A comparison of coulometric detectors for catecholamine analysis by LC-EC

An ion-pairing chromatographic method which uses a controlled potential coulometric detector is described. Two coulometric detectors with different electrolytic cell designs have been investigated. The resulting sensitivity can be comparable to the conventional amperometric detector. This technique has been applied to the analysis of catecholamines.     

A Semiautomated Analysis Method for Catecholamines, Indoleamines, and Some Prominent Metabolites in Microdissected Regions of the Nervous System: An Isocratic HPLC Technique Employing Coulometric Detection and Minimal Sample Preparation

The application of a commercially available coulometric electrochemical detector to the automated HPLC analysis of some monoamines and their metabolites in microdissected areas of the rat nervous system is described. Apart from the stability and high sensitivity of the system, other appealing features of the technique are the facile sample preparation and long-term sample storage characteristics which show minimal analyte degradation. Basal values of some regional monoamine and metabolite concentration are listed together with a brief appendix that serves as a user's guide to the operation and maintenance of the detection system.     

Molecular imaging of amyloid beta peptides in mouse brain sections using mass spectrometry

In this paper, a reverse-phase high-performance liquid chromatographic method using a chemically modified electrode coupled with microdialysis was developed to study the effect of electromagnetic impulse (EMI) on monoamine neurotransmitter metabolism in nerve cells. To detect the monoamines and their metabolites, a poly (para-aminobenzoic acid) (P-pABA)-modified electrode was prepared. The modified electrode exhibited efficiently electrocatalytic oxidation for monoamines and their metabolites with relatively high sensitivity, stability, and long life. Nerve cells were primarily cultured. EMI was radiated to three experimental model nerve cells: (i) on mature nerve cells, (ii) on the culture medium, and (iii) on juvenile nerve cells for various periods of time. Then the levels of monoamines in the culture medium were detected by high-performance liquid chromatography-electrochemical detection. The data indicated that electromagnetic fields could influence neurotransmitter metabolism by direct effect on nerve cells or effect on the nutrient medium and that the effect was not only relevant with the length of radiation time, but also with the growing state of the nerve cells.     

Quantitation of daunorubicin and its metabolites by high-performance liquid chromatography with electrochemical detection

A selective and sensitive high-performance liquid chromatographic method was developed for the separation and quantitation of daunorubicin and its metabolites in serum, plasma, and other biological fluids. Daunorubicin and metabolites in human plasma were injected directly into the high-performance liquid chromatography system via a loop-column to pre-extract the drugs from the plasma, and quantitated against a multilevel calibration curve with adriamycin as the internal standard. The column effluent was monitored with an electrochemical detector at an applied oxidative potential of 0.65 V and by fluorescence. Daunorubicin and four metabolites were separted and characterized by this method. In a blinded evaluation of accuracy and precision, the mean coefficients of variation were 3.8, 3.6 and 9.8% at concentrations of 150, 75 and 15 ng/ml, respectively, and blank samples gave negligible readings. The amperometric sensitivity was greater than achieved by fluorescence detection, and offers an alternative method for quantitation of these compounds. The new method has a limit of detection of less than 2 ng on column, allowing quantitation of < 10 ng/ml in plasma samples without organic extraction prior to chromatographic analysis.     

Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection

We describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their aglycone structures, 2-methyl-3-(3'-3'-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone) and 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentenyl)-1,4-naphthoquinone (7C-aglycone), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C18) solid-phase extraction (SPE), and the predominantly conjugated vitamin K metabolites were hydrolyzed with methanolic HCl. The resulting carboxylic acid aglycones were quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis was by reversed-phase (C18) HPLC with a methanol-aqueous mobile phase. Metabolites were detected by amperometric, oxidative ECD of their quinol forms, which were generated by postcolumn coulometric reduction at an upstream electrode. The assay gave excellent linearity (typically, r2 > or = 0.999) and high sensitivity with an on-column detection limit of < 3.5 fmol (< 1 pg). The interassay precision was typically 10%. Metabolite recovery was compared with that of an internal standard [2-methyl-3-(7'-carboxy-heptyl)-1,4-naphthoquinone] added to urine samples just before analysis. Using this methodology, we confirmed that the 5C- and 7C-aglycones were major catabolites of both phylloquinone (vitamin K1) and menaquinones (vitamin K2) in humans. We propose that the measurement of urinary vitamin K metabolite excretion is a candidate noninvasive marker of total vitamin K status.     

