Reductive Electrochemical Detection Research Articles

Comparison of HPLC with electrochemical detection and LC–MS/MS for the separation and validation of artesunate and dihydroartemisinin in animal and human plasma

High-performance liquid chromatography with reductive electrochemical detection (HPLC-ECD) method has been used for assaying artemisinins since 1985. Although the methods have been remarkably improved, tandem mass spectrometry (LC–MS/MS) systems with significant advantages have gradually replaced HPLC-ECD to analyze artesunate and dihydroartemisinin in plasma. In the present study, the two methods were evaluated for linearity, quantitation limits, selectivity, precision, and accuracy. The HPLC-ECD performed well in terms of various validation parameters, and showed a good agreement with the LC–MS/MS when calibrated in plasma. However, the major benefit of LC–MS/MS is that it requires only one-tenth the plasma volume needed by HPLC-ECD assay.

COMPARISON OF THE BIOEQUIVALENCE OF THREE ORAL FORMULATIONS OF DIHYDROARTEMISININ BASED ON EX VIVO BLOOD SCHIZONTOCIDAL ACTIVITIES AGAINST PLASMODIUM FALCIPARUM

Sera collected at various time intervals from healthy Thai male subjects after the administration of the three oral formulations of dihydroartemisinin (Cotecxin) manufactured in the People's Republic of China, a formulation manufactured by Arenco n.v. Pharmaceutica in Belgium, and a formulation manufactured by the Faculty of Pharmacy of Mahidol University in Thailand) were investigated for their ex vivo blood schizontocidal activities against the K1 strain of Plasmodium falciparum. Blood schizontocidal activities of sera were evaluated using the maximum inhibitory dilution as a parameter. Sera obtained following the administration of the three formulations of dihydroartemisinin showed significantly distinct degree of ex vivo antimalarial activities. The differences may reflect the bioinequivalence between these three formulations of dihydroartemisinin. The ex vivo blood schizontocidal activity profiles generally coincided with plasma concentration-time profiles. Thus, the ex vivo model might be the useful tool for evaluating and comparing the bioequivalence of the interesting drugs especially where high-performance liquid chromatography with reductive electrochemical detection for drug analysis is not available. The effect of inoculum size of P. falciparum was shown in the ex vivo model as presented in the in vitro sensitivity test. To determine the effect of the inoculum size on the drug activity, the ex vivo model might be superior to the in vitro model since the pharmacokinetic profiles can be considered.

High performance liquid chromatography in pharmaceutical analyses

High performance liquid chromatography in pharmaceutical analyses

Determination of methyldibromoglutaronitrile in cosmetic products by high-performance liquid chromatography with electrochemical detection: Method validation

An increased frequency of contact allergy to methyldibromoglutaronitrile (MDBGN), a commonly used preservative in cosmetics and other consumer products, has been reported in recent years. A high-performance liquid chromatography (HPLC) method for the determination of MDBGN in cosmetic products has been validated in the present study. The identification is performed by reductive electrochemical detection of the bromine present in the molecule. The method is suitable for compliance testing of cosmetic products as well as for the research to support clinical and epidemiological studies.

Determination of oxygen permeability/transmissibility and storage of contact lenses using HPLC with reductive electrochemical detection in combination with a specifically designed sampling unit.

A new analytical technique for the determination of oxygen permeability/transmissibility of contact lenses is presented in this paper. The method is based on high performance liquid chromatography (HPLC) with reductive electrochemical detection at -750 mV (vs Ag/AgCl) in combination with a patented sampling chamber that was designed especially for the purpose to determine oxygen at nanomolar levels. Compared to conventional method, the new technique exhibits higher sensitivity, selectivity, and versatility. The method permits the selective determination of oxygen permeability (Dk/L)/transmissibility (Dk) of soft as well as rigid contact lenses with good agreement with the Dk/L (Dk) values reported in the literature. Precision was determined by repeated measurements and yielded relative standard deviations of 3-8% for hydrophile lenses and 5-13% for rigid contact lenses. Because of the extraordinarily high sensitivity of the chromatographic oxygen sensor for the first time, the capability of hydrogel lenses to store oxygen could also be directly monitored.

