Coulometric Array Detection Research Articles

Novel and sensitive high-performance liquid chromatographic method based on electrochemical coulometric array detection for simultaneous determination of catecholamines, kynurenine and indole derivatives of tryptophan

A novel and simple method has been developed for the simultaneous quantification of tryptophan, kynurenine and indole derivatives as well as four catecholamines, including dopamine, noradrenaline, homovanillic acid and 3,4-dihydroxyphenylacetic acid. The method utilises isocratic reversed-phase high-performance liquid chromatography with electrochemical coulometric array detection. The influence of various parameters on chromatographic performance, such as the composition and the pH of the mobile phase and the detection potentials, was investigated. Separation of 13 compounds was achieved by a mobile phase consisting of 10% methanol in 50 m M sodium phosphate–acetate buffer, pH 4.10, containing 0.42 m M octanesulphonic acid. The calibration curve was linear over the range 12 pg to 300 ng on-column. The detection limits ( S/ N 3) depended on the working potential and were found to be between 10 and 100 pg injected. The method was reproducible with intra-day RSDs of 0.3 to 1.5% and inter-day RSDs of 0.5 to 4%.

Determination of phenolic xenoestrogens in water by liquid chromatography with coulometric-array detection

A sensitive method for the simultaneous determination of phenolic xenoestrogens such as bisphenol A, 2,4-dichlorophenol, 4- tert.-butylphenol, 4- n-pentylphenol, 4- n-hexylphenol, 4- n-heptylphenol, 4-octylphenol, 4-nonylphenol was developed using reversed-phase LC and coulometric-array detection. Stepwise gradient elution with phosphoric acid in water–acetonitrile was used. The calibration curves were linear in the range of 5.0 (or 10.0)–1000 ng ml −1 with correlation coefficients of 0.9978–0.9999, the limits of detection were 0.01–0.02 ng ml −1. Sample clean-up was performed by solid-phase extraction (SPE) using 3M Empore™ extraction disks. Three commercial sorbents, C 18, SDB-XD (styrene–divinylbenzene polymer) and SDB-RPS (sulfonated styrene–divinylbenzene polymer) were compared. The highest recoveries were obtained with SDB-RPS. They were above 70% with a relative standard deviation of less than 15%. The proposed method was applied to the determination of phenolic xenoestrogens in various water samples.

Validated method for the determination of hydroquinone in human urine by high-performance liquid chromatography–coulometric-array detection

The paper describes the computer aided method development and validation for the determination of hydroquinone in human urine from a clinical study on renal excretion of hydroquinone metabolites and the release of free hydroquinone in the urinary tract in order to evaluate the proposed urine disinfecting concept. The presented method uses high-performance liquid chromatography on reversed-phase material with a polar endcapping (Aqua-C 18, 250×4.6 mm). Selective and sensitive determination (LOQ=12.5 ng on-column) of the target compound was achieved by electrochemical array detection (CoulArray). Gradient and parameter optimization were supported by DryLab software in order to minimize efforts of the expensive and time-consuming method development. Specificity and selectivity were carried out by separation experiments involving the prodrug arbutin and the metabolites hydroquinone, hydroquinone glucuronide, and hydroquinone sulfate, respectively. Hydroquinone glucuronide reference standard was obtained from in vitro glucuronidation in a rat liver microsomes assay. The method was validated according to the criteria for validation of pharmaceutical bioanalytical methods as drafted by the US Department of Health and Human Services, 1998.

Analysis of lycopene geometrical isomers in biological microsamples by liquid chromatography with coulometric array detection

Methods of analysis for determining low quantities of lycopene cis– trans isomers in biological tissues are needed. Development of two liquid chromatography (LC) methods based on the polymeric C 30 stationary phase equipped with coulometric electrochemical array detection (ED) is described. Separation of 13 lycopene isomers including prolycopene, (a novel tetra- cis-lycopene found in Tangerine tomatoes) was accomplished with both isocratic and gradient methods using different proportions of methanol, methyl tert.-butyl ether, water and 1 M ammonium acetate buffer. Carotenoids were detected at potential settings between 200 and 620 mV. Differences in generated current–voltage curves aided in tentative identification of trans carotenoid species and select cis isomers of lycopene. These methods were successfully applied in the analysis of small quantities of plasma, buccal mucosal cells, prostate and cervical tissues. Limits of detection for trans-lycopene by ED were found to be 50 fmol representing a 10- to 100-fold increase over conventional UV–Vis absorbance methods.

