Electrochemical detectors: functional group analysis.

In addition to chromatographic resolution of enantiomeric compounds, electrochemical detection may be used to delineate structure. This information can be obtained via voltammetric measurement at various electrode materials. This paper focuses on isolated electrode arrays as used for the resolution of redox-active compounds in liquid chromatography. For polar aliphatic compounds, electrocatalytic detection at noble metals using pulsed voltammetric detection can be used to identify certain functional groups (e.g., aldehydes, alcohols, amines, and thiols). Electrochemical systems are also versatile, rugged, and highly amenable to miniaturization, which are all important factors for consideration for inclusion in extraterrestrial exploration vehicles.

Experimental determination and calculations of redox potential descriptors of compounds directed against retroviral zinc fingers: Implications for rational drug design.

A diverse set of electrophilic compounds that react with cysteine thiolates in retroviral nucleocapsid (NC) proteins and abolish virus infectivity has been identified. Although different in chemical composition, these compounds are all oxidizing agents that lead to the ejection of Zn(II) ions bound to conserved structural motifs (zinc fingers) present in retroviral NC proteins. The reactivity of a congeneric series of aromatic disulfides toward the NC protein of the human immunodeficiency virus type 1 (HIV-1), NCp7, has been characterized by HPLC separation of starting reagents from reaction products. We calculated the absolute redox potentials of these compounds in the gas phase and in aqueous solvent, using a density functional theory method and a continuum solvation model. Pulsed polarography experiments were performed and showed a direct correlation between calculated and experimentally determined redox propensities. A dependence between protein reactivity and redox potential for a specific compound was shown: Reaction with NCp7 did not take place below a threshold value of redox potential. This relationship permits the distinction between active and nonactive compounds targeted against NCp7, and provides a theoretical basis for a scale of reactivity with retroviral zinc fingers. Our results indicate that electrophilic agents with adequate thiophilicity to react with retroviral NC fingers can now be designed using known or calculated electrochemical properties. This may assist in the design of antiretroviral compounds with greater specificity for NC protein. Such electrophilic agents can be used in retrovirus inactivation with the intent of preparing a whole-killed virus vaccine formulation that exhibits unaffected surface antigenic properties.

Pulsed electrochemical detection of sulfur-containing antibiotics following high performance liquid chromatography.

Pulsed electrochemical detection (PED) following reversed-phase chromatography has been applied to the direct detection of sulfur-containing antibiotics, specifically, penicillins, cephalosporins, and lincomycin. The compounds are detected sensitively and selectively without the need for derivatization. Integrated pulsed amperometric detection (IPAD) yields limits of detection lower than UV detection for these compounds. Detection limits using an optimized IPAD waveform are typically 10 ppb or less. The high selectivity of PED for thiocompounds reduces sample preparation. This work is applied to the determination of penicillin and related analogues in various pharmaceutical formulations/preparations, including a chicken feed.

Assay for cephapirin and ampicillin in raw milk by high-performance liquid chromatography--integrated pulsed amperometric detection.

The FDA has issued guidelines governing the use of antibiotics in cattle and routinely tests for the presence of antibiotics in milk. Unfortunately, these compounds are often difficult to detect by direct methods because they often lack a chromophore or fluorophore. Integrated pulsed amperometric detection (IPAD) following reversed-phase liquid chromatography is well-suited for this analysis because it is selective, sensitive, and direct; i.e., derivatization is not required. This work involves the development of a simple, rapid assay for the determination of beta-lactam antibiotic residues in milk using HPLC-IPAD, specifically, ampicillin and cephapirin. Since the analyst studied here are detectable by UV detection, a comparison between IPAD and UV detection will be made. Sample preparation schemes that involve the extraction of antibiotics of interest from the milk matrix and subsequent cleanup are an important aspect of this project. These procedures will be discussed in detail. In addition, analytical figures of merit and IPAD wave form optimization will be addressed.

Pulsed electrochemical detection of thiols and disulfides following capillary electrophoresis.

Pulsed electrochemical detection (PED) following capillary electrophoresis (CE) has been applied to the direct detection of thiocompounds. Both reduced and oxidized thiol moieties are detected without the need of derivatization. Thiocompounds can be detected over a wide range of pH conditions (i.e., pH 0-14), and except for pH, their response is relatively unperturbed by buffer composition. Integrated pulsed amperometric detection (IPAD) results in more stable baselines, eliminates oxide-induced artifacts, and yields lower limits of detection than other PED waveforms. Mass detection limits using optimized IPAD waveforms are typically 2 pg (5 fmol) or less. The high selectivity of PED for thiocompounds reduces sample preparation and produces simpler electropherograms of complex samples containing these biologically significant compounds.

Pulsed electrochemical detection of nonchromophoric compounds following capillary electrophoresis.

Pulsed electrochemical detection (PED) following capillary electrophoresis (CE) has been applied to the direct detection of polar aliphatic compounds. These compounds typically have poor optical detection properties and are considered to be electroinactive under constant applied potentials. The voltammetric responses at microelectrodes of model compounds were studied under various buffers and pH conditions. The detection of unsubstituted carbohydrates requires highly alkaline conditions, whereas amine-containing compounds (e.g., glycopeptides, peptides, and amino acids) and thiocompounds can best be detected at mildly alkaline (i.e., pH 9.0) and mildly acidic (i.e., pH 5.5) conditions, respectively. In a comparative study of pulsed amperometric detection (PAD) and integrated pulsed amperometric detection (IPAD), IPAD is determined to be better suited to manage the large oxide-formation backgrounds, which accompany oxide-catalyzed detections of amine- and sulfur-containing compounds. IPAD results in more stable baselines, eliminates oxide-induced artifacts, and often yields lower limits of detection. Mass detection limits under optimal conditions are typically 10 fmole or less. This paper delineates PED waveform parameters for the optimal detection of underivatized nonchromophoric compounds of biological significance in various operating buffers and electrolytes.