The Effects of Nitric Oxide on the Oxidations ofl-Dopa and Dopamine Mediated by Tyrosinase and Peroxidase

The effects of nitric oxide (NO) on both tyrosinase/O(2)- and horseradish peroxidase/H(2)O(2)-mediated oxidations of dopamine and its o-dihydric phenol precursor l-dopa were compared with autoxidative processes and quantitatively assessed by oxidative and reductive electrochemical detection systems. In peroxidase/H(2)O(2)/NO-catalyzed reactions, significantly more substrate was oxidized than in the corresponding control incubations lacking NO. In tyrosinase/O(2)/NO-promoted reactions the total amounts of l-dopa and dopamine oxidized were significantly less than the amounts of the substrates oxidized by enzyme alone. These data indicate that the activity of the heme protein peroxidase was enhanced by NO, whereas tyrosinase, a copper-containing monoxygenase, was inhibited. The NO-mediated reduction of tyrosinase/O(2) activity may be attributed to the formation of an inhibitory copper.nitrosyl complex. An oxidized nitrodopamine derivative, considered to be either the quinone or semiquinone of 6-nitrosodopamine, was generated in peroxidase/H(2)O(2)/NO-mediated reactions with dopamine along with two oxidized melanin precursors, dopamine quinone and dopaminechrome. No corresponding nitroso compound was formed in reactions involving l-dopa or in any of the tyrosinase-mediated reactions. The formation of such a noncyclized nitrosodopamine represents an important alternative pathway in catecholamine metabolism, one that by-passes the formation of cytoprotective indole precursors of melanin. The results of this investigation suggest that cellular integrity and function can be adversely affected by NO-promoted oxidations of dopamine and other catechols, reactions that not only accelerate their conversion to reactive quinones but also form potentially cytotoxic noncyclized nitroso derivatives. Reduced levels of dopamine in the brain through NO-enhanced oxidation of the catecholamine will almost certainly be manifested by diminished levels of the dopamine-derived brain pigment neuromelanin.

Selective and sensitive assay for the determination of benzodiazepines by high-performance liquid chromatography with simultaneous ultraviolet and reductive electrochemical detection at the hanging mercury drop electrode

An isocratic chromatographic method for the simultaneous determination of 10 benzodiazepines is presented. The selectivity of the assay was optimized by variation of stationary phase, temperature, as well as ionic strength, composition and pH of the mobile phase and the dependence of the detector response on the applied potential was investigated. The best results with respect to resolution at moderate retention times were obtained with a mixture of 0.02 mol/l phosphate buffer (pH 6) and acetonitrile in a volume ratio of 55:45 (v/v) on a LiChrospher-100 RP-8ec column (150×4.6 mm I.D.). Considerable improvement of selectivity was achieved if the column temperature was kept constant at 12°C. Two detection modes were applied, UV detection at 250 nm, inserted upstream to the electrochemical detector, and reductive electrochemical detection at the hanging mercury drop electrode at −1.4 V (vs. Ag/AgCl), which proved to be especially sensitive in case of nitrophenyl-containing benzodiazepine species. The elaborated assay showed to be linear up to at least 2 mg/l for each compound. Detection limits generally were in the range of 6.5–123 ng/ml (130 pg–2.46 ng on-column, using a 20-μl loop) with relative standard deviations between 1.1 and 8.6% depending on the actual benzodiazepine investigated.

Separation of negatively charged isomeric quinones in acidic solution by capillary electrophoresis with reductive electrochemical detection

A capillary electrophoresis method with reductive electrochemical detection was developed for the separation of the novel enzyme cofactor pyrroloquinoline quinone (PQQ) and three isomeric analogues. Tuning the reduction potential of the o-quinone moiety to a value more positive than the reduction potential for oxygen was accomplished by adjusting the capillary buffer to pH 2, thus eliminating the need for deoxygenation. To counter the suppression of the electroosmotic flow (EOF) at pH 2, a negative separation voltage of –22.5 V was applied to a 25 µm id capillary resulting in migration of the anionic isomers toward the electrochemical detector. Fast and efficient separation was achieved in 0.15 mM phosphate buffer at pH 2. A mass detection limit for PQQ of 2 fmol was obtained with end-column detection. This approach may find utility for the separation and sensitive detection of a wide range of reducible quinones.

