bed bulk electrolysis flow cell. The electrochemical characterization of the parent p-substituted aromatic amine and the N-oxidized derivatives was achieved through systematic comparison with previously well described aromatic amine and nitro systems using cyclic voltammetry and liquid chromatography with electrochemical detection (LC-EC). Chromatographically assisted hydrodynamic voltammetry indicated current limiting plateau potentials of 0.45 and -0.2 V versus Ag/AgCl, respectively, for synthetically prepared procainamide hydroxylamine and electrolytically prepared nitrosoprocainamide. Reaction characterization and binding behaviour is described for each of the procainamide metabolites following in vitro incubations with cysteine, glutathione, ascorbic acid and mouse haemoglobin.

Determination of Acetylcholine and Choline in Human Plasma Using High-Performance Liquid Chromatography Combined with an Immobilized Enzyme Reactor

Abstract A method for assaying Acetylcholine (ACh) and Chloine (Ch) simultaneously in human plasma by high-performance liquid chromatography with electrochemical detection (LCEC) combined with an immobilized enzyme reactor is described for the first time in this paper. Human plasma samples were obtained from 12 volunteers free of neurological and psychological disease. The volunteers were from 22 to 41 years old (mean 30.1 years), eight being males. Extraction of ACh and Ch from plasma was performed according to the liquid cation-exchange method. Concentrations of ACh ranged from 57.0 to 179.6 pmol/ml (mean and S.D. of 121.6 and 40.9 pmol/ml respectively). The present method, LCEC combined with an immobilized enzyme reactor is raped, simple and sensitive compared with the biological methods, GC-MS and RIA for analyzing ACh and Ch in plasma.

Origin of noradrenergic innervation of the spleen in rats

Noradrenergic (NA) sympathetic innervation of the spleen was examined in young adult Sprague-Dawley rats (200–250 g) following surgical removal of the superior mesenteric-celiac ganglia (SM-CG) and/or bilateral transection of the subdiaphragmatic vagus nerve. Sham-operated and unoperated rats served as controls. NA sympathetic innervation of spleens from sham-operated and unoperated controls, and from vagotomized rats, was qualitatively similar, with fibers distributing to the capsule, trabeculae, vasculature, and parenchyma of the white pulp. Complete ganglionic extirpation resulted in almost total denervation of NA fibers in all compartments of the spleen. High-performance liquid chromatography with electrochemical detection (LCEC) for catecholamines (CA) and quantitative morphometry of the density of NA varicosities confirmed these observations. LCEC revealed a greater than 85% depletion of norepinephrine (NE) in the spleen following superior mesenteric-celiac ganglionectomy. Stereological evaluation of NA varicosities with a point counting method revealed a decline of 99% in the volume density of NA terminals that occurred uniformly in all compartments of spleens from ganglionectomized rats. In addition, stereological analysis revealed a loss of total NA varicosities (approximately 31% decrease) in spleens from sham-operated rats. This loss in volume density occurred largely due to a loss in parenchymal fibers (approximately 45% decrease). Bilateral subdiaphragmatic vagotomy blocked the effect on NA innervation produced by the surgical stress of sham operation. Retrograde tracing following injection of either fluorogold or true blue into the spleen, coupled with immunocytochemical localization of tyrosine hydroxylase (TH), demonstrated abundant fluorogold (true blue)-labeled neurons in the SM-CG; many, but not all, of these neurons also were TH-positive. These findings indicate that the SM-CG neurons supply NA innervation to the spleen, providing sympathetic innervation as the second neuron in the classical two-neuron sympathetic chain, and suggest additional non-NA innervation of the spleen as well. This study also suggests that surgical stress of sham operation may alter directly the NA innervation of the spleen, possibly by inducing temporary retraction of NA fibers of the parenchymal compartment, which is likely to reduce the availability of NE for interaction with cells of the immune system that possess adrenoceptors and are present adjacent to NA varicosities in this region. Bilateral vagotomy ameliorated the effects of sham operation on NA innervation; since the vagal nerve does not distribute fibers to the spleen, this effect is likely to occur through altered feedback circuits effecting sympathetic outflow, or through altered neuroendocrine outflow.