Reverse iontophoresis: noninvasive glucose monitoring in vivo in humans.

PURPOSE: To demonstrate that "reverse iontophoresis" can be used to noninvasively obtain information about systemic glucose levels in vivo in humans. METHODS: The passage of current across the skin in vivo drives ions into the tissue, from the electrode chambers positioned on the skin surface, and simultaneously pulls ions from the body in the opposite direction. Because of the net negative charge on the skin, under normal conditions, the membrane is permselective to cations, and a potential gradient also results, therefore, in electroosmotic convection of solvent in the direction of counterion flow (i.e., from anode to cathode). Thus, it is also possible to enhance the transport of polar, yet uncharged, species using iontophoresis. In an earlier study, the in vitro extraction of glucose, by "reverse iontophoresis" was established, and extension of the approach to an in vivo model was indicated. The idea has therefore been further explored in vivo in humans. RESULTS: Using small, simple, prototypical electrode chambers, attached to the ventral forearm surface, direct current iontophoresis at 0.25 mA/cm2 for periods of up to 1 hour, and a sensitive analytical procedure to measure the quantities of glucose extracted, it has been shown that iontophoretic sampling of glucose is feasible. However, the shorter periods (15 minutes or less) of extraction considered yield results which are "contaminated" (it is believed) by glucose that is a product of lipid metabolism within the skin. While this material is expected to complicate the initial calibration of the approach, the problem is effectively resolved within one hour, by which time the glucose arriving in the electrode chambers on the skin surface is expected to directly reflect the subcutaneous tissue concentration. CONCLUSIONS: Based upon these initial observations, further investigation can now be directed towards optimization of electroosmotic flow and sampling time, improved reproducibility and the development of a practical assay methodology.

A comparison of pulsed amperometric detection and conductivity detection of underivatized amino-acids in liquid chromatography.

Pulsed amperometric detection (PAD) in tandem with conductivity detection (CD) has been applied to the direct detection of amino-acids by liquid chromatography. Although sensitive, PAD has a limited linear range of response. Sequential use of conductimetric detection and then PAD extends the dynamic range of amino-acid determination.

Comparison of pulsed coulometric detection and potential-sweep pulsed coulometric detection for underivatized amino acids in liquid chromatography.

Pulsed coulometric detection (PCD) and potential-sweep pulsed coulometric detection (PS-PCD) are applied to the direct detection of amino acids in protein hydrolyzates. The detection mechanisms are based upon surface-catalyzed oxidation of the amine functionalities activated by the transient formation of surface oxide on Au electrodes. PCD uses a triple-step potential waveform in which the integration of electrode current at a constant potential is followed by anodic and cathodic polarizations to clean and reactivate the electrode surface. PS-PCD incorporates a cyclic potential sweep into the triple-step waveform which proceeds through the formation and subsequent removal of the surface oxide with simultaneous current integration. Reactions catalyzed by the formation of the noble metal oxide can be monitored in PS-PCD with the automatic rejection of the surface oxide background. A significant decrease is obtained also in the fluctuation and drift in the base line resulting from gradient elution and variations of the electrode surface. PCD and PS-PCD following gradient elution chromatography are demonstrated to allow for the direct detection of 20 amino acids including secondary amino acids. Detection limits for lysine are ca. 220 ppb (11 ng, 75 pmol) by PCD and 60 ppb (3 ng, 19 pmol) by PS-PCD applied at gold electrodes.

Determination of iodide and thiosulfate by paired-ion, reversed-phase high-performance liquid chromatography with ultraviolet absorbance, electrochemical, and conductimetric detection.

Inorganic anions are typically determined by ion chromatography, ion-selective electrodes, or by "wet chemical" procedures. An alternative to these approaches is afforded by paired-ion, reversed-phase high-performance liquid chromatography (HPLC), and the application of this technique to the determination of iodide and thiosulfate is discussed. Each analyte can be determined using ultraviolet (UV) absorbance detection and oxidative, amperometric electrochemical detection (ED). Furthermore, in the case of iodide, an additional and quite selective ED scheme is available through the use of series-configured dual glassy carbon electrodes. The dual-series ED approach utilizes an upstream electrode which is maintained at an oxidative potential. This results in the formation of an electroactive species, which may be detected at a downstream electrode held at a reductive potential. Finally, it is demonstrated that non-suppressed conductimetric detection is also perfectly viable within the context of paired-ion, reversed-phase HPLC, provided the overall conductance of the mobile phase is not excessive.

Liquid chromatographic determination of sodium salinomycin in feeds, with post-column reaction.

A method for the detection and quantitation of sodium salinomycin in feeds by a liquid chromatographic, post-column reaction system is presented. Sodium salinomycin is leached from feed with hexane, isolated as a dried residue, and dissolved in mobile phase for analysis. The drug is separated from interfering substances on a silica LC column, combined with vanillin reagent in a mixing tee, reacted with vanillin at elevated temperature in a coil, and detected in the effluent stream at 527 nm. Response, as peak area, is linear with concentration. Feed components, other common veterinary drugs, and closely related compounds do not interfere. Average recovery and coefficient of variation (CV) values for liquid spiked feeds and supplements were 101 +/- 3.5% and 100 +/- 0.3%, respectively. Results for laboratory blends and commercial scale feeds were within 6% of intent with CV values ranging from 0.4 to 5.1%.