Simple high-performance liquid chromatographic method with electrochemical detection for the simultaneous determination of artesunate and dihydroartemisinin in biological fluids

A simple, rapid, sensitive, selective and reproducible high-performance liquid chromatographic method with reductive electrochemical detection is described for the simultaneous quantification of artesunate (ARS) and dihydroartemisinin (DHA) in plasma. The procedure involved the extraction of ARS, DHA and the internal standard (artemisinin, ARN) with a mixture of dichloromethane and tert.-methyl butyl ether (8:2, v/v). Chromatographic separation consisted of the mobile phase (acetonitrile–water containing 0.1 M acetic acid, pH 4.8; 45:55, v/v) running through the column (Nova-Pak C 18, 150 cm×3.9 mm I.D., 5 μm particle size). The retention times of α-DHA, β-DHA, ARS and ARN were 2.9, 4.2, 4.5 and 6.0 min, respectively. The average recoveries of ARS, α-DHA and ARN in the concentration range of 10–800 ng/ml were 81.9, 88.2, 101.1 and 84.3%, respectively. The coefficients of variation (precision and repeatability) were below 10% for all three compounds at concentrations of 50, 200, 400 and 800 ng/ml, and below 20% at a concentration of 10 ng/ml. The limits of quantification for both ARS and α-DHA in spiked plasma samples were 5 and 3 ng/ml, respectively. The method was found to be suitable for application to pharmacokinetic studies of both ARS and DHA.

Plasma levels of artesunate and dihydroartemisinin in children with Plasmodium falciparum malaria in Gabon after administration of 50-milligram artesunate suppositories.

A thermostable suppository of artesunate (artesunic acid) has been developed. In Gabon, 12 children with Plasmodium falciparum malaria received two administrations of this suppository in a 4-hr interval. Parasitemia and fever were then measured and the plasma levels of artesunate and its active metabolite, dihydroartemisinin, were determined by means of a reversed phase high-pressure liquid chromatography method using reductive electrochemical detection. Substantial parasite clearance (97-100%) was noted 24 hr after the beginning of the treatment and body temperature had returned to normal. Absorption, metabolism, and elimination of artesunate were rapid. Mean values of maximum plasma levels (Cmax) and maximum concentration peak times (tmax) were evaluated. The Cmax of dihydroartemisinin (0.18 +/- 0.10 microg/ml [mean +/- SE]) was higher than the Cmax of artesunate (0.09 +/- 0.04 microg/ml) and the tmax of dihydroartemisinin (1.13 +/- 0.58 hr) was higher than the tmax of artesunate (0.58 +/- 0.19 hr). Plasma levels 30 min after the second suppository administration were not consistently higher than those found 30 min after the first administration.

Validation of an improved reversed-phase high-performance liquid chromatography assay with reductive electrochemical detection for the determination of artemisinin derivatives in man.

For the determination of artemisinin (ART) and analogs, a reversed-phase high-performance liquid chromatography method using reductive electrochemical detection (ED) was set up with some important modifications as compared to previously published assays. A different technique of deoxygenating resulted in a factor 2-3 lower background current. A Spectroflow 400 liquid chromatograph in combination with a Triathlon autoinjector coupled to a Decade electrochemical detector was used. The detector was operated in the reductive mode as a closed system under chromatography grade helium to exclude any access of oxygen. The Decade has a glassy carbon electrode and a reference Ag/AgCl electrode. Infrequent electropolishing was required implicating a very stable system. By increasing acetonitril or lowering the pH of the mobile phase, the various derivatives could be determined in the same chromatogram. The assay was validated using artemether (ATM) and dihydroartemisinin (DHA) as test substances. In the concentration range seen in people after usual doses (5 to 220 ng/ml), the assay performs with adequate accuracy and precision. The interassay and intraassay precision are < 6% for ATM. For DHA, the interassay and intraassay precision are < 9%. The accuracy expressed as the deviation from the expected concentration varies from -1% to +4.5% for the intraassay ATM-determinations and from +1% to +6.3% for the interassay measurements. For DHA, the accuracy is somewhat less, varying from -0.3% to -9.5% for the intraassay measurements and -0.6% to +2.6% for the interassay measurements. The reproducibility of the assay, measured over a time period of 3 months, is good for ATM and DHA with an interassay precision of < 18% in 70 repetitive samples and an accuracy varying from -0.6% to +7.6%. In a cross-check with two other reference laboratories who used comparable methods of determination, a strong correlation (correlation coefficient > 0.98) was achieved. The method was applied in a study in which artemether was administered orally to healthy white subjects. We consider high-performance liquid chromatography with electrochemical detection an accurate and precise method for quantitative determination of artemisinin derivatives in pharmacokinetic studies.

