Coulometric Detection Research Articles

Interference from Indian diet on the internal standard in a commercial method for the measurement of urinary metanephrines by high-performance liquid chromatography with electrochemical detection

Urinary metanephrines are widely used in the diagnosis of catecholamine secreting tumours. Over the past two years we have been using the commercial Recipe(®) ClinRep(®) Complete Kit for Metanephrines in Urine coupled with high-performance liquid chromatography and coulometric detection. It was noticed that the internal standards on the patient chromatograms were sporadically raised due to interference. The interference had identical chromatographic and electrochemical properties to the Recipe(®) internal standard (undisclosed identity). Inspection of the patient names showed it seemingly had a higher frequency and magnitude in patients of Indian origin. The source of the interference was tracked by dietary observation and intervention to curry leaves, a common component of Indian foods. The interference was chromatographically and electrochemically indistinguishable from the internal standard. The mass spectrum of the pentafluoropropionate derivative of the interference matched the Recipe(®) internal standard and was identified as methoxyhydroxybenzylamine by library match. The component co-elutes exactly with internal standard and artifactually decreases the metanephrine and normetanephrine results. It is surprising that it has not been described previously. Patients being assessed for catecholamine secreting tumours should be advised to withdraw from eating Indian foods at least 24 h prior to commencement of urinary collection.

Evaluation of herb and fruit juice adulteration and authenticity by coulometric array detection and pattern recognition analysis

Evaluation of herb and fruit juice adulteration and authenticity by coulometric array detection and pattern recognition analysis

Determination of Olanzapine and N-desmethyl-olanzapine in Plasma Using a Reversed-Phase HPLC Coupled with Coulochemical Detection: Correlation of Olanzapine or N-desmethyl-olanzapine Concentration with Metabolic Parameters

BackgroundOlanzapine (OLZ) is one of the most prescribed atypical antipsychotic drugs but its use is associated with unfavorable metabolic abnormalities. N-desmethyl-olanzapine (DMO), one of the OLZ metabolites by CYP1A2, has been reported to have a normalizing action on metabolic abnormalities, but this remains unclear. Our aim was to explore the correlation between the concentrations of OLZ or DMO with various metabolic parameters in schizophrenic patients.MethodsThe chromatographic analysis was carried out with a solvent delivery system coupled to a Coulochem III coulometric detector to determine OLZ and DMO simultaneously in OLZ-treated patients. The correlation between the concentration of OLZ or DMO and the metabolic parameters was analyzed by the Spearman rank order correlation method (r s).Principal FindingsThe established analytical method met proper standards for accuracy and reliability and the lower limitation of quantification for each injection of DMO or OLZ was 0.02 ng. The method was successfully used for the analysis of samples from nonsmoking patients (n = 48) treated with OLZ in the dosage range of 5–20 mg per day. There was no correlation between OLZ concentrations and tested metabolic parameters. DMO concentrations were negatively correlated with glucose (r s = –0.45) and DMO concentrations normalized by doses were also negatively correlated with insulin levels (r s = –0.39); however, there was a marginally positive correlation between DMO and homocysteine levels (r s = +0.38).ConclusionsThe observed negative correlations between levels of DMO and glucose or insulin suggest a metabolic normalization role for DMO regardless of its positive correlation with a known cardiovascular risk factor, homocysteine. Additional studies of the mechanisms underlying DMO’s metabolic effects are warranted.

Synthesis, HPLC Measurement and Bioavailability of the Phenolic Amide Amkamide

Amkamide is a phenolic amide whose analogues were recently reported to have potent mitochondria protective activity, although their bioavailability is still unknown. Therefore, in this study, amkamide was synthesized and confirmed by nuclear magnetic resonance, and a high-performance liquid chromatography (HPLC) method was developed for analyzing the amides in biological samples. HPLC separation was performed on a Nova-Pak C18 column using a gradient condition and a coulometric electrochemical detector. The HPLC method was able to produce excellent and reproducible separations of amkamide from other amides (oretamide, becatamide, enferamide and veskamide). All five amides yielded outstanding peak resolutions with detection limits as low as 100 fmol. Therefore, the bioavailability of amkamide together with becatamide was determined in a mouse model. Their plasma concentrations were measured following two oral administrations (2 and 4 mg per 30 g body weight) using the HPLC method. As expected, relatively high amounts of amkamide and becatamide (51-58 µM) were detected at the time after administration when the maximum concentration was reached (approximately 20 and 25 min) at the high dose. The data indicate that HPLC methods with amide standards may be utilized to measure amkamide and its analogues in biological samples with excellent resolution and detection limits.