Liquid chromatography method for plant and mammalian lignans in human urine

Recently new mammalian lignan precursors were identified but no analysis methods are available for assay of those compounds in human urine. Previously published methods were developed for GC-MS about only two plant lignans were included. Consequently, a method for HPLC equipped with a coulometric electrode array detector was developed to measure plant and mammalian lignans in human urine. The plant lignans, secoisolariciresinol (Seco), matairesinol (Mat), lariciresinol (Lar), pinoresinol (Pin), syringaresinol (Syr) and isolariciresinol (IsoL) were included into the new method together with two mammalian lignans, enterolactone (Enl) and enterodiol (End). Validation of the method demonstrated that it could be applied to normal urine containing low amounts of plant lignans and moderate amounts of mammalian lignans, but the method was also applicable for samples from study subjects in supplementation studies, i.e. sample with very high concentrations of mammalian lignans. The method was found to be a useful tool for studies on plant lignan intake and the activity of micro flora in the metabolism of plant lignans.     

Simple and selective assay of 4-hydroxymephenytoin in human urine using solid-phase extraction and high-performance liquid chromatography with electrochemical detection and its preliminary application to phenotyping test

A simple and selective HPLC method for the determination of 4-hydroxymephenytoin (4-OH-M) in human urine, using a controlled potential coulometric detector equipped with a dual working electrode cell of fully porous graphite, has been developed. After acid hydrolysis of urine, 4-OH-M and the internal standard (I.S.), 5-hydroxy-1-tetralone, were extracted from urine by means of a Bond Elut Certify LRC column. The extracts were chromatographed on a reversed-phase μBondapak C₁₈ column using methanol-50 mM KH₂PO₄ (pH 4.0) (30:70, v/v) as the mobile phase at a flow-rate of 1.0 ml/min. Electrochemical detection at applied potential of 800 mV resulted in a limit of quantitation of 0.76 μg/ml. The method showed a satisfactory sensitivity, precision, accuracy, recovery and selectivity. The present method was applied to the phenotyping test in thirteen Japanese healthy volunteers who recieved an oral 10-mg racemic mephenytoin. The phenotypes determined by the present method were found to be in agreement with those obtained with the reported customary assay based on gas chromatography.     

Flow injection analysis of blood L-lactate by using a Prussian Blue-based biosensor as amperometric detector

An amperometric l-lactate biosensor was fabricated by confining lactate oxidase in a Prussian Blue-modified electrode with a Nafion membrane. The detector was assembled in a flow injection apparatus and operated at -0.1 V. Conditions for optimal electrode response were determined by investigating the influence of the amount of immobilized enzyme, the sample volume, and the flow rate. At the established operational conditions, the biosensor exhibited negligible response from interfering species usually present in biological fluids. The stability of the biosensor was also investigated, and its sensitivity was maintained unchanged at certain experimental conditions. l-Lactate was determined in blood samples, and the influence of physical exercise on the results was clearly evidenced, demonstrating that the proposed amperometric detector is suitable for monitoring changes in the l-lactate levels in biological fluids.     

Sensitive high-performance liquid chromatographic determination of cyclizine and its demethylated metabolite,norcyclizine, in biological fluids using coulometric detection

An accurate, sensitive, selective and reproducible high-performance liquid chromatographic method with coulometric detection for the determination of cyclizine and its inactive demethylated metabolite, norcyclizine, in biological fluids has been developed. The drugs were separated using a custom packed reversed-phase C₁₈ analytical column and phosphate buffer (0.05 M, pH 3)-acetonitrile (7:3) as mobile phase. The dual electrode coulometric detector was operated in the “oxidative-screen” mode with the upstream electrode (detector 1) set at 0.55 V and the downstream electrode (detector 2) set at 0.90 V. Serum and urine samples were prepared for analysis by solid-phase extraction, followed by a simple phase-separation step. The limit of quantitation was 1 ng/ml for both cyclizine and norcyclizine in serum and urine.     

