Reductive Mode Electrochemical Detection Research Articles

Novel reductive–reductive mode electrochemical detection of Rohypnol following liquid chromatography and its determination in coffee

Rohypnol (flunitrazepam) has been successfully determined in coffee by high performance liquid chromatography dual electrode detection (LC-DED) in the dual reductive mode. Initial studies were performed to optimise the chromatographic conditions and these were found to be 50% acetonitrile, 50% 50mM pH 2.0 phosphate buffer at a flow rate of 0.75mLmin−1, employing a Hypersil C18, 5μm, 250mm×4.6mm column. Cyclic voltammetric studies were made to ascertain the redox behaviour of Rohypnol at a glassy carbon electrode over the pH range 2–12. Hydrodynamic voltammetry was used to optimise the applied potential at the generator and detector cells; these were identified to be −2.4V and +0.8V for the redox mode and −2.4V and −0.1V for the dual reductive mode respectively. A linear range of 0.5–100μgmL−1, with a detection limit of 20ngmL−1 was obtained for the dual reductive mode. Further studies were then performed to identify the optimum conditions required for the LC-DED determination of Rohypnol in beverage samples. A convenient and rapid method for the determination of Rohypnol in beverage samples was developed using a simple sample pre-treatment procedure. A recovery of 95.5% was achieved for a sample of white coffee fortified at 9.6μgmL−1 Rohypnol.

Voltammetric Behavior ofp-Nitrophenol and Its Trace Determination in Human Urine by Liquid Chromatography with a Dual Reductive Mode Electrochemical Detection System

The determination of trace p-nitrophenol (PNP) concentrations in human urine has been successfully achieved by high performance liquid chromatography dual electrode detection (LC-DED) in the reduction-reduction mode. Initial cyclic voltammetric studies were undertaken to investigate the electrochemical behavior of PNP at a glassy carbon electrode over a wide pH range; the redox processes giving rise to the signals have been deduced. Further, deductions regarding the behavior in the flow cells were made from hydrodynamic voltammetric data. PNP eluting from the analytical LC column is first reduced to p-hydroxylaminophenol, at the first generator electrochemical cell. This species then undergoes chemical oxidation to give a quinoneimine species which is then detected at the downstream detector electrode using an applied potential of −0.1 V. The optimum chromatographic mobile phase consisted of 40% acetonitrile, 60% water, containing 25 mM o-phosphoric acid, at a flow rate of 0.5 mL/min; this was used in conjunction with a Hypersil C18 column. Hydrodynamic voltammetric studies were undertaken to investigate the dual electrode behavior of PNP, and an applied potential of −2.0 V at the generator cell and −0.10 V at the detector cell were found to be optimum. The response was found to be linear over the range 7.0 ng to 500 ng on column, with an associated R2 value of 0.9981; the limit of detection was found to be 1.0 ng PNP on column. No interferences were seen for a number of common drugs or for the principal electrochemically active components of human urine or serum. The developed assay was successfully applied to the determination of trace concentrations of PNP in human urine samples, exhibiting coefficients of variation of 7.1% (n=7), with a mean recovery of 94.7% for urine fortified at 522 ng mL−1.

Separation and quantitation of peroxidized phospholipids using high-performance thin-layer chromatography with tetramethyl- p-phenylenediamine detection

A simple method for the selective determination of phospholipid hydroperoxide (PLOOH) families in complex lipid populations has been developed. Referred to as HPTLC-TPD, the method is based on PLOOH separation by normal-phase high-performance thin-layer chromatography, followed by spray detection with N, N, N ′, N ′-tetramethyl- p-phenylenediamine and densitometric scanning of the purple bands. Parental phospholipids and alcohol analogues are unreactive. Calibration curves, dynamic ranges, and detection limits were established for hydroperoxide standards prepared from phospatidylcholine, phosphatidylserine, phosphatidylethanolamine, and cardiolipin. For all PLOOH classes, responsiveness was linear out to at least 10 nmol of sample load, the detection limit being 0.1–0.2 nmol. HPTLC-TPD data were validated by subjecting duplicate samples to more complex column chromatography with reductive-mode electrochemical detection. General applicability of the new technique was demonstrated using lipid extracts from two test systems: (i) photoperoxidized liposomal membranes and (ii) tumor cells that had been oxidatively stressed with the respiratory inhibitor antimycin A. HPTLC-TPD provides a convenient, specific, and highly sensitive means for quantifying individual PLOOH families in complex natural mixtures.