Carbon-based Electrode Materials for DNA Electroanalysis

This review addresses recent studies of newly developed carbon-based electrode materials and their use for DNA electroanalysis. Recently, new carbon materials including carbon nanotubes (CNT), graphene and diamond-based nanocarbon electrodes have been actively developed as sensing platforms for biomolecules, such as DNA and proteins. Electrochemical techniques using these new material-based electrodes can provide very simple and inexpensive sensing platforms, and so are expected to be used as one of the "post-light" DNA analysis methods, which include coulometric detection, amperometric detection with electroactive tags or intercalators, and potentiometric detection. DNA electroanalysis using these new carbon materials is summarized in view of recent advances on electrodes.

Evaluation of Herb and Fruit Juice Adulteration and Authenticity by Coulometric Array detection and Pattern Recognition Analysis

Evaluation of Herb and Fruit Juice Adulteration and Authenticity by Coulometric Array detection and Pattern Recognition Analysis

Plasma Cysteine/Cystine Reduction Potential Correlates with Plasma Creatinine Levels in Chronic Kidney Disease

Background/Aims: Oxidative stress has been considered a nontraditional risk factor for cardiovascular disease in the chronic kidney disease (CKD) population, possibly triggered by uremic toxicity. Methods: A chromatographic method with coulometric detection was adapted to directly and simultaneously determine cysteine (Cys) and cystine (Cyss) in plasma samples. Healthy subjects and CKD subjects in different stages were analyzed. The free Cys and free Cyss levels in their plasma were determined, and the reduction potential [E_h(Cyss/2Cys)] was calculated with the Nernst equation. Results: Healthy plasma presented E_h(Cyss/2Cys) of –123 ± 7 mV. Plasma E_h(Cyss/2Cys) correlated significantly with creatinine levels (p < 0.0001, r = 0.62). Conclusion: Plasma E_h(Cyss/2Cys) correlated with increased levels of plasma creatinine, supporting the view that uremia triggers oxidative stress. In addition, it may be used as a quantitative oxidative stress biomarker in uremic conditions.

Analytical approach to determine biogenic amines in urine using microextraction in packed syringe and liquid chromatography coupled to electrochemical detection

The goal of this work was to develop and validate an analytical method for the detection and quantification of the biogenic amines serotonin (5-HT), dopamine (DA) and norepinephrine (NE), using microextraction in packed syringe (MEPS) and liquid chromatography coupled to electrochemical detection (HPLC-ED) in urine. The method was validated according to internationally accepted guidelines from the Food and Drug Administration. Linearity was established between 50 and 1000 ng/mL for 5-HT and between 5 and 1000 ng/mL for DA and NE, with determination coefficients (R(2)) >0.99 for all compounds. The limits of quantification and detection were respectively 50 and 20 ng/mL for 5-HT, and 5 and 2 ng/mL for DA and NE. Within- and between-run precision ranged from 0.84 to 9.41%, while accuracy ranged from 0.79 to 12.76% for all compounds. The intermediate precision and accuracy were 1.50-8.36 and 0.54-13.51%, respectively. The method was found suitable for clinical routine analysis of the studied compounds, using a sample volume of 0.5 mL. This is the first study employing a commercially available MEPS column for the simultaneous detection and quantification of 5-HT, DA and NE in urine by coulometric detection.

Abstract IA24: Biomarkers of diet predict disease risk.