Development and performance of an enzyme-linked amperometric immunosensor for the detection of Staphylococcus aureus in foods

An amperometric enzyme-linked immunosensor was developed to detect and quantify levels of Staphylococcus aureus electrically in pure cultures and in foods. The assay was a modification of a 'sandwich' ELISA for the protein A of Staph. aureus, employing catalase-labelled anti-protein A antibody. On addition of hydrogen peroxide to the assay system the catalase released O2 which was monitored using an amperometric oxygen electrode. The rate of current increase was proportional to the antigen concentration (protein A or Staph. aureus). Protein A was detected reliably at 0.1 ng/ml representing a 20-fold increase in sensitivity over the conventional ELISA that used horseradish peroxidase. Pure cultures of Staph. aureus were detected at 10(-3)-10(-4) cfu/ml with the amperometric electrode (cf greater than 10(5)/ml for conventional ELISA). The same level of sensitivity was achieved for inoculated food samples. Low levels of contamination (1 cfu/g) of Staph. aureus were detected after incubation at 37 degrees C for 18 h, and the immunosensor could from the basis of a test for screening and identification of protein A-bearing Staph. aureus in 24 h, although natural variations in protein A content between different strains could make the system unreliable in accurate quantification of cell numbers.     

Comparison of results from different laboratories in measuring 8-oxo-2'-deoxyguanosine in synthetic oligonucleotides

The European Standards Committee on Oxidative DNA Damage (ESCODD) was set up in 1997 to resolve methodological problems and to reach agreement on the basal level of 8-oxo-2'-deoxyguanosine (8-oxodG) in biological samples. In the present ESCODD trial 6 samples of 8-oxodG-containing oligonucleotides with different ratios of 8-oxodG/2'-deoxyadenosine (dAdo) were sent to 25 laboratories throughout Europe. The methods used were HPLC with electrochemical detection (amperometric or coulometric), GC-MS or LC-MS-MS. The LC-MS-MS and the coulometric HPLC analyses gave 8-oxodG concentrations within 53 and 73% of expected values, respectively, whereas the amperometric HPLC and GC-MS consistently overestimated the 8-oxodG concentration by several fold. As the oligonucleotides contained no 2'-deoxyguanosine (dGuo), this was not due to artificial oxidation. On the contrary, in most cases the concentrations of dAdo and thymidine (dThd), used as estimates for non-oxidised DNA bases were underestimated, though a few laboratories overestimated the lowest concentration samples containing 8 and 20 μM, respectively. In one-third of the reported results, the ratio of 8-oxodG/10 5 dAdo was within 25% of the calculated value in the oligonucleotide samples and in half of the results the coefficient of variation in duplicate samples was less than 10%. The coefficients of variation were higher for the dAdo concentrations than for 8-oxodG. Our findings strongly indicate that careful quality control must be applied to the analytical procedures for 8-oxodG and very importantly also to the procedures for non-modified 2'-deoxyribonucleosides. We recommend the use of synthetic oligonucleotides for this purpose.     

Comparison of Results from Different Laboratories in Measuring 8-oxo-2′-deoxyguanosine in Synthetic Oligonucleotides

The European Standards Committee on Oxidative DNA Damage (ESCODD) was set up in 1997 to resolve methodological problems and to reach agreement on the basal level of 8-oxo-2'-deoxyguanosine (8-oxodG) in biological samples. In the present ESCODD trial 6 samples of 8-oxodG-containing oligonucleotides with different ratios of 8-oxodG/2'-deoxyadenosine (dAdo) were sent to 25 laboratories throughout Europe. The methods used were HPLC with electrochemical detection (amperometric or coulometric), GC-MS or LC-MS-MS. The LC-MS-MS and the coulometric HPLC analyses gave 8-oxodG concentrations within 53 and 73% of expected values, respectively, whereas the amperometric HPLC and GC-MS consistently overestimated the 8-oxodG concentration by several fold. As the oligonucleotides contained no 2'-deoxyguanosine (dGuo), this was not due to artificial oxidation. On the contrary, in most cases the concentrations of dAdo and thymidine (dThd), used as estimates for non-oxidised DNA bases were underestimated, though a few laboratories overestimated the lowest concentration samples containing 8 and 20 &#119 M, respectively. In one-third of the reported results, the ratio of 8-oxodG/10 5 dAdo was within 25% of the calculated value in the oligonucleotide samples and in half of the results the coefficient of variation in duplicate samples was less than 10%. The coefficients of variation were higher for the dAdo concentrations than for 8-oxodG. Our findings strongly indicate that careful quality control must be applied to the analytical procedures for 8-oxodG and very importantly also to the procedures for non-modified 2'-deoxyribonucleosides. We recommend the use of synthetic oligonucleotides for this purpose.     