Determination of glutathione in human plasma using high-performance liquid chromatography with electrochemical detection with a carbon-epoxy resin composite electrode chemically modified with cobalt phthalocyanine.

High-performance liquid chromatography with electrochemical detection (LCEC), incorporating a novel carbon-epoxy resin working electrode modified with cobalt phthalocyanine, has been employed for preliminary studies directed towards the determination of normal circulating levels of reduced glutathione (GSH) in human plasma. The mobile phase consisted of 0.05 M phosphate buffer (pH 3) containing 0.1% m/m ethylenediaminetetraacetic acid (EDTA); the calibration graph was linear in the range 0.24-30.7 ng of GSH injected. The mean recovery of GSH added to a control serum over the physiological concentration range (0.38-3.07 ng ml-1) was 99%; this was achieved following a simple sample pre-treatment method, prior to LCEC, involving chelation of divalent cations with EDTA and subsequent acidification with orthophosphoric acid. Using the LCEC method, the mean circulating level of GSH in plasma, found in three normal subjects, was 2.69 microM, GSH; this indicates that the method might be applicable to the determination of depressed circulating levels of GSH.

Development of an improved carbon electrode chemically modified with cobalt phthalocyanine as a re-usable sensor for glutathione

Cyclic voltammetry, differential-pulse voltammetry (DPV), amperometry and flow injection with electrochemical detection were used to investigate the electrochemical behaviour of reduced glutathione (GSH) at a carbon paste electrode and also at a carbon paste electrode doped with cobalt phthalocyanine (CPC). The modified electrode was found to reduce greatly the overpotential necessary for the oxidation of GSH at the electrode surface; the process was dependent on the ionic strength and pH of the supporting electrolyte. The oxidation of GSH in the presence of CPC was found to occur at two potentials, 0.27 and 0.75 V versus a saturated calomel electrode. Using DPV the first wave was found to give optimum sensitivity for the measurement of GSH concentations in the range 2.5 × 10–6–6.25 × 10–5M; whereas the second was preferential for GSH concentrations of 1.25 × 10–4–2.5 × 10–3M. Electrode deterioration rendered the other techniques unsuitable for routine quantitative analyses. Similar investigations were also carried out on a carbon-epoxy resin composite electrode containing CPC; the various techniques were used to optimise the electrode response for the determination of GSH in biological fluids. This electrode was found to be superior to the graphite paste electrode as GSH could be determined in flowing or stirred solutions. The modified composite electrode was stable and could be used over a period of at least 10 h. The limits of detection were 10 ng ml–1 and 450 pg injected on column for amperometry and high-performance liquid chromatography with electrochemical detection (LCEC), respectively. Preliminary studies using LCEC have shown that the method is suitable for the determination of circulating levels of GSH in human plasma.

Maintenance of noradrenergic sympathetic innervation in the involuted thymus of the aged Fischer 344 rat