Characterization of Diet-Dependent Metabolic Serotypes: Analytical and Biological Variability Issues in Rats

This report, the first in a series on diet-dependent changes in the serum metabolome (metabolic serotype), describes validation of the use of high performance liquid chromatography (HPLC) separations coupled with Coulometric array detectors to characterize changes in the metabolome. The long-term aim of these studies is to improve understanding of the effects of significant variation in nutritive status on physiology and on disease processes. Initial studies focus on identifying the effects of dietary (or caloric) restriction on the redox-active components of rat serum. Identification of compounds of interest is being carried out using HPLC separations coupled with coulometric array analysis, an approach allowing simultaneous examination of nearly 1200 serum compounds. The technical and practical issues discussed in this report are related to both analytical validity (HPLC running conditions, computer-automated peak identification, mathematical compensation for chromatographic drift, etc.) and biological variability (individual variability, cohort-cohort variability, outliers). Attention to these issues suggests approximately 250 compounds in serum are sufficiently reliable, both analytically and biologically, for potential use in building mathematical models of serotype.

Migration of 4-nonylphenol from polyvinyl chloride food packaging films into food simulants and foods

Migration of 4-nonylphenol (NP) from polyvinyl chloride (PVC) films for food packaging into food simulants and foods has been studied in domestic applications such as wrapping of food and reheating in a microwave oven. The migration of NP from the PVC films was determined by high-performance liquid chromatography with electrochemical coulometric-array detection (LC/ED). Twelve PVC films intended for commercial use and ten for domestic applications (total: 22 samples) were analysed. Some of the PVC films (two home-use and ten retail-use) contained NP at concentrations of between 500 and 3300 µg/g. Migration of NP from the films was influenced by the test conditions (n-heptane at 25°C for 60min, distilled water at 60°C for 30min and 4% acetic acid at 60°C for 30min). The amount of NP migrating from the PVC films into n-heptane (0.33-1.6 µg/cm2) was higher than the amount migrating into distilled water or 4% acetic acid (up to 9.7ng/cm2) for the 11 films in which NP was detected. Up to 0.23% of the NP migrated into distilled water and 4% acetic acid and up to 62.5% into n-heptane. In addition, we investigated NP migration into cooked rice samples wrapped in PVC film. Using spiked samples the method gave an average recovery of 83.7% (n = 5) with a standard deviation of 2.5%. Migration of NP ranged from not detectable (<1.0ng/g) to 410.0ng/g by reheating samples in a microwave oven for 1min and from not detectable to 76.5ng/g by keeping samples at room temperature for 30min.

Screening of homocysteine from newborn blood spots by high-performance liquid chromatography with coulometric array detection

Homocystinuria, due to a deficiency of cystationine-β-synthase, refers to the rare inborn error of the metabolism of homocysteine. The identification and prompt treatment of homocystinuria during the neonatal period can prevent or greatly reduce the severity of the clinical consequences. We report a new method for homocystinuria diagnosis from dried blood spots on newborn screening cards, based on high-performance liquid chromatography with electrochemical coulometric array detection. This method shows an excellent linearity ( y=10.36 x+0.04; r=0.999), precision (RSDs ranged from 2.7 to 5.8%), recovery (87%) and appears to be a cost-effective approach, being simple, rapid, sensitive and cheap.

