Electrochemical Liquid Chromatography Research Articles

Effects of Bone Marrow Mesenchymal Stem Cells on Myelin Repair and Emotional Changes of a Cuprizone-Induced Demyelination Model

Multiple sclerosis (MS) is an autoimmune disease for which bone marrow mesenchymal stem cells (BM-MSCs) have become one of the most promising tools for treatment. Cuprizone(CPZ) induces demyelination in the central nervous system and its use has established a demyelination sheath animal model which is particularly suitable for studying the effects of BM-MSCs on the remyelination and mood improvement of a demyelinating model mice. 70 C57BL/6 male mice were selected and divided into 4 groups: the normal control (n = 20), chronic demyelination (n = 20), myelin repair (n = 15) and cell-treated groups (n = 15). Mice in the normal control group were given a normal diet; the chronic demyelination group mice were given a 0.2% CPZ mixed diet for 14 weeks, mice in the myelin repair and cell-treated groups mice were given a 0.2% CPZ diet for 12 weeks and normal diet for 2 weeks, while the cell-treated group mice were injected with BM-MSCs from the 13th week. The cuprizone-induced demyelination model was successfully established and BM-MSCs extracted, behavioural changes of the mice were detected by open field test, elevated plus maze test and tail suspension test, demyelination and repair of the corpus callosum and astrocyte changes were observed by immunofluorescence and electron microscopy and the concentrations of monoamine neurotransmitters and their metabolites detected by enzyme-linked immunosorbent assay (ELISA) and high performance liquid chromatography-electrochemistry (HPLC-ECD). Results suggest BM-MSCs were successfully extracted and cultured, and migrated to the demyelinating area of brain tissue after cell transplantation. Compared with the normal control group, the mice in the chronic demyelination group showed obvious anxiety and depression behaviours (p < 0.05); compared with the chronic demyelination group, the anxiety and depression behaviours of the cell-treated group mice were improved (p < 0.05); compared with the normal control group, the demyelination of the corpus callosum region of the chronic demyelination group mice was significant (p < 0.01), while the myelin sheath of the cell-treated and myelin repair groups was repaired when compared with the chronic demyelination group (p < 0.05), and the cell-treated group had a more significant effect than the myelin repair group (p < 0.05). Compared with the normal control group, the number of astrocytes in the corpus callosum of the chronic demyelination group mice was significantly increased (p < 0.01), and the expression of glial fibrillary acidic protein (GFAP) in the cell-treated group was lower than that in the chronic demyelination and myelin repair groups (p < 0.05); the serum concentrations of norepinephrine (NE), 5-hydroxytryptamine (5-HT) and 5-Hydroxyindole-3-acetic acid (5-HIAA) between the normal control and the chronic demyelination groups were significantly different (p < 0.05). The CPZ-induced model can be used as an experimental carrier for MS combined with anxiety and depression, and BM-MSC transplantation promotes the repair of myelin sheath and the recovery of emotional disorders in the model.

Effect of Jin-3-needling therapy on plasma corticosteroid, adrenocorticotrophic hormone and platelet 5-HT levels in patients with generalized anxiety disorder

To observe the therapeutic efficacy of Jin-3-needling therapy (J3N) on generalized anxiety disorder (GAD) through clinical global impression scale (CGI), and to explore the mechanism by measuring the plasma levels of corticosteroid (CS), adrenocorticotrophic hormone (ACTH), and platelet 5-hydroxytryptamine (5-HT) before and after treatment. Eighty-six GAD patients with the diagnosis agreeing with the inclusion criteria were assigned, according to the sequence of visiting time, to three groups. The 29 patients in the Western medicine group were treated mainly with fluoxetine or paroxetine, Alprazolam might be given additionally in severe conditions if necessary; the 29 patients in the needling group received J3N therapy with Sishenzhen, Dingshenzhen, Neiguan (PC6), Shenmen (HT7) and Sanyinjiao (SP6) as the chief acupoints selected; and the 28 patients in the combined treatment group were treated with both drugs and needling in the same way as applied in the above two groups. The therapeutic course for all was 6 weeks. Conditions of patients were evaluated before and after treatment with CGI, and levels of CS, ACTH as well as 5-HT were measured by high performance liquid chromatography-electrochemistry. By CGI scoring, the scores of severity index and the general index were not different significantly in the three groups, but the efficacy index proved to be the highest in the needling group, the second in the combined trentment group, and the lowest in the drug group. Plasma level of ACTH and platelet content of 5-HT were lowered in all the three groups after treatment, showing statistical significance (P<0.05), but no significant change was found in CS level (P>0.05). The therapeutic efficacy of J3N in treating GAD is equivalent to, but with the efficacy index significantly higher than, that of conventional treatment. Moreover, when combined with drugs, needling might effectively prevent the side effect of the routinely used Western drugs. The regulatory action of needling on platelet 5-HT and plasma ACTH is probably one of the acting pathways for J3N treatment on GAD.

