Integrated Pulsed Amperometric Detection Research Articles

Determination of sulfur‐containing antibiotics using high‐performance liquid chromatography with integrated pulsed amperometric detection

AbstractSulfur‐containing antibiotics (e.g., ampicillin, penicillin, lincomycin, cephalosporin, and sulfanilamide) are a general class of compounds that have antibacterial properties. Some of these antibiotics lack a strong chromophore, and consequently are poorly detected using absorbance detection. The presence of oxidizable sulfur within an antibiotic’s molecular structure allows its direct detection by pulsed amperometry. Pulsed amperometry is an electrochemical detection method that uses a pulsed potential‐time waveform at a noble metal electrode surface to electrocatalytically oxidize analytes in proportion to concentration. The current for sulfur compounds on a gold (Au) electrode is caused by the desorption of the analyte previously adsorbed at the oxide‐free Au surface. Current integration in conjunction with rapidly changed voltage potentials is termed integrated pulsed amperometric detection (IPAD). Recent publications using high‐performance liquid chromatography (HPLC) with IPAD have demonstrated the capability to measure sulfur antibiotics. Our investigations of HPLC‐IPAD showed poor long‐term peak area reproducibility for these drugs. In this article, we present a new IPAD waveform that we demonstrate has good long‐term (>2 months) peak area reproducibility (4–5% relative s.d.). We show that a broad spectrum of sulfur‐antibiotic chemical structures can be detected, and we compare IPAD to absorbance detection. For nonchromophoric sulfur‐containing antibiotics, IPAD showed greater sensitivity than absorbance detection. The lower limits of detection, linearities, and peak area precision are compared for both IPAD and absorbance detection methods using seven antibiotics representing a diversity of chemical structures, including one nonsulfur‐containing antibiotic. The stabilities of cephradine and cephapirin were monitored using both IPAD and absorbance detections. Cephradine showed high chemical stability in aqueous ambient conditions at 10 μg/ml, whereas cephapirin showed a decomposition rate of 10% per day by both detection techniques. Some decomposition products could only be detected by IPAD. High recoveries were demonstrated for two antibiotics (sulfamethoxazole and trimethoprim) in a pharmaceutical tablet formulation. For the same formulation, the feasibility of conducting a dissolution study was also demonstrated. IPAD is a good primary detection method to complement or replace absorbance detection for nonchromophoric sulfur‐containing antibiotics and their sulfur‐containing impurities and decomposition products. HPLC‐IPAD is also a good confirming technique for sulfur antibiotics with chromophoric properties, and their impurities and decomposition products. Drug Dev. Res. 53:268–280, 2001. © 2001 Wiley‐Liss, Inc.

Integrated pulsed amperometric detection of glufosinate, bialaphos and glyphosate at gold electrodes in anion-exchange chromatography

A rapid and practical method for direct detection of the herbicides (glufosinate, bialaphos and glyphosate) in anion-exchange chromatography has been developed with integrated pulsed amperometric detection (IPAD). The electrochemical behavior of these herbicides showed catalytic currents based on the oxidation of amines in their structures. Waveform in IPAD was similar to that for amino acids, which exhibited adsorption/desorption catalytic features at gold electrode surface in alkaline solution. Under optimized conditions, detection limits (signal-to-noise ratio of 3) for glufosinate, bialaphos and glyphosate were 20, 65 and 50 ng ml −1, respectively, with correlation coefficients of 0.995, 0.997 and 0.996 over concentration ranges of 0.1–45, 0.3–32 and 0.1–50 μg ml −1, respectively. The relative standard deviations ( n=5) were 1.7–3.0%. The present method was successfully applied to the determination of glyphosate in urine and serum.

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.

Improved ion chromatography–integrated pulsed amperometric detection method for the evaluation of biogenic amines in food of vegetable or animal origin and in fermented foods

An improved method for the simultaneous determination of underivatized biogenic amines, cadaverine, putrescine, spermidine, histamine, tyramine and some amino acids precursors, histidine and tyrosine, in food products, based on ion-exchange chromatography (IC) with integrated pulsed amperometric detection (IPAD) has been developed. The method was successfully used for the analysis of biogenic amines and amino acids in food both of vegetable (kiwi, Actinidia chinensis) and animal origin, (fish, pilchard), as well as in fermented foods, such as cheese (Emmenthal) and dry sausages (salami). The method was also successfully used to study the changes in biogenic amines during the ripening of dry fermented sausages (salami). The analytes were extracted from foods with perchloric acid and the extracts were purified by liquid–liquid partition using n-hexane. Determination of biogenic amines was performed through cation-exchange chromatography with isocratic elution and IPAD. The detection limits for the analytes under investigation were found to range from 1.25 to 2.50 ng, at a signal-to-noise ratio of 3:1. Average recoveries ranged from 85.5 to 97.4% and R.S.D. values ranged from 3.4 to 8.8. The proposed method offers a number of advantages over our previous IPAD method, such as the application to a larger number of analytes and matrices, a simpler extraction procedure and clean-up, isocratic elution using low acid and base concentrations, an improved chromatographic separation and a lower detection limit.

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.

Pulsed electrochemical detection of thiocompounds following microchromatographic separations

Pulsed electrochemical detection (PED) following microchromatographic separations has been applied to the determination of thiocompounds (e.g., methionine, cystamine, glutathione, glutathione disulfide) under typical reversed-phase conditions. Both reduced and oxidized thiol moieties are detected with high selectivity and sensitivity without the need of derivatization. The anodic responses of model thiol redox couples are shown to be based upon oxide-catalyzed anodic oxygen transfer reactions at noble metal electrodes. The mechanism of detection and optimization of PED waveform parameters are studied using cyclic and pulsed voltammetry at rotated disk electrodes. 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 (background) currents, which accompany the Faradaic signal of the analyte. IPAD results in more stable baselines, eliminates oxide-induced artifacts, and yields lower limits of detection. The higher frequency waveforms (1 Hz) needed to define the narrow peaks of microchromatographic separations and the fast scan rates (1000 mV s −1) of the IPAD waveform require the use of microelectrodes (≤ 1 mm), which exhibit reduced capacitance effects. Mass detection limits for thiocompounds are typically 0.2–3 pmole for microbore chromatography and 0.1-0.3 pmole for capillary liquid chromatography. The high selectivity of PED for thiocompounds reduces sample preparation and produces simpler chromatograms of complex mixtures, such as liver extracts.