Electrochemical methods liquid chromatography detection

Monitoring enzyme catalyzed reactions by voltammetry and amperometry is an extremely active area of bioelectrochemical interest. Whereas liquid chromatography provides selectivity, the use of enzymes to generate electroactive products provides specificity to electroanalytical techniques. In essence, enzymes are used as a derivatiz-ing agent to convert a nonelectroactive species into an electroactive species. Alternatively, electrochemistry has been used as a sensitive method to follow enzymatic reactions and to determine enzyme activity. Enzyme-linked immunoassays with electrochemical detection have been reported to provide even greater specificity and sensitivity than other enzyme linked electrochemical techniques. 

Imperato, A., and Di Chiara, G. Transtriatal dialysis coupled to reverse-phase high performance liquid chromatography with electrochemical detection A new method for the study of the in vivo release of endogenous dopamine and metabolites. J Neurosci A.966-911, 1984. 

Wang, J., Electrochemical detection for liquid chromatography, in HPLC Detection Newer Methods, Patonay, G., Ed.,VCH Publishers, New York, 1992, chap. 5. 

MTHF was initially measured by microbiological and radioisotope dilution assay [12, 13], and later by high-pressure liquid chromatography (HPLC) using electrochemical (EC), ultraviolet, or fluorescence detection [14-16]. Compared to other methods, EC detection is more sensitive. 

Electrochemical detection is better suited to the analysis of erythromycin and lincomycin. This method of detection has been applied for the determination of erythromycin A (139) and lincomycin (154) residues in salmon tissues. Liquid chromatography coupled with mass spectrometry is particularly suitable for confirmatory analysis of the nonvolatile macrolides and lincosamides. Typical applications of this technique are through thermospray mass spectrometry, which has been used to monitor pirlimycin in bovine milk and liver (141,142), and chemical ionization, which has been applied for identification of tilmicosin (151) in bovine muscle, and for identification of spiramycin, tylosin, tilmicosin, erythromycin, and josamycin residues in the same tissue (150). 

P Hayes, MR Smyth, I McMurrough. Comparison of electrochemical and ultraviolet detection methods in high-performance liquid chromatography for the determination of phenolic compounds commonly found in beers. Part 1. Optimization of operating parameters. Analyst 112 1197-1203, 1987. 

Acworth IN, Bogdanov MB, McCabe DR, Beal MF (1999) Estimation of hydroxyl free radical levels in vivo based on liquid chromatography with electrochemical detection. Methods Enzymol... 

Synaptic neurotransmission in brain occurs mostly by exocytic release of vesicles filled with chemical substances (neurotransmitters) at presynaptic terminals. Thus, neurotransmitter release can be detected and studied by measuring efflux of neurotransmitters from synapses by biochemical methods. Various methods have been successfully employed to achieve that, including direct measurements of glutamate release by high-performance liquid chromatography of fluorescent derivatives or by enzyme-based continuous fluorescence assay, measurements of radioactive efflux from nerve terminals preloaded with radioactive neurotransmitters, or detection of neuropeptides by RIA or ELISA. Biochemical detection, however, lacks the sensitivity and temporal resolution afforded by electrophysiological and electrochemical approaches. As a result, it is not possible to measure individual synaptic events and apply quantal analysis to verify the vesicular nature of neurotransmitter release. 

In conclusion, we have tried to present the principles of LCEC, describe the present applications that have been made, and survey the areas of potential utility by reviewing pertinent chemistry and related methods. It is our opinion that the success of LCEC in neurochemistry can carry over to pesticides owing not only to the advantages of electrochemical detection but also to the tremendous potential of its adjunct, liquid chromatography. 

Duan et al. reported the use of a rapid and simple method for the determination of acetylcholine and choline in mouse brain by high performance liquid chromatography, making use of an enzyme-loaded post column and an electrochemical detector [144]. Perchloric acid extracts of small brain tissue were injected onto the HPLC system with no prior clean-up procedure. Detection limits for both compounds were 1 pmol, and this method was successfully applied to the measurement of acetylcholine in discrete brain areas of the mouse. 

Mayer determined acetylcholine and choline by enzyme-mediated liquid chromatography with electrochemical detection [195]. The two compounds were separated by passing the eluted fractions through a post-column reactor containing immobilized Acetylcholineesterase and choline oxidase. In the presence of either compound, the dissolved oxygen was converted into hydrogen peroxide, which was detected amperometrically at a platinum electrode. This method was used to determine choline in rat brain homogenates. 

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