Determination of some heterocyclic aromatic amines in soup cubes by ion-pair chromatography with coulometric electrode array detection

An analytical method based on ion-pair chromatography with coulometric electrode array detection was developed for determination of four heterocyclic aromatic amines (IQ, MeIQx, 4,8-DiMeIQx and 7,8-DiMeIQx). These compounds are eluted isocratically (960 ml of a solution containing 30 mM trichloroacetic acid and 50 mM sodium acetate adjusted with glacial acetic acid to pH 2.5 are mixed with 40 ml tetrahydrofuran) from a phenyl–hexyl phase and are detected in a coulometric array detector. The working potentials of the four cells are set at +350, +690, +160 and +10 mV against modified palladium electrodes. The detection limits ( S/ N=3) depend on the working potential and are found between 0.1 and 1.1 ng injected. In commercial soup cubes, IQ, MeIQx and 4,8-DiMeIQx are present in concentrations between 0.3 and 2.8 ng/g. The recovery (54.8–100.9%) is influenced by the food matrix. The limits of quantitation are between 0.2 and 2.3 ng/g ( S/ N=5).

An Improved Method for the Determination of Green and Black Tea Polyphenols in Biomatrices by High-Performance Liquid Chromatography with Coulometric Array Detection

Tea polyphenols are strong antioxidants and are believed to have beneficial health effects. However, the blood and tissue levels of these compounds are not well characterized because of a lack of suitable analytical methods for the biological resolution of these compounds. Previously, we developed methods for the analysis of three green tea catechins. Now we report an improved method for the measurement of the levels of the different catechins and theaflavins in biological fluids and tissues. The method includes digestion of the plasma, urine, or tissue samples with β-d-glucuronidase and sulfatase, followed by extraction with ethyl acetate and subsequent separation by reversed-phase high-performance liquid chromatography (HPLC). The polyphenols are identified on the basis of their retention times, spectral analysis, and electrochemical behavior across an array of electrodes. In a single HPLC run, it is possible to determine the major catechins and theaflavins as well as some of the catechin metabolites. The detection limits for catechins and theaflavins are from 5 to 10 ng/ml of saliva, plasma, or urine.

Determination of 4-nonylphenol and 4-octylphenol in human blood samples by high-performance liquid chromatography with multi-electrode electrochemical coulometric-array detection

Alkylphenols can affect human health because they disrupt the endocrine system. In this study, an analytical method for determining trace amounts of 4-nonylphenol (NP) and 4-octylphenol (OP) in human blood samples was developed. Reversed-phase HPLC with multi-electrode electrochemical coulometric-array detection was used for the determination of NP and OP in plasma and serum samples prepared with a solid-phase extraction method. The separation was achieved using an isocratic mobile phase of 0.7% phosphoric acid-acetonitrile with a C18 reversed phase column. The detection limits of NP and OP were 1.0 and 0.5 ng ml-1, respectively. The recoveries of NP and OP added to human plasma samples were above 70.0% with a relative standard deviation of less than 15.5%. The method was found to be applicable to the determination of NP and OP in various human blood samples such as serum and plasma.

Method for the Detection of Nucleosides, Bases, and Hydroxylated Adducts Using Gradient HPLC with Coulometric Array and Ultraviolet Detection

Free radicals are compounds with an unpaired electron capable of independent existence. These highly reactive species have been implicated in many disease states and can react with cell membranes, lipids, proteins, and DNA. When an oxygen radical reacts with DNA, base damage, cross-linking (e.g., DNA-DNA or DNA-protein), or DNA backbone damage (e.g., single- or double-strand breaks) can occur and often result in cell death. The field of oxidative metabolism as it relates to DNA damage has grown tremendously, with more DNA adducts being identified as biomarkers. These biomarkers are indicative of DNA damage. Measurement of these biomarkers has proved to be a challenge because of their relatively low occurrence (1 per 10(5)-10(6) bases). Methodologies for the measurement of DNA damage include thin-layer chromatography, enzyme-linked immunosorbent assay, gas chromatography-mass spectrometry, DNA sequencing, high-performance liquid chromatography (HPLC)-ultraviolet, and HPLC-ECD. HPLC-ECD (electrochemical detection) is a powerful technique that is both sensitive and selective. However, HPLC-ECD is generally not amenable to gradient analyses, so its utility is restricted. In addition, many of the bases and nucleosides are not electrochemically active. Gradient HPLC separation coupled to both a coulometric electrochemical array detector and an ultraviolet detector overcomes these limitations. Presented here is a gradient HPLC method that measures a wide variety of nucleosides, bases, and hydroxylated adducts using the inherent stability, sensitivity, and wide dynamic range of a coulometric electrochemical array detector and the universal detection qualities of an ultraviolet detector. Linear ranges, limits of detection, and detailed methods development are presented.