Abstract Over-nutrition and suboptimal dietary macronutrient choices are arguably the major environmental stressor in individuals living in Western societies. Obesity and poor diet are estimated to cause or contribute to as many as 25% of all cancer. One of the most clear examples of this, dietary or caloric restriction (CR), is the most potent and reproducible known means of increasing longevity and reducing morbidity in mammals. CR is also accepted to reduce cancer risk in animals. As one example, risk of breast cancer is generally decreased by more than 90% in CR rodents, and the CR-mediated effects are usually dominant to those induced by genetic risk factors, carcinogens, or co-carcinogens. The robust observations of reduced morbidity in CR animals is directly analogous to studies in humans that link obesity with poor health outcomes, including increased risk of neoplastic disease. We therefore proposed to test the general concept that biomarkers of diet in rats will predict risk of future disease in humans. Methods: Metabolomics measurements in sera/plasma were conducted HPLC coupled with coulometric detector arrays (N∼600 rats, ∼1700 humans). Classification and predictive power were tested, optimized, and subsequently validated using a series of megavariate data analysis approaches in sequential blinded cohorts. Results: Exploratory studies identified 93 redox-active small molecules from sera (measured by) with potential to distinguish dietary groups in both male and female rats (60 6 month old FBFN1 rats/group, AL/CR/male females in primary set). Partial Least Squares Projection to Latent Structures Discriminant Analysis, a projection method optimized for class separation built models with &amp;gt;95% accuracy in distinguishing groups. Data processing choices of transformation, scaling, and winsorizing (outlier removal) each affected strength of the models, and, in some cases, revealed distinct metabolites to be of importance in building these models, often in gender-specific ways. Diets varying in extent and duration of CR were used to develop models for intermediate caloric intakes, which are more relevant for human studies (total N=∼180 males, 180 females). Markers were adapted for human study, analytically validated at both the instrumentation (N=30; 100% accuracy in blinded splits) and at the sample collection levels (N=34; majority stable under worst case shipping conditions), then biologically validated (N∼200, metabolites and profiles had intra-class correlation coefficients from ∼0.65-0.85). We will present these modeling approaches, the models and their ability to distinguish sera based on caloric intake, as well as data from the initial application of these markers to address risk of breast cancer in case-control studies nested within the Nurses' Health Study. We will address some of the checks and cross-checks used to evaluate these data. Conclusion: Metabolomics profiles offer a potential biochemical approach to validate nutritive status and contribute to epidemiological investigations. Citation Format: Bruce S. Kristal. Biomarkers of diet predict disease risk. [abstract]. In: Proceedings of the AACR Special Conference on Post-GWAS Horizons in Molecular Epidemiology: Digging Deeper into the Environment; 2012 Nov 11-14; Hollywood, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2012;21(11 Suppl):Abstract nr IA24.

An in vitro study on the pharmacological ascorbate treatment of influenza virus

To investigate the effects and mechanism of pharmacological ascorbate against Influenza A/CA/7/09 (H1N12009). NHBE cells (≈ 95% confluent monolayer) in 12-well plates (Corning) were kept at 37°C at all times. NHBE cells were exposed to A/CA/7/09 (H1N12009) influenza virus at MOI of 0.01 for 1 h, rinsed with NHBE medium, and incubated with NHBE medium containing 20 mmol/L ascorbate or 20 mmol/L ascorbate +600 IU/ml Catalase. The cells were then incubated for an additional 4 - 12 h and the culture medium was harvested for titration. Viral titers were determined as log(10) 50% tissue culture infective doses (TCID₅₀) assay in MDCK cells. Ascorbate in NHBE medium was determined using HPLC separation coupled with coulometric electrochemical detection. Hydrogen peroxide was detected indirectly by Clark-type oxygen electrode. In vitro experiments showed that pharmacological ascorbate killed not only isolated viruses, but also viruses from normal human bronchial epithelial cells. The antiviral effect of ascorbic acid appeared to be dose-dependent. 2.5 mmol/L ascorbic acid was able to eliminate 90% of the viruses and 20 mmol/L ascorbic acid totally blocked viral replication in vitro. The antiviral effect of pharmacological ascorbate varied at different phases of infection. Pharmacological ascorbate eliminated viral infectivity with treatment times as short as 4 hours at early stage of infection. But the effect was reversed by catalase. Pharmacological ascorbate (vitamin C) as a pro-drug eliminates or kills influenza virus, probable by producing steady-state concentrations of hydrogen peroxide (H₂O₂) in extracellular fluid.