High-performance liquid chromatography with mercury cathode electrochemical detection: application to lipid hydroperoxide analysis

Lipid hydroperoxide species can be analyzed with high sensitivity and specificity, using reversed-phase high-performance liquid chromatography with reductive mode electrochemical detection on a mercury drop cathode [HPLC-ED(Hg)]. The purpose of this study was to examine different variables in the operation of HPLC-ED(Hg) and to select optimal conditions for the analysis of several biologically relevant peroxides, including species derived from cholesterol, cholesteryl linoleate, oleate, linoleate, and two synthetic phosphatidylcholines. Parameters such as operating potential and mobile-phase solvent proportions, electrolyte composition, and ionic strength were evaluated for each peroxide class. Under optimal conditions, we have achieved baseline separation of four cholesterol hydroperoxide species, not only from one another, but also from phospholipid hydroperoxides; detection limits were <0.3 pmol and <30 pmol for the cholesterol and phospholipid hydroperoxides, respectively.

Characterization of lipid hydroperoxides generated by photodynamic treatment of leukemia cells.

A new technique, high-performance liquid chromatography with reductive mode electrochemical detection on a mercury drop (HPLC-EC), has been used for analyzing lipid hydroperoxide (LOOH) formation in photooxidatively stressed L1210 leukemia cells. Highly specific and sensitive for peroxides (detection limits < 0.5 pmol for cholesterol hydroperoxides and < 50 pmol for phospholipid hydroperoxides), this approach allows different classes of LOOH to be separated and determined in minimally damaged cells. L1210 cells in serum-containing growth medium were irradiated in the presence of merocyanine 540 (MC540), a lipophilic photosensitizing dye. Lipid extracts from cells exposed to a light fluence of 0.11 J/cm2 (which reduced clonally assessed survival by 30%) showed 12-15 well-defined peaks in HPLC-EC. None of these peaks was observed when cells were irradiated without MC540 or when dye/light-treated samples were reduced with triphenylphosphine prior to analysis. Three peaks of relatively low retention time (< 12 min) were assigned to the following species by virtue of comigration with authentic standards: 3 beta-hydroxy-5 alpha-cholest-6-ene-5-hydroperoxide (5 alpha-OOH), 3 beta-hydroxycholest-4-ene-6 beta-hydroperoxide (6 beta-OOH), and 3 beta-hydroxycholest-5-ene-7 alpha/7 beta-hydroperoxide (7 alpha/7 beta-OOH). Formation of 5 alpha-OOH and 6 beta-OOH (single oxygen adducts) was confirmed by subjecting [14C]cholesterol-labeled cells to relatively high levels of photooxidation and analyzing extracted lipids by HPLC with radiochemical detection. Material represented in a major peak at 18-22 min on HPLC-EC was isolated in relatively large amounts by semipreparative HPLC and shown to contain phospholipid hydroperoxides (predominantly phosphatidylcholine species, PCOOH) according to the following criteria: (i) decay of 18-22 min peak during Ca2+/phospholipase A2 treatment, with reciprocal appearance of fatty acid hydroperoxides; (ii) reduction of peroxide during treatment with reduced glutathione and phospholipid hydroperoxide glutathione peroxidase, but not glutathione peroxidase; and (iii) comigration with PCOOH standards in thin-layer chromatography. HPLC-EC analysis revealed quantifiable amounts of PCOOH and ChOOH at a light fluence that clonally inactivated < 10% of the cells, which allows for the possibility that photoperoxidative damage plays a causal role in cell killing.

Analysis of Cholesterol and Phospholipid Hydroperoxides by High-Performance Liquid Chromatography with Mercury Drop Electrochemical Detection

High-performance liquid chromatography (HPLC) with reductive mode electrochemical detection on a mercury drop has been employed for the separation and determination of lipid hydroperoxides. Under the conditions used, baseline separation is achieved for three cholesterol hydroperoxide (ChOOH) standards, not only from one another, but also from two different phosphatidylcholine hydroperoxide (PCOOH) standards. Applying this method to a test system, photodynamically treated murine leukemia cells, we have identified and quantified a major family of overlapping PCOOHs and three ChOOHs, two of which are characteristic singlet oxygen adducts. In a typical separation, the detection limit is <0.5 pmol for ChOOHs and <50 pmol for more slowly eluting PCOOHs. In this respect, mercury drop detection outperforms all previously described electrochemical detection methods for lipid hydroperoxides and compares favorably with other HPLC-based approaches. However, in terms of equipment cost, relative simplicity of operation, and fewer potential artifacts, this method has a clear advantage over all other existing high-sensitivity methods.