Decreased expression of serotonin in the jejunum and increased numbers of mast cells in the terminal ileum in patients with irritable bowel syndrome

To investigate if there are changes in serotonin (5-HT) levels, enterochromaffin (EC) cells and mast cells in small intestinal mucosa of patients with irritable bowel syndrome (IBS). Diarrhea-predominant (IBS-D, n = 20), or constipation-predominant (IBS-C, n = 18) IBS patients and healthy controls (n = 20) underwent colonoscopy and peroral small intestinal endoscopy, and mucosal samples were obtained at the descending part of the duodenum, proximal end of jejunum and terminal ileum. High-performance liquid chromatography-electrochemistry and immunohistochemical methods were used to detect 5-HT content, EC cells and mast cells. (1) There were no differences in the number and distribution of EC cells between IBS patients and the normal group. (2) The mucosal 5-HT contents at the duodenum, jejunum and ileum in IBS-C patients were 182 +/- 90, 122 +/- 54, 61 +/- 35 ng/mg protein, respectively, which were all lower than those in the normal group (256 +/- 84, 188 +/- 91, and 93 +/- 45 ng/mg protein, respectively), with a significant difference at the jejunum (P < 0.05). There were no differences in the small intestinal mucosal 5-HT contents between IBS-D patients and the normal group. The mucosal 5-HT contents at the duodenum were significantly higher than those at the ileum in the three groups (P < 0.001). (3) The numbers of mast cells in patients with IBS-C and IBS-D at the ileum were 38.7 +/- 9.4 and 35.8 +/- 5.5/high power field (hpf), respectively, which were significantly more than that in the normal group (29.8 +/- 4.4/hpf) (P < 0.001). There was no significant difference in the numbers of mast cells at the other two parts between IBS patients and the normal group. The numbers of mast cells in IBS-C, IBS-D, and normal groups were all significantly higher at the ileum (38.7 +/- 9.4, 35.8 +/- 5.5, 29.8 +/- 4.4/hpf, respectively) than at the duodenum (19.6 +/- 4.7, 18.5 +/- 6.3, 19.2 +/- 3.3/hpf, respectively, P < 0.001). The changes in the 5-HT signaling pathway at the jejunum of IBS-C patients and the increase in mast cells in patients with IBS at the terminal ileum may offer evidence to explain the pathogenesis of IBS.

Liquid chromatography with multichannel electrochemical detection for the determination of trans-resveratrol in rat blood utilizing an automated blood sampling device

A sensitive and selective multichannel liquid chromatography–electrochemistry method was developed for the determination of the natural product trans-resveratrol (resveratrol) in rat blood. After administration of resveratrol, blood samples were periodically collected by an automated blood sampling device. Resveratrol was extracted from 150 μl of diluted blood (blood and saline at a ratio of 1:1) with acetonitrile containing 1% of trichloroacetic acid. Chromatographic separation was achieved within 12 min using a C 18 (100×2.0 mm) 3 μm column with a mobile phase containing 20 m M sodium acetate, 0.5 m M EDTA, pH 4.5 and 21% acetonitrile at a flow-rate of 0.4 ml/min. A multichannel detector with glassy carbon electrodes was used, which can control up to four working electrodes simultaneously with applied potentials of +800, 700, 600, 500 mV vs. Ag/AgCl. The limit of detection was 2 ng/ml at a signal-to-noise ratio of 3:1 and the limit of quantitation was 4 ng/ml. The linearity of the calibration curve was obtained over the analytical range of 5–1000 ng/ml. The intra- and interassay precision was in the range of 2.5–4.4% and 1.2–4.3%, respectively. Using this method it was possible to quantify blood concentration following a single dose of resveratrol to rats with good accuracy and precision. Thus the pharmacokinetic properties of resveratrol in rats can be examined for intraperitoneal, oral and intravenous dosing.