A novel test using dried blood spots for the chromatographic assay of methadone

A novel test has been developed for the analysis of methadone in dried blood spot specimens from patients undergoing methadone maintenance treatment. An isocratic reversed-phase high-performance liquid chromatography method with coulometric detection has been optimized for the determination of methadone. The clean-up of dried blood spots was performed by means of an original microextraction by packed sorbent procedure after microwave-assisted extraction of the drug with a suitable solvent. Extraction yields were satisfactory, always being higher than 90.0%. The calibration curve was linear over the 4-500ngmL(-1) concentration range. The method had satisfactory sensitivity (limit of quantitation of 4ngmL(-1)), precision (relative standard deviation less than 5.8%), selectivity and accuracy (recovery greater than 87.0%). It was successfully applied to dried blood spot samples collected from heroin-addicted patients undergoing methadone maintenance therapy at dosages between 40 and 240mgday(-1). The statistical analysis (Bland-Altman plot) showed that the results were in good agreement with those found from the analysis of plasma samples obtained from the same patients. Thus, the method has proved to be suitable for the monitoring of methadone by means of dried blood spots.

Selection of Pharmaceutical Antioxidants by Hydrodynamic Voltammetry

AbstractAntioxidant compounds are routinely included in pharmaceutical formulations in order to minimize the oxidative degradation of the active pharmaceutical ingredient(s) (API). The effectiveness of the antioxidants being considered is often assessed by creating several formulations with the API(s) and single or multiple antioxidants in order to place each of these formulations on stability under accelerated conditions. This trial and error selection process is costly and time consuming, often taking weeks before the oxidation is observed at a detectable level. Using a flow injection coulometric detector based system, a potential tool has been developed to guide initial antioxidant selection for hindering the oxidative degradation for a given drug substance in its formulation environment. The relative potential for the API to be oxidized can be measured by matching the conditions of the coulometric experiment to the formulation matrix. Using this selection criterion, the choice of antioxidants to study for individual formations can be greatly narrowed, thus leading to a significant savings in development time and cost.

Interference Compensation for Thin Layer Coulometric Ion-Selective Membrane Electrodes by the Double Pulse Technique

Ion-selective membranes operated in a thin layer coulometric detection mode have previously been demonstrated to exhibit attractive characteristics in view of realizing sensors without the need for frequent recalibration. In this methodology, the analyte ion is exhaustively removed across an ion-selective membrane by an applied potential, and the resulting current is integrated to yield the coulomb number and hence the amount of analyte originally present in the sample. This exhaustive process, however, places greater demands on the selectivity of the membrane compared to direct potentiometry, since the level of interference will increase as the analyte depletes. We evaluate here a double pulse protocol to reduce the level of interference, in which the sample is electrolyzed once again after the initial coulometric detection pulse. Since the analyte ion is no longer present at significant concentrations during the second pulse, but an interfering ion of high concentration did not appreciably deplete, the second electrolysis step may be used to partially compensate for undesired interference. These processes are here evaluated by numerical simulation for ions of the same charge, demonstrating that the resulting coulomb number may indeed be reduced for systems of limited selectivity. The improvement in operational selectivity relative to uncompensated coulometry is found to be ca. 6-fold. The methodology is successfully demonstrated experimentally with a calcium selective membrane and tetraethylammonium as a model interfering agent, and the observed relative errors after background compensation can be favorably compared to that in direct potentiometry where no sample depletion occurs.