Determination of catecholamines in tissue and body fluids using microbore HPLC with amperometric detection

Performance of microbore reverse phase HPLC coupled with amperometric detection is detailed for the analysis of catecholamines in small tissue samples and human blood plasma and cerebrospinal fluid. Extraction procedures for pre-concentration and clean-up of these samples are described. Marked signal enhancement is observed due to the smaller column volume as well as the increased coulometric yield which results from the lower flow rates used with this technique. Detection limits of 0.2 to 0.5 picograms are obtained allowing analysis of catecholamines in extremely small tissue samples or small volumes of cerebrospinal fluid or plasma.     

Bio-medical applications of high performance liquid chromatography (HPLC) with amperometric detection

Electrochemical detection of eluted solutes in HPLC is now well established as a selective and highly sensitive technique. Measurement of catecholamines and metabolites has been the most popular application with an exponential increase in literature. Recent improvements include the use of diphenylborate for extractions, the introduction of smaller diameter HPLC materials which improve resolution and sensitivity, and the replacement of carbon paste with glassy carbon which now gives a more stable electrode with similar sensitivity. Applications of HPLC with amperometric detection to the determination of drugs, amino acids and peptides are discussed.     

Simultaneous use of electrochemistry and chemiluminescence to detect reactive oxygen species produced by human neutrophils

A novel approach for the simultaneous optical and electrochemical detection of biologically produced reactive oxygen species has been developed and applied. The set-up consists of a luminol-dependent chemiluminescence assay combined with two amperometric biosensors sensitive to superoxide anion radicals (O(2)(-)) and hydrogen peroxide (H(2)O(2)), respectively. The method permits direct, real-time in vitro determination of both extra- and intracellular O(2)(-) and H(2)O(2) produced by human neutrophil granulocytes. The rate of O(2)(-) production by stimulated neutrophils was calculated to about 10(-17)mol s(-1) per single cell. With inhibited NADPH oxidase, a distinct extracellular release of H(2)O(2) instead of O(2)(-) was obtained from stimulated neutrophils with the rate of about 3 x 10(-18)mol s(-1) per single cell. When the H(2)O(2) release was discontinued, fast H(2)O(2) utilisation was observed. Direct interaction with and possibly attachment of neutrophils to redox protein-modified gold electrodes, resulted in a spontaneous respiratory burst in the population of cells closely associated to the electrode surface. Hence, further stimulation of human neutrophils with a potent receptor agonist (fMLF) did not significantly increase the O(2)(-) sensitive amperometric response. By contrast, the H(2)O(2) sensitive biosensor, based on an HRP-modified graphite electrode, was able to reflect the bulk concentration of H(2)O(2), produced by stimulated neutrophils and would be very useful in modestly equipped biomedical research laboratories. In summary, the system would also be appropriate for assessment of several other metabolites in different cell types, and tissues of varying complexity, with only minor electrode modifications.     

Gradient liquid chromatography of leucine-enkephalin peptide and its metabolites with electrochemical detection using highly boron-doped diamond electrode