Determination of Melatonin in Commercially Available Products by LCEC and LC/MS/MS

Simple and reliable methods for liquid chromatography/electrochemistry (LCEC) and LC/MS/MS with atmospheric pressure chemical ionization (APCI) have been developed for the determination of melatonin in commercially available products. The dissolution of melatonin from the melatonin-containing formulations was simply obtained by dissolving the tablets and capsules in 20% acetonitrile in 0.1 N perchloric acid with ultrasonication. The filtrate of the resultant solution was separated on a 1 × 150 mm C

Improved detection limit for catecholamines using liquid chromatography-electrochemistry with a carbon interdigitated array microelectrode.

The detection limit of catecholamines can be lowered by using a carbon-based interdigitated array (IDA) microelectrode as a detector for liquid chromatography (LC). The IDA electrode is more sensitive than conventional glassy carbon electrodes due to the high current density caused by radial diffusion at each microband, and redox cycling between two microband arrays. Since the number of redox cycles increases at lower flow-rates, the carbon IDA is particularly useful for microbore LC. In an LC system with a 1-mm microbore column and a carbon IDA electrode, the peak height of dopamine (DA) and DOPAC did not decrease with decreasing flow-rate because of this redox cycling. A low detection limit of 5 fg (32 amol) and 9.6 fg (57 amol) was obtained for DA and DOPAC due to the high current density and low background noise level (0.1 pA) at the carbon IDA electrode. The total charge generated by oxidizing DA at the anodic array was more than the value calculated by assuming that all the DA molecules were oxidized.

Detection of basal acetylcholine in rat brain microdialysate

A liquid chromatography-electrochemistry (LC-EC) method is described for the determination of basal acetylcholine (ACh) in microdialysate from the striatum of freely moving rats. This method is based on the separation of ACh and choline (Ch) by microbore liquid chromatography followed by passage of the effluent through a post-column immobilized enzyme reactor (IMER), containing acetylcholinesterase (AChE) and choline oxidase (ChO), and then the electrochemical detection of the hydrogen peroxide produced. Instead of the conventional platinum electrode used for the anodic detection of hydrogen peroxide, a peroxidase-redox polymer modified glassy carbon electrode operated at + 100 mV vs. Ag/AgCl has been used to detect the reduction of hydrogen peroxide. With this method, a detection limit of 10 fmol (injected) for ACh ( S/ N = 3:1) was obtained and the basal ACh concentration in striatal microdialysate was determined without using esterase inhibitors.

Peptidergic, catecholaminergic and morphological properties of avian chromaffin cells are modulated distinctively by growth factors

Most neurons and endocrine cells are known to co-express a ‘classical neurotransmitter’ with one or more neuropeptides. Although their expression has been shown to be modulated by differentiation factors, it is not known if particular combinations of neurotransmitter/neuropeptide(s) are co-regulated. We have analyzed the effect of nerve growth factor (NGF), neurotrophin-3 (NT-3), brain derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF), basic fibroblast growth factor (bFGF) and transforming growth factor β1 (TGF-β1) on the modulation of neuroactive substances co-expressed by avian chromaffin cells. The content of the neuropeptides neuropeptide Y (NPY), Enkephalin (ENK) and somatostatin (SS) was measured by radioimmunoanalysis, and the content of the catecholamines norepinephrine (NE) and epinephrine (E) by.high pressure liquid chromatography-electrochemistry (HPLC-EC). In addition, the morphological differentiation of chromaffin cells in response to the growth factors was assessed. All of the studied factors had distinct effects on the chromaffin content of neuropeptides and catecholamines. Our results show that the modulation of CAs and neuropeptides, and among the neuropeptides themselves is completely dissociated. Moreover, the cellular responses to the different growth factors show that neurochemical properties are modulated independently of morphological ones.

Isolation and identification of seven metabolites of a water-soluble platelet aggregation inhibitor in rat urine.