Simultaneous Analysis of the Majority of Low-Molecular-Weight, Redox-Active Compounds from Mitochondria

Studies of the interaction between oxidative stress and mitochondrial dysfunction are complicated by analytical limitations, especially the need to assess multiple parameters in relatively small samples. We have addressed this problem by developing a methodology for the simultaneous analysis of the majority of low-molecular-weight, redox-active compounds from mitochondria using HPLC separations followed by coulometric array detection. The method described should also be applicable for the study of redox-active compounds in other subcellular organelles as well as in intact cells and tissues. The protocol described enables simultaneous measurement of antioxidants (e.g., tocopherols, ascorbate, lipoates, uric acid, and glutathione), markers of oxidative stress (e.g.,o-tyrosine,m-tyrosine, nitrotyrosine, dityrosine, glutathione disulfide, and 8-hydroxydeoxyguanosine) as well as other metabolites (e.g., purines and indoles). In all, ca. 600 redox active compounds can be detected, most with a limit of detection of ∼5 pg on column. Results, including analytical parameters, from a study of liver mitochondria from control and diabetic rats are presented to demonstrate utility of this methodology.

Analysis of phytoestrogens and polyphenols in plasma, tissue, and urine using HPLC with coulometric array detection.

The study of phytoestrogens in food sources and their metabolism, effects, and mechanism of action in animals requires very selective and often sensitive analytical techniques. We have applied coulometric array detection, which uses a series of flow-through electrochemical sensors each providing 100% electrolytic efficiency, for measurement of a variety of phytochemicals in complex matrices. Recent work has involved the resolution of coumestrol (COM), daidzein (DE), daidzin (DI), diethylstilbestrol (DES), enterodiol (ED), enterolactone (EL), equol (EQ), estradiol (E2), estriol (E3), estrone (E), genistein (GE), and quercetin (QE). Binary gradient reversed-phase (C18) chromatography was used with a sodium acetate buffer (pH 4.8)-methanol-acetonitrile solvent system. Eight coulometric sensors were set at 260, 320, 380, 440, 500, 560, 620, and 680 mV (vs Pd reference). Compounds were resolved in 30 min via both their oxidation/reduction characteristics and chromatographic behavior. Respective maximal oxidation potentials (mV) were: COM = 380; DE = 500; DI = 620; DES = 440; ED = 620; EL = 620; EQ = 560; E2 = 560; E3 = 560; E1 = 560; GE = 500; and QE = 260 with limits of detection of 5-50 pg. Uterine tissue homogenates (30 mg/ml in Tris-EDTA) and plasma from Sprague-Dawley rats sacrificed 1 hr after sc injection with either vehicle, dimethylsulfoxide, 10 microg DES, or 1.0 mg EQ were analyzed before and after enzymatic hydrolysis with beta-glucuronidase/sulfatase. Urine samples from humans receiving a Boston-area diet with or without soy protein isolate supplements were also analyzed. Ethanol extracts were evaporated and reconstituted in 20% methanol before HPLC analysis. DE, ED, EL, EQ, and GE were determined in urine with less than 5% (R.S.D.) intraassay imprecision and 85%-102% recovery. Levels (ng/ml) of GE (1.8), QE (11.2), and EQ (1.7) were found in control plasma before hydrolysis and GE (293), QE (183), and EQ (22) after hydrolysis. Higher concentrations, corresponding to sc injection, in free and total EQ were found in both tissue and plasma.