Boron-doped diamond thin film (BDD) electrodes have been used to study the oxidation reactions and to detect leucine-enkephalinamide (LEA) and its metabolites, tyrosine (T), tyrosyl-alanine (TA), tyrosyl-alanine-glycine (TAG) and leucine-enkephalin (LE) using cyclic voltammetry (CV), flow-injection analysis (FIA), and gradient liquid chromatography (LC) with amperometric detection. At diamond electrodes, well-defined and highly reproducible cyclic voltammograms were obtained with signal-to-background (S/B) ratios 5-10 times higher than those observed for glassy carbon (GC) electrodes. The analytical peaks of LC for LEA and its metabolites were well resolved. No deactivation of BDD electrodes was found after several experiments with standard as well as plasma samples, indicating high stability of the electrode. Calibration curves were linear over a wide range from 0.06 to 30 microM with regression coefficients of 0.999 for all compounds. The limits of detection obtained based on a signal-to-noise ratio of 3:1 were 3, 2.2, 2.7, 20 and 11 nM for T, TA, TAG, LE and LEA, respectively. These values were at least one order lower than those obtained at GC electrodes, which has given limits of detection of 22.88, 20.64, 89.57, 116.04 and 75.67 for T, TA, TAG, LE and LEA, respectively. Application of this method to real samples was demonstrated and validated using rabbit serum samples. This work shows the promising use of conducting diamond as an amperometric detector in gradient LC, especially for the analysis of enkephalinamide and its metabolites.     

Monoamine concentrations in Hydra magnipapillata

Several monoamines and related metabolites were quantified in Hydra magnipapillata using a three dimension HPLC system with multiple coulometric electrochemical detectors. Serotonin (5HT), N-methylserotonin (N-MET) and dopamine were detected, being reported in a coelenterate for the first time. It is suggested that monoamine levels were associated with the interstitial cell lineage. 5HT and N-MET were more concentrated in the body column where nematoblasts and nematocytes develop than in the tentacles and hypostome. It is concluded that the main indoleamine in Hydra is not 5HT but N-MET and a new metabolic pathway in Hydra is suggested: tryptophan-5HT-N-MET.     

Chiral analysis of amino acids using electrochemical composite bienzyme biosensors.

The construction and performance of bienzyme amperometric composite biosensors for the selective determination of l- or d-amino acids is reported. D- or L-Amino acid oxidase, horseradish peroxidase, and the mediator ferrocene were coimmobilized by simple physical inclusion into the bulk of a graphite-70% Teflon electrode matrix. Working conditions including amino acid oxidase loading and pH were optimized. Studies on the repeatability of the amperometric response obtained at +0.00 V, with and without regeneration of the electrode surface by polishing, on the useful lifetime of one single biosensor and on the reproducibility in the fabrication of different biosensors illustrate the robustness of the bioelectrodes design. Calibration plots by both amperometry in stirred solutions and flow injection with amperometric detection were obtained for L-arginine, L-phenylalanine, L-leucine, L-methionine, L-tryptophan, D-leucine, D-methionine, D-serine, and D-valine. Differences in sensitivity were discussed in terms of the hydrophobicity of the substrate and of the electrode surface. The bienzyme composite electrode was applied to the determination of L- and D-amino acids in racemic samples, as well as to the estimation of the L-amino acids content in muscatel grapes.     

Improved assay of reaction products to quantitate catechol-O-methyltransferase activity by high-performance liquid chromatography with electrochemical detection

We applied coulometric detection (three electrochemical electrodes in series) to quantitate vanillic acid and isovanillic acid using reversed-phase HPLC. The formation of these reaction products from dihydroxybenzoic acid was used as a precise and reproducible measure of catechol-O-methyltransferase (COMT) activity in striatal homogenates and recombinant membrane-bound COMT protein. This detection system has a higher sensitivity (0.5 pmol per injection) than a single-cell amperometric detection. As in a previous method, the deproteinized supernatants of the COMT assay could be injected directly onto the HPLC system allowing the handling of a large number of samples in one day.     

Determination of urine catecholamines by capillary electrophoresis with dual-electrode amperometric detection

Demonstrated in this study is that without pretreatment and preconcentration nanomolar-level catecholamines in human urine samples can be quantitatively determined with ease by utilizing capillary electrophoresis coupled with amperometric detection. The detector employs a parallel-opposed dual-electrode scheme assembled with an on-capillary electrode and a disk electrode and takes advantage of the redox cycling of analytes between the two working electrodes to improve the limit of detection. The matrix effect of urine samples significantly decreases the detection sensitivity from that obtained in standard solutions. Therefore, calibration curves derived from standard solutions cannot be used in quantitative determination of catecholamines. Methods of standard addition and internal standard have been studied. The results suggest that isoproterenol is a good internal standard to facilitate the measurements of dopamine, epinephrine, and norepinephrine in human urine samples.