1. Seven metabolites of 7-piperidino-1,2,3,4,5-tetrahydroimidazo[2,1- b]quinazolin-2-one dihydrochloride monohydrate (DN-9693) were isolated from rat urine by extraction with Amberlite XAD-2 and purification by silica gel and Sephadex LH-20 open-column chromatography and preparative high-performance liquid chromatography (hplc). The structure assignment of the metabolites was performed by field desorption mass spectrometry and 200-MHz Fourier transform nmr spectroscopic analysis and comparison with authentic standards when available. 2. DN-9693 underwent metabolism mainly at the piperidine ring to give the 4-hydroxypiperidine derivative (III) and 2-hydroxy-piperidine derivative, which is further metabolized to lactam (II) or delta-aminovaleric acid (V). The acyl side chain of V was shortened by beta-oxidation to form the 3-aminopropionic acid derivative (VII). V and/or VII underwent oxidative dealkylation to give the 7-amino derivative, which was conjugated with acetic acid to form the 7-acetylamino derivative (IV). DN-9693 also underwent hydrolysis of its lactam moiety to give VI. 3. The urinary excretion of III, V and VII was determined by liquid chromatography/electrochemistry (LC/EC) and V proved to be the major metabolite in rat urine. 4. A procedure is also presented for the identification of DN-9693 metabolites using LC/EC.

Electrocatalytic oxidation and amperometric determination of sulfhydryl compounds at a copper hexacyanoferrate film glassy carbon electrode in liquid chromatography

AbstractElectrocatalytic oxidation of sulfhydryl compounds was effective on a copper hexacyanoferrate (CuHCF) film glassy carbon electrode, at a significantly reduced overpotential (0.55 to 0.65 V) and for a broader pH range (2.0 to 7.0). The electrocatalysis was evaluated in terms of several preliminary experimental parameters, such as buffer pH, surface adsorbent concentration, operating potential, mobile‐phase flow rate, and concentration dependence in batch and flow systems. Electrochemical liquid chromatography (LCEC) of sulfhydryl compounds gave a linearity over two orders of magnitude and detection limits at picomole level. Overlaying the CuHCF film electrode surface with a Nafion layer is essential for improving the catalysis stability and offers enhancement of the liquid chromatography electro chemistry (LCEC) response.

Retinoblastoma aqueous humor: aromatic amino acids.

Aqueous humor from children with retinoblastoma obtained at enucleation and from eyes with adult cataracts were assayed with electrochemical liquid chromatography (Model 5500 Coulochem electrode array system) for metabolites of tyrosine, tryptophan metabolic pathways, catecholamine degradation pathways and ascorbate. More than 20 metabolites were identified in human aqueous for the first time. High levels of ascorbate were found in aqueous of eyes with adult cataracts (254, 336 ng/ml). Tyrosine metabolism in both sets of eyes was through dopamine. Vandylmandelic acid (VMA), homovanillic acid (HVA), and 3-methoxy, 4-hydroxyphenylglycol (MHPG) were all detected in retinoblastoma eyes. Although eyes with either adult cataracts or childhood retinoblastoma convert tryptophan through the serotonin pathway, retinoblastoma eyes metabolize tryptophan through the kynurenine pathway to a greater degree than adult cataract eyes.

Liquid chromatography—electrochemistry procedure for the determination of chlorophenolic compounds in pulp mill effluents and receiving waters

An isocratic, reversed-phase liquid chromatography procedure is described for the separation and determination of chlorophenolic compounds, including several priority pollutant chlorophenols. The separation was experimentally optimised, and selective and sensitive detection was obtained by dual electrode amperometry. The most suitable combination was a silica-based column with phenyl functional groups, and a mobile phase of acetonitrile-acetate buffer (40:60, pH 5). Dual glassy-carbon electrodes were used in a parallel configuration in a thin-layer flow cell, with differential current measurement (+0.90 V relative to +0.60 V). A solid-phase extraction scheme using a polymeric sorbent (Chromosorb 102) was devised for the extraction and preconcentration of selected chlorophenols, chloroguaiacols, and a chlorosyringol from aqueous samples. Use of tribromophenol as an internal standard was also investigated. The total procedure —preconcentration and chromatography— was applied to the analysis of effluents discharged by a paper mill, and a pulp mill.

Electrochemical detection of proteins in high-performance liquid chromatography using on-line, postcolumn photolysis

Direct detection of proteins in high-performance liquid chromatography electrochemistry (LCEC) is difficult. By using on-line, postcolumn photolysis, proteins now can be detected by LCEC at microgram per milliliter levels. The compatibilities of size exclusion chromatography (SEC), reversed-phase chromatography (RPC), ion-exchange chromatography (IEC), and hydrophobic interaction chromatography (HIC) with photolysis-electrochemical detection is described for proteins together with the analytical figures of merit. Inherent from the advantages of electrochemical detection, the method is sensitive and selective.