Sympathetic postganglionic noradrenergic (NA) fibers innervate both primary and secondary lymphoid organs in the rat. In secondary lymphoid organs such as the spleen and lymph nodes, this innervation diminishes with age. This study was undertaken to determine whether a similar decline in NA innervation occurs in the thymus. Thymuses from Fischer 344 (F344) rats were examined at 3, 8, 12, 17, 21, and 27 months of age with fluorescence histochemistry for catecholamines, and with high-performance liquid chromatography with electrochemical detection (LCEC) for quantitation of monoamines. In adult animals of all age groups, the compartmentation of NA innervation was maintained; NA fibers entered the thymus with the vasculature and arborized in the cortex, particularly in the subcapsular region and around the vasculature at the corticomedullary junction. NA nerve fibers were sparse in the thymuses from 3-month-old rats and increased in density at 8 and 12 months of age. The density of NA nerve fibers in the thymuses from 17, 21, and 27 months of age increased further, in parallel with thymic involution. Quantitation of monoamines revealed stable thymic norepinephrine (NE) and 5-hydroxytryptamine (5-HT) content from 8 to 27 months of age. The lower levels of NE detected at 3 months of age may reflect a slow maturation and reorganization of this fiber system as a developmental phenomenon. Measurement of 3-methoxy-4-hydroxy-phenyleneglycol (MHPG), a major metabolite of NE, showed a more than twofold rise from 3 to 12 months of age, and remained high through 27 months of age. The resultant MHPG NE ratios, as an index of NE availability for interaction with target cells, showed a decline from 3 months of age through 17–21 months of age, followed by a marked rise at 27 months of age. These findings suggest that the thymus is able to maintain NA innervation in the face of involution, and that these fibers provide a NA-enriched microenvironment for interaction with adrenergic receptors on thymocytes.

Electrochemical enzyme-immunoassay

Immunoassay defines the body of techniques which use the antibody (Ab) macromolecule, usually of the IgG class, for the detection and quantitation of an enormous range of simple and complex antigen (Ag) molecules. The success of the methods relies on both the specificity and formation constant of the Ab used, and the ability to detect the interaction between Ab and Ag. Sensitive assays in complex matrices require some kind of label to be present to provide this ability. The development of immunoassay methods has in large part been driven by the available technology. Thus, although one of the first immunoassays using a label was electrochemically based [1], the state of that art in 1951 was relatively primitive, and could not meet the analytical demands of the Ag. Some years later, Yalow and Berson [2] developed immunoassays based on the radioisotopic label (RIA). For the first time, Ab selectivity was married to very low detection limit technology, and the result was an enormous growth rate in the use of RIA in both the clinical and research laboratory. RIA has the significant advantage that the label used is not a normal constituent of physiological samples, and interferences of this type are therefore absent. RIA, however, has the disadvantages that accompany radioisotope handling, together with an inability to distinguish label which is bound from that which is not. There has therefore been an enormous effort to find suitable replacements for the radiolabel. Where the low detection limit of RIA was not required for a successful assay, labels detectable by spectroscopic methods have become important. Where very low detection limits are demanded, then the concept of amplification of, or by, the label has been developed. The most successful of these have been enzyme linked using an immunosorbent phase for Ag extraction (ELISA), although assays based on the lysis of label-containing liposomes are also exhibiting very low detection limits. In our hands, electrochemically based immunoassays at very low detection limits have also used enzyme amplification and ELISA, with either NAD/NADH and glucose-6-phosphate dehydrogenase [3], or phenyl phosphate/phenol and alkaline phosphatase [4]. The greatest sensitivity thus far has been obtained with ELISA coupled with liquid chromatography with electrochemical detection (LCED) [5]. The power of this approach can be readily illustrated by examining the evolution of its use in an assay for IgG, itself an important analyte. This evolution covers three principal stages of development and a reduction in detection limit of five orders of magnitude. The basic methodology is shown in figure 1. The assay is essentially a sandwich ELISA, using an alkaline phosphatase labelled second Ab for amplification. Oxidative flow amperometry at +875 mV

LC-EC determination of nucleotides and nucleic acids: Application to enzyme assays and the analysis of DNA fragments