Combined liquid chromatography–coulometric detection and microextraction by packed sorbent for the plasma analysis of long acting opioids in heroin addicted patients

The sublingual combination of buprenorphine and naloxone (Suboxone ®) and Methadone Maintenance Therapy have been found effective in treating heroin addiction. A new analytical method suitable for the simultaneous determination of buprenorphine, norbuprenorphine, methadone and naloxone in human plasma by means of liquid chromatography with coulometric detection has been developed. The chromatographic separation was achieved with a phosphate buffer–acetonitrile mixture as the mobile phase on a cyano column. The monitoring cell of the coulometric detector was set at an oxidation potential of +0.600 V. A rapid clean-up procedure of the biological samples using a microextraction by packed sorbent technique has been implemented, employing a C8 sorbent inserted into a syringe needle. The extraction yield values were satisfactory for all analytes (>85%). The calibration curves were linear over a range of 0.25–20.0 ng mL −1 for buprenorphine and norbuprenorphine, 3.0–1000.0 ng mL −1 for methadone and 0.13–10.0 ng mL −1 for naloxone. The sensitivity was also high with limits of detection of 0.08 ng mL −1 for both buprenorphine and norbuprenorphine, 0.9 ng mL −1 for methadone and 0.04 ng mL −1 for naloxone. The intraday and interday precision data were always satisfactory. The method was successfully applied to plasma samples obtained from former heroin addicts treated with opioid replacement therapy.

Coulometric Detection of Irreversible Electrochemical Reactions Occurring at Pt Microelectrodes Used for Neural Stimulation

The electrochemistry of 50 μm diameter Pt electrodes used for neural stimulation was studied in vitro by reciprocal derivative chronopotentiometry. This differential method provides well-defined electrochemical signatures of the various polarization phenomena that occur at Pt microelectrodes and are generally obscured in voltage transients. In combination with a novel in situ coulometric approach, irreversible H(2) and O(2) evolution, Pt dissolution and reduction of dissolved O(2) were detected. Measurements were performed with biphasic, charge-balanced, cathodic-first and anodic-first current pulses at charge densities ranging from 0.07 to 1.41 mC/cm(2) (real surface area) in phosphate buffered saline (PBS) with and without bovine serum albumin (BSA). The extent to which O(2) reduction occurs under the different stimulation conditions was compared in O(2)-saturated and deoxygenated PBS. Adsorption of BSA inhibited Pt dissolution as well as Pt oxidation and oxide reduction by blocking reactive sites on the electrode surface. This inhibitory effect promoted the onset of irreversible H(2) and O(2) evolution, which occurred at lower charge densities than those in PBS. Reduction of dissolved O(2) on Pt electrodes accounted for 19-34% of the total injected charge in O(2)-saturated PBS, while a contribution of 0.4-12% was estimated for in vivo stimulation. These result may prove important for the interpretation of histological damage induced by neural stimulation and therefore help define safer operational limits.

Improved method for morphine determination in biological fluids and tissues: rapid, sensitive and selective

Morphine was assayed using a simple two step solvent extraction--acid back extraction sample preparation method, coupled with normal phase high-performance liquid chromatography (HPLC) and dual electrode coulometric detection. HPLC is performed with a 1.0 M Tris-methanol (5:95) mobile phase with subtle pH adjustments to separate morphine and internal standard from any interfering compounds. The use of normal phase HPLC (silica column) substantially reduces problems from interfering lipophilic substances sometimes encountered with reverse phase HPLC following solvent extraction and which would otherwise require more time-consuming sample preparation. Dual electrode detection further improves the selectivity for morphine and gives excellent sensitivity (0.5 ng mL-1), reproducibility and stability for automated sample injection. This method has proven suitable for pharmacokinetic studies of morphine.

Rapid assays of clozapine and its metabolites in dried blood spots by liquid chromatography and microextraction by packed sorbent procedure

A novel analytical approach has been developed for the determination of clozapine and its metabolites in dried blood spots on filter paper, using a chromatographic method coupled with a microextraction by packed sorbent procedure. The analytes were separated on a RP-C18 column using a mobile phase composed of 20% methanol, 16% acetonitrile and 64% aqueous phosphate buffer. Coulometric detection was used, setting the guard cell at +0.050 V, the first analytical cell at −0.200 V and the second analytical cell at +0.500 V. Clozapine and its metabolites were extracted from dried blood spots with phosphate buffer and, then, a microextraction by packed sorbent procedure for the sample clean-up was implemented obtaining good extraction yields. The calibration curve was linear over the 2.5–1000 ng mL −1 blood concentration ranges for all the analytes. The method validation gave satisfactory results in terms of sensitivity, precision, selectivity and accuracy. The analytical method was successfully applied to dried blood spots from several psychiatric patients for therapeutic drug monitoring purpose.