Forensic Applications of the Determination of Benzodiazepines in Blood Samples by Microcolumn Cleanup and High-Performance Liquid Chromatography with Reductive Mode Electrochemical Detection

Recently described microcolumn cleanup and high-performance liquid chromatography with electrochemical reductive mode detection have facilitated the determination of benzodiazepines in contaminated and degraded blood samples. Examples from an extensive application of the procedure to British forensic science casework are given here.

Determination of menadione in an animal feed using supercritical fluid extraction and HPLC with electrochemical detector.

Menadione (vitamin K3) is extracted from spiked rat chow using supercritical fluid carbon dioxide at 8000 psi and 60 degrees C. Quantitative extraction requires only 20 min. The extraction does not suffer from the problems associated with conventional solvent extraction of lipophilic materials from animal feeds. Menadione is determined in the extract, which does not require further cleanup, using reversed-phase high-performance liquid chromatography (HPLC) with reductive mode electrochemical detection at a silver electrode at -0.75 V vs. calomel. The minimum detectable quantity by the detector is 125 pg of menadione, and the response is linear over at least 4 orders of magnitude; however, the minimum quantity extractable is about 20 micrograms/g of feed. Repetitive extracts of a spiked feed sample over a five-day period show an average recovery of 90.5% with a relative standard deviation of 2.2% at the 1 mg/g level.

Determination of Vitamin K1 in Powdered Infant Formulas, Using Supercritical Fluid Extraction and Liquid Chromatography with Electrochemical Detection

Vitamin K1 (phylloquinone) is extracted from commercial soy protein-based and milk-based powdered infant formulas by using supercritical fluid extraction with CO2 at 8000 psi and 60 degrees C. Quantitative extraction requires only 15 min, and does not suffer from the problems associated with conventional solvent extraction of lipophilic materials from media such as formulas. Vitamin K1 is determined in the extracts by using reverse-phase liquid chromatography (LC) with reductive mode electrochemical detection at a silver electrode polarized at -1.1 V vs SCE. LC run time is 9 min. The minimum detectable quantity is 80 pg, and response is linear over at least 5 orders of magnitude. Recovery of vitamin K1 from a milk-based powdered formula was 95.6% with RSD of 7.4%, and from a soy protein-based product, 94.4% recovery with RSD of 6.5%.

Detection and differentiation of nitrocellulose traces of forensic science interest with reductive mode electrochemical detection at a pendent mercury drop electrode coupled with size-exclusion chromatography

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTDetection and differentiation of nitrocellulose traces of forensic science interest with reductive mode electrochemical detection at a pendent mercury drop electrode coupled with size-exclusion chromatographyJ. B. F. LloydCite this: Anal. Chem. 1984, 56, 11, 1907–1912Publication Date (Print):August 1, 1984Publication History Published online1 May 2002Published inissue 1 August 1984https://doi.org/10.1021/ac00275a033RIGHTS & PERMISSIONSArticle Views151Altmetric-Citations19LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (784 KB) Get e-Alerts Get e-Alerts

Determination of misonidazole and desmethylmisonidazole in plasma by high-performance liquid chromatography with reductive electrochemical detection

A reversed-phase high-performance liquid chromatographic method with reductive mode electrochemical detection was developed for the determination of misonidazole and desmethylmisonidazole in plasma. A thin-layer amperometric detector with glassy carbon working electrode was used to detect the nitroimidazoles at a potential of -0.60 V. The calibration curves were linear. The within-day and day-to-day coefficients of variation were below 3% for plasma misonidazole concentrations of 6-60 mg/1 and 1-15 mg/1 for desmethylmisonidazole. Electrochemical detection limits were between 2 and 4 pg, which is about 10-20 times lower than that obtained by detection at 323 nm. Limits of quantitation of the nitroimidazoles in plasma were in the order of 1-2 μg/l. Under the described conditions other nitroimidazoles and nitro compounds can also be detected with ultimate sensitivity by reductive mode electrochemical detection.

Deoxygenation of small samples for reductive mode electrochemical detection in high-performance liquid chromatography

Description de cette technique de desoxygenation ne necessitant qu'une seringue hypodermique