Pre- and post-column derivatization reactions for liquid chromatography—electrochemistry

Derivatization reactions are frequently employed to improve analyte detection. In order to take advantage of the selectivity and sensitivity of liquid chromatography—electrochemistry, a variety of pre- and post-column reaction schemes have been developed. These include derivatization by chemical, electrochemical, photochemical and enzymatic means. Examples of each of these are presented.

RECENT ASPECTS OF LIQUID CHROMATOGRAPHY/ELECTROCHEMISTRY

In this paper we just report some recent aspects of liquid chromatography/electrochemistry (LC/EC) in this laboratory. In addition to convenient detection design, multiple working electrode and microelectrodes in thin-layer flow cell, ion transfer across the water/solidified nitrobenzene interface in flow system, we concentrte our attention recently to study the use of chemically modified electrodes (CME) for CL/EC dealing with promotor catalytic reaction, electrode surface pretreatment and charge selectivity

Electroanalytical approaches to understanding benzene metabolism

Electrochemical techniques are ideally suited to the study of the metabolism of aromatic xenobiotics because the metabolites are frequently easier to oxidize than the parent compounds. In many cases, the trace metabolites have the lowest oxidation potentials and hence electrochemical methods have the greatest selectivity for these compounds. The sensitivity of dual-electrode liquid chromatography-electrochemistry for the detection and identification of trace metabolites was demonstrated by the detection of the secondary metabolite, hydroquinone, in a microsomal incubation containing benzene and ascorbic acid. The use of an electrochemical detector in a series configuration provides increased selectivity for chemically reversible metabolites such as hydroquinone. Electrochemical methods can also be used to generate metabolites. The products of the electrochemical oxidation of phenol and biphenol are compared with those generated in a peroxidase incubation.

A method for the measurement of S-adenosylmethionine in small volume samples of cerebrospinal fluid or brain using high-performance liquid chromatography-electrochemistry

A method for the measurement of S-adenosylmethionine in cerebrospinal fluid and brain using high-performance liquid chromatography with electrochemical detection is described. The method is based upon the catechol O-methyltransferase-catalyzed methylation of dihydroxybenzylamine to its 3- and 4-methoxy derivatives and measurement of the latter. Detection limits are 0.1 pmol for 3- and 4-methoxyhydroxybenzylamine, recovery is 100%, and variability is less than 8%. HPLC measurement of 3- and 4-methoxyhydroxybenzylamine is linear up to at least 100 pmol injected. The use of a nonbiological catechol in the assay minimizes interference from endogenous and exogenous catechols and the wide limits of linearity allows the assay of small volumes of cerebrospinal fluid or brain extract without further concentration or dilution.

Biomedical applications of liquid chromatography-electrochemistry

Liquid chromatography-electrochemistry has proven to be a viable tool for solving a wide variety of practical analytical problems, primarily in biomedical research. Over 2000 papers have appeared and over 20 manufacturers participate in this area. In spite of these obvious measures of acceptance, there continues to be a general lack of understanding of how these systems work. This naturally stems from the unfamiliarity of chromatographers with electrochemistry and electrochemists with chromatography. Furthermore, there are aspects of both which are not yet understood in molecular detail by anyone. While there is more work to do to optimize the technique, liquid chromatography-electrochemistry clearly is very useful at its present state of development. This article presents an overview of the technology and more recently developed applications. Emphasis is placed on the chromatograph as a whole, dual-channel detectors, microdialysis sampling for in vivo determinations, derivatization and post-column reactions, and studies of xenobiotic metabolism.

Application of Liquid Chromatography/Electrochemistry in Pharmaceutical and Biochemical Analysis: A Critical Review

Abstract In the last 20 years, research efforts have increased the number of separation and detection techniques available for the analyses encountered in the development of pharmaceutical compounds. From a holistic point of view, the analytical effort required in the study of a bioactive moiety involves development of analytics to evaluate not just to determine and analyze the compound of interest, but, just as important, the effect the compound has on the biological system into which it is introduced. Therefore, in evaluating an analytical technique such as liquid chromatography/electrochemistry (LCEC) for its applicability to the study of bioactive compounds, it usually becomes apparent that techniques useful to the scientist studying a drug's metabolism are also useful to the scientist studying the physical and chemical properties of the drug.

Highly sensitive determination of catecholamine and serotonin concentrations in plasma by liquid chromatography-electrochemistry.

Highly sensitive determination of catecholamine and serotonin concentrations in plasma by liquid chromatography-electrochemistry.