Determination of glutathione and glutathione disulfide in human whole blood using HPLC with coulometric detection: A comparison with fluorescence detection

We describe a relatively simple method for the determination of glutathione (GSH) and glutathione disulfide (GSSG) in human whole blood. We have used an HPLC with coulometric electrochemical detection for the simultaneous measurement of GSH and GSSG. Diluted and filtered trichloroacetic acid extracts were injected directly into the HPLC system and were eluted isocratically on a Polaris 5u C18-A, 250 × 4.6 mm analytical column. Glutathione in samples extracted with trichloroacetic acid and diluted with 1.0 mMhydrochloric acid was stable at 4 °C for at least 8 h. The analytical performance of this method is satisfactory: the intra-assay and inter-assay coefficients of variation were below 10%. Quantitative recoveries from spiked whole blood samples were at intervals 91.6–97.6% for GSH and 85.0–104.4% for GSSG. The linear range is 5.0–2000.0 μmol/l, with a detection limit of 2.1 μmol/l (signal-to-noise ratio = 3) for GSH and 2.0–250.0 μmol/l, with a detection limit of 0.9 μmol/l for GSSG.

The use of experimental design in the development of an HPLC–ECD method for the analysis of captopril

An accurate, sensitive and specific high performance liquid chromatography–electrochemical detection (HPLC–ECD) method that was developed and validated for captopril (CPT) is presented. Separation was achieved using a Phenomenex ® Luna 5 μm (C 18) column and a mobile phase comprised of phosphate buffer (adjusted to pH 3.0): acetonitrile in a ratio of 70:30 (v/v). Detection was accomplished using a full scan multi channel ESA Coulometric detector in the “oxidative-screen” mode with the upstream electrode ( E 1) set at +600 mV and the downstream (analytical) electrode ( E 2) set at +950 mV, while the potential of the guard cell was maintained at +1050 mV. The detector gain was set at 300. Experimental design using central composite design (CCD) was used to facilitate method development. Mobile phase pH, molarity and concentration of acetonitrile (ACN) were considered the critical factors to be studied to establish the retention time of CPT and cyclizine (CYC) that was used as the internal standard. Twenty experiments including centre points were undertaken and a quadratic model was derived for the retention time for CPT using the experimental data. The method was validated for linearity, accuracy, precision, limits of quantitation and detection, as per the ICH guidelines. The system was found to produce sharp and well-resolved peaks for CPT and CYC with retention times of 3.08 and 7.56 min, respectively. Linear regression analysis for the calibration curve showed a good linear relationship with a regression coefficient of 0.978 in the concentration range of 2–70 μg/mL. The linear regression equation was y = 0.0131 x + 0.0275. The limits of detection (LOQ) and quantitation (LOD) were found to be 2.27 and 0.6 μg/mL, respectively. The method was used to analyze CPT in tablets. The wide range for linearity, accuracy, sensitivity, short retention time and composition of the mobile phase indicated that this method is better for the quantification of CPT than the pharmacopoeial methods.

Electrochemical characterization of repaglinide and its determination in human plasma using liquid chromatography with dual-channel coulometric detection

A simple, fast and sensitive HPLC method employing dual-channel coulometric detection for the determination of repaglinide in human plasma is presented. The assay involved extraction of repaglinide by ethyl acetate and isocratic reversed-phase liquid chromatography with dual-channel coulometric detection. The mobile phase composition was 50 mM disodium hydrogen phosphate/acetonitrile (60:40, v/v), pH of the mobile phase 7.5 set up with phosphoric acid. For all analyses, the first cell working potential was +380 mV, the second was +750 mV ( vs. Pd/H 2). Calibration curve was linear over the concentration range of 5–500 nmol L −1. Rosiglitazone was used as an internal standard. The limit of detection (LOD) was established at 2.8 nmol L −1, and the lower limit of quantification (LLOQ) at 8.5 nmol L −1. The developed method was applied to human plasma samples spiked with repaglinide at therapeutical concentrations. It was confirmed that the method is suitable for pharmacokinetic studies or therapeutic monitoring.