Nucleoside, base and adduct measurement using gradient HPLC with coulometric array and UV detection: [formula omitted], ESA Inc, Chelmsford, MA 01824. Kenneth Hensley, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104. Douglas R McCabe, ESA Inc, Chelmsford, MA 01824

Nucleoside, base and adduct measurement using gradient HPLC with coulometric array and UV detection: [formula omitted], ESA Inc, Chelmsford, MA 01824. Kenneth Hensley, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104. Douglas R McCabe, ESA Inc, Chelmsford, MA 01824

High-Performance Liquid Chromatography Coupled with Coulometric Array Detection of Electroactive Components in Fruits and Vegetables: Relationship to Oxygen Radical Absorbance Capacity

An HPLC procedure utilizing reversed-phase chromatography coupled with a coulometric array detection system was developed for the characterization of overall antioxidant status in fruits and vegetables. The method was reliable and sensitive (20 pg to 1 ng detection limit) and can also be used to identify and simultaneously quantify multi-antioxidants including vitamin C, glutathione, phenolic acids, and flavonoids in fruits and vegetables. Each fruit and vegetable aqueous extract showed an unique distribution of chromatographic peaks that could serve as a “fingerprint” for the fruit or vegetable. A significant positive linear correlation was demonstrated in fruit and vegetable aqueous extracts between the total antioxidant activities determined by using oxygen radical absorbance capacity (ORACROO•) assay and the electrochemical data generated from the coulometric array detectors. From these data, the ORACROO• assay appears to be a valid and useful procedure for measuring total antioxidant activity in extracts of fruits and vegetables. Keywords: Fruit; vegetable; antioxidant; phenolic acid; flavonoid; anthocyanin

Urinary metanephrine and normetanephrine determined without extraction by using liquid chromatography and coulometric array detection

We describe a procedure for the direct measurement of metanephrine (MN) and normetanephrine (NMN) in hydrolyzed urine, using HPLC with coulometric array detection. Acid-hydrolyzed samples were diluted and filtered before separation by isocratic reversed-phase ion-pair chromatography. Eight serial coulometric sensors, set at incrementally increasing anodic potentials, were used to screen lower-oxidizing interferences and provide stepwise oxidation of the metanephrines. Voltammetric behavior across three adjacent sensors was used to assess resolution and aid in peak identification. Values obtained in commercial controls were consistently within the specified target range. Variability, expressed as CV, was 5.45-9.22% between runs and 1.60-4.52% within-run for both compounds. The limit of detection was 2.6 micrograms/L for MN and 2.8 micrograms/L for NMN, with a linear response to 15.0 mg/L for both analytes. Results from patients' samples correlated well with those by a method involving dual ion-exchange extraction (r = 0.963, n = 82 for MN; r = 0.9768, n = 83 for NMN). This procedure provided high selectivity and objective peak purity information while greatly simplifying sample preparation.

Analysis of phenolic and flavonoid compounds in juice beverages using high-performance liquid chromatography with coulometric array detection

Analysis of phenolics and flavonoids in juice beverages using reversed-phase HPLC with coulometric array detection is described. Sixteen serial coulometric detectors were used for on-line resolution of co-eluting compounds and generation of voltammetric data. Within each class of compounds, oxidation potential corresponded to specific substitution patterns where: catechol < methoxycatechol < monohydroxyl < methoxyl. Twenty-seven standard compounds were resolved in a 45-min run. The limits of detection were in the low ng/ml range with a linear response range of at least three orders of magnitude. Intra-run retention time variation was < 1% (R.S.D.) and adjacent sensor response ratios varied by <5% (R.S.D.). The utility of this technique in generating multivariate data for differentiation of juices and juice mixtures is shown.

Determination of neurochemicals in biological fluids by using an automated high-performance liquid chromatographic system with a coulometric array detector

The chromatographic separation of 33 neurochemicals was achieved by using a combined gradient of organic modifier, pH and counter-ion. A secondary separation of unresolved analytes was obtained by using electrochemical detection with a coulometric array of sixteen electrodes. The stability of the analytes was studied and data on analytical performance are reported in addition to a list of neurochemicals detected in a normal plasma sample.