High-performance liquid chromatography (HPLC) coupled to an electrochemical detector in an oxidative mode was used to analyze purine bases, nucleosides, and nucleotides as well as restriction fragments of nucleic acids. Ligands were separated by liquid chromatography with electrochemical detection (LC-EC) using size exclusion, ion-exchange, or reverse phase techniques. Using an amperometric electrochemical detector the determination was characterized with respect to sensitivity, selectivity, and capacity factor. It was observed from hydrodynamic and cyclic voltammetry that the optimum oxidation potential differed for the three major classes of purines, permitting an enhancement in selectivity when compared to detection. Guanylyl moieties demonstrated a half-wave potential at 0.800 V vs Ag AgCl , while those for the adenylyl and inosylyl groups are above 1.000 V vs Ag AgCl . The facility of the method to analyze components of a complex biological milieu was demonstrated by examining the purine pools of crude and partially purified eye lens homogenates as well as by comparing the traditional hexokinase assay to the newly developed LC-EC technique. Additionally, LC-EC was compared to detection for determination of the purine-metabolizing enzyme activities, adenylate deaminase and adenylosuccinate synthetase from crude cellular lysates of the cellular slime mold, Dictyostelium discoideum. Finally, the technique was used to assay the fragments from λ-DNA cut with the restriction endonuclease Pst-1.

Voltammetric behaviour of all-trans-retinol (vitamin A1) at a glassy carbon electrode and its determination in human serum using high-performance liquid chromatography with electrochemical detection.

Cyclic voltammetry has been used to investigate the oxidation of all-trans-retinol (vitamin A1) at a planar glassy carbon electrode. The electrode reaction was found to be dependent on the percentage of methanol, pH and ionic strength of the acetate buffer. The maximum electrochemical signal was obtained with a supporting electrolyte containing 95% methanol-0.075 M acetate buffer (pH 5.0). The oxidation was found to occur in two steps and to be irreversible; the reactant was found to undergo adsorption at the glassy carbon surface. The final product may also be adsorbed; this did not affect the signal obtained with a glassy carbon electrode in the wall-jet configuration, but did affect seriously that obtained with a thin-layer cell. Liquid chromatography with electrochemical detection (LCEC) was performed with the optimum supporting electrolyte as the mobile phase; the calibration graph was linear in the range 0.05–22.0 ng injected. The LCEC method was shown to be about four times more sensitive than liquid chromatography with UV detection; the former was applied successfully to the determination of normal and depressed circulating levels of the vitamin in human serum samples.

Simultaneous analysis of methionine- and leucine-enkephalin from rat brain: quantification by liquid chromatography-electrochemistry

This study focuses on the application of liquid chromatography with electrochemical detection (LC-ED) for the analysis of methionine-enkephalin (ME) and leucine-enkephalin (LE) extracted from rat brain regions. The high applied potentials necessary for enkephalin detection required the development of an efficient sample processing protocol. Brain extracts were processed using chromatographic mode sequencing (CMS). The decrease in electroactive interfering substances by CMS improved the chromatographic resolution of ME and LE and the electrode performance. Other qualitative and analytical methods were used to evaluate the enkephalin data obtained by LC-ED for rat brain regions. This study demonstrates that LC-ED provides both the sensitivity and specificity necessary for the analysis of enkephalins from rat brain regions.

Depletion of brainstem epinephrine stores by α-methyldopa: Possible relation to attenuated sympathetic outflow

The antihypertensive effect of α-methyldopa (MD) is believed to be critically dependent on its ability to deplete endogenous catecholamines or cause the synthesis of false neurotransmitters. We used liquid chromatography with electrochemical detection (LCEC) and negative chemical ionization gas chromatography-mass spectrometry (GC-MS) for quantitation of catecholamines and MD metabolites in rat. MD intraperitoneally (100 mg/kg q12 hr × 12 days), significantly increased α-methylnorepinephrine (MNE) in brain (1.02 ± 0.33 μg/g), heart (1.67 ± 0.57 μg/g) and adrenal glands (114.93 ± 50.47 μg/g) Endogenous norepinephrine (NE), epinephrine (E) and dopamine (DA) were reduced. ME levels were 2.19 ± 0.44 μg/g (n=6) in the adrenal gland but only 99 ± 26 pg/g (n=3) in the brainstem. MD-induced endogenous brainstem NE depletion was more than compensated by MNE production, but brainstem E depletion was not compensated for by a stoichiometric production of brainstem ME.