On-line deoxygenation for reductive electrochemical detection of artemisinin and dihydroartemisinin in liquid chromatography

A straightforward method for the deoxygenation of mobile phases and samples required for reductive detection in liquid chromatography is described. The method, which is based on the use of a commercially available on-line deoxygenator placed between the column and the detector, eliminates the problems associated with separate deoxygenations of the mobile phase and samples and restrictions imposed by work in an oxygen-free environment. The method is compatible with determinations of artemisinin and its main metabolite, dihydroartemisinin, and also artelinate, in liquid samples. With the deoxygenator, efficiencies of the order of 4000–10 000 theoretical plates were obtained for artemisinin using 3.0, 4.0 and 4.6 mm diameter columns. The extra-column band broadening due to the deoxygenator was not found to be a limiting factor. Amperometric detection with a gold electrode gave rise to lower background currents and better peak current reproducibilities than that with glassy carbon and gold amalgam electrodes. The relative standard deviations in the peak current for 10 injections of 100 pmol of artemisinin, dihydroartemisinin and artelinate were 3.0, 1.4 and 3.3%, respectively. The peak currents for the compounds depended linearly on the amount injected in the range 10–200 pmol and the detection limits for artemisinin and dihydroartemisinin were calculated to be 2 and 3 pmol, respectively.

Supercritical Fluid Carbon Dioxide Extraction and Liquid Chromatographic Separation with Electrochemical Detection of Methylmercury from Biological Samples

Using the coupled methods presented in this paper, methylmercury can be accurately and rapidly extracted from biological samples by modified supercritical fluid carbon dioxide and quantitated using liquid chromatography with reductive electrochemical detection. Supercritical fluid carbon dioxide modified with methanol effectively extracts underivatized methylmercury from certified reference materials Dorm-1 (dogfish muscle) and Dolt-2 (dogfish liver). Calcium chloride and water, with a ratio of 5:2 (by weight), provide the acid environment required for extracting methylmercury from sample matrices. Methylmercury chloride is separated from other organomercury chloride compounds using HPLC. The acidic eluent, containing 0.06 mol L–1 NaCl, insures the presence of methylmercury chloride and facilitates the reduction of mercury on a glassy carbon electrode. If dual glassy carbon electrodes are used, a positive peak is observed at –0.65 to –0.70 V and a negative peak is observed at –0.90V with the organomercury compounds that were tested. The practical detection limit for methylmercury is 5 × 10–8 mol L–1 (1 × 10–12 mol injected) when a 20 μL injection loop is used.

Dual-electrode detection for capillary electrophoresis/electrochemistry.

The extremely low sample volumes required for capillary electrophoresis and the high sensitivity and selectivity of electrochemical detection make capillary electrophoresis/electrochemistry (CEEC) a very useful method for bioanalysis. In this paper, two types of dual-electrode detectors for CEEC are described. The first employs a ring-disk microelectrode placed in a wall-jet configuration and is used for the selective detection of substances undergoing chemically reversible oxidations. Collection efficiencies obtained for catecholamines with this configuration were between 25 and 35%. The second electrode design consists of two adjacent carbon fibers embedded in an epoxy matrix and is analogous to the parallel dual-electrode configuration used in liquid chromatography/electrochemistry. This configuration can be used to confirm peak identity and purity by operating the electrodes at two different potentials. Alternatively, it is possible to perform simultaneous oxidative and reductive electrochemical detection.

Reductive Electrochemical Detection for Capillary Electrophoresis

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTReductive Electrochemical Detection for Capillary ElectrophoresisMichael A. Malone, Paul L. Weber, Malcolm R. Smyth, and Susan M. LunteCite this: Anal. Chem. 1994, 66, 21, 3782–3787Publication Date (Print):November 1, 1994Publication History Published online1 May 2002Published inissue 1 November 1994https://doi.org/10.1021/ac00093a039RIGHTS & PERMISSIONSArticle Views104Altmetric-Citations18LEARN 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 (642 KB) Get e-Alerts Get e-Alerts

Measurement of artemisinin and its derivatives in biological fluids

The development of selective analytical methods for the determination of artemisinin and its analogues and metabolites in biological fluids poses challenging problems. All are thermally labile, lack ultraviolet (u.v.) absorbent or fluorescent chromophores and do not possess functional groups for derivatization. Two chemical approaches have been used; acid (or alkali) catalysed decomposition to u.v. absorbing compounds followed by high-performance liquid chromatography (HPLC) of the decomposition products, and HPLC with reductive electrochemical detection. Each has its difficulties. The first, although sensitive, may lack specificity if drug metabolites are also converted to identical products, but a recently developed method surmounts this problem. The second, while providing good sensitivity and specificity, requires rigorous deoxygenation of the sample and mobile phase and an electrochemical detector, which is expensive, and may prove difficult to operate under field conditions. As an alternative to a chemical method, a bioassay may be suitable for plasma level monitoring in such circumstances. However its lack of selectivity means it is of questionable value in the determination of the pharmacokinetic parameters for individual drug-related species.

Determination of oxygenated and nitro-substituted polycyclic aromatic hydrocarbons by HPLC and electrochemical detection

A method is described for the measurement of oxy- and nitro-substituted polynuclear aromatic hydrocarbons (OXY- and NITRO-PAHs) using high performance liquid chromatography with reductive electrochemical detection. A series of reference OXY- and NITRO-PAHs were separated in a reversed-phase column, conditions for electrochemical detection were established and the compounds were quantified with a sensitivity of 3-0.3 ng injected. Samples of air particulate matter were collected for the analysis of these PAH derivatives and the presence of 9,10-anthraquinone, benz[a]anthracen-7, 12-dione at levels of between 75 and 398 pg/m 3 in the air samples has been confirmed.

Reductive electrochemical detection in liquid chromatography with a zinc amalgam scrubber column

The dissolved oxygen in an acidic mobile phase can be reduced and eliminated by a scrubber column packed with zinc amalgam particles. The zinc amalgam scrubber column is mounted between the pump and the injection device and enables sensitive amperometric detection at potentials down to −0.65 V vs. Ag/AgCl. The high overvoltage of hydrogen on mercury allows the use of acidic mobile phases without problems with evolution of hydrogen gas. The accessible potential range is limited by the reduction of Zn 2+ dissolved from the scrubber column. The possibilities for trace amount determinations of nitrocompounds are demonstrated in the detection of trinitrophenyl derivatives of amino acids from reaction with trinitrobenzenesulphonic acid. The detection was focused on γ-aminobutyric acid, and a detection limit of 0.1 pmol at −0.6 V vs. Ag/AgCl was obtained.

Development of a sensor for the detection of nitrite using a glassy carbon electrode modified with the electrocatalyst [Os(bipy) 2(PVP) 10Cl]Cl

The electrocatalytic polymer [Os(bipy) 2(PVP) 10Cl]Cl, where bipy = 2,2′-bipyridyl and PVP = poly(4-vinylpyridine), was used to modify glassy carbon for use in planar flow cells and conventional electrochemical cells for the reductive electrochemical detection of nitrite. The electrocatalyst accelerates a slow electrochemical reduction of nitrite via a two-step reduction process resulting in a 70-fold increase in response compared with the unmodified electrode. The detector response was evaluated with regard to flow-rate, applied potential, electrolyte pH and surface coverage of the polymer film. A mechanism for the two-step reduction process is proposed. The detector response appears to be limited by the rate constant of the mediated electrochemical reaction. A linear response range extending from 5 × 10 −6 to 1 × 10 −2 M nitrite was obtained, with a detection limit of 0.23 ng ml −1 for the planar flow cell and 0.23 ng ml −1 for a conventional electrochemical cell. The performance of the sensor was assessed with respect to stability, selectivity and interference. The sensor was applied to the determination of nitrite in saliva.

Qualitative and quantitative high performance liquid chromatographic analysis of monoamine neurotransmitters and metabolites in cerebrospinal fluid and brain tissue using reductive electrochemical detection

The application of reductive coulometric electrochemical detection for analysis of the monoamine neurotransmitters norepinephrine, dopamine, and serotonin and their common metabolites in brain and cerebrospinal fluid following separation by isocratic high performance liquid chromatography is described. The high sensitivity and screening capabilities of coulometric electrodes permits the accurate quantitation of as little as 3-5 pg of these compounds in tissue following a simple single step purification procedure. Moreover, comparison of peak height ratios obtained from analysis of authentic reference standards and tissue samples at selected multiple electrode potentials provides a straightforward means for qualitative evaluation of peak identification and purity during analysis of biological samples. The method is comparatively inexpensive and precise within and between day coefficients of variation for most compounds range from 2-5%. Thirty samples can be run in duplicate in a 24 h period.