Amino acids electrochemical detection

Relative to the UV-spectroscopic and/or fluorometric determination of derivatized amino acids, electrochemical detection of amino acids seems to be more complex and problematic. Furthermore, electrochemical detection has not gained sufficient popularity to warrant an extensive examination here. For those readers specifically interested in electrochemical detection of amino acids, an excellent review article has been written by Dou et al. (143). 

To analyze potential interference of amino acids in monosaccharide analysis, each of the 20 amino acids (10 /xg each, each injected separately) was subjected to the chromatography conditions used for separating, detecting, and quantifying monosaccharides. In addition to PAD detection, we monitored UV detection at 215 nm after the electrochemical detector to verify amino acid electrochemical detection. Ten amino acids (R, K, Q, V, N, A, I, L, T and C) eluted between 2 and 25 min and were both PAD and UV active. Of these ten, two amino acids could potentially interfere with monosaccharide analysis. Glutamine was found to elute as a shoulder on mannose. However, acid hydrolysis conditions used to release monosaccharides from glycoproteins likely would oxidize glutamine. 

Only a few amino acids are detected by the UV or visible spectrophotometers, fluorometers, or electrochemical detectors that are routinely used with HPLC analyzers. Consequently, amino acids typically are postcolumn derivatized for analysis by HPLC. The most widely used reagent for this purpose is ninhydrin. A number of colored products are formed, but the major one is presumed to result from deamination and condensation as follows ... 

Dennany L, O Reilly EJ, Keyes TE, Eorster RJ (2006) Electrochemiluminescent monolayers on metal oxide electrodes Detection of amino acids. Electrochem Comm 8(10) 1588-1594. doi 10.1016/j. elecom.2006.07.022... 

Argine and valine can be detected electrochemically with a detection limit for argine of ca. 100 ng (signal noise of 4 1). The detector is rather less sensitive towards valine. The conditions used to chromatograph and detect these amino acids were a Lichrosorb RP-8 column eluted with O.IM aqueous phosphate buffer (pH 6.7) with an applied potential of +1.62 V vs Ag/AgCl. The literature contains reports [22,23] of other amino acids being detected electrochemically and this method may be generally applicable for amino acids. 

Because LCEC had its initial impact in neurochemical analysis, it is not, surprising that many of the early enzyme-linked electrochemical methods are of neurologically important enzymes. Many of the enzymes involved in catecholamine metabolism have been determined by electrochemical means. Phenylalanine hydroxylase activity has been determined by el trochemicaUy monitoring the conversion of tetrahydro-biopterin to dihydrobiopterin Another monooxygenase, tyrosine hydroxylase, has been determined by detecting the DOPA produced by the enzymatic reaction Formation of DOPA has also been monitored electrochemically to determine the activity of L-aromatic amino acid decarboxylase Other enzymes involved in catecholamine metabolism which have been determined electrochemically include dopamine-p-hydroxylase phenylethanolamine-N-methyltransferase and catechol-O-methyltransferase . Electrochemical detection of DOPA has also been used to determine the activity of y-glutamyltranspeptidase The cytochrome P-450 enzyme system has been studied by observing the conversion of benzene to phenol and subsequently to hydroquinone and catechol... 

Multiple electrodes have been used to obtain selectivity in electrochemical detection. An early example involved the separation of catecholamines from human plasma using a Vydac (The Separation Group Hesperia, CA) SCX cation exchange column eluted with phosphate-EDTA.61 A sensor array using metal oxide-modified surfaces was used with flow injection to analyze multicomponent mixtures of amino acids and sugars.62 An example of the selectivity provided by a multi-electrode system is shown in Figure 2.63... 

Weber, S. G., Tsai, H., and Sandberg, M., Electrochemical detection of dipeptides with selectivity against amino acids, ]. Chromatogr., 638, 1, 1993. 

Progress has been made in developing electrochemical methods for detection of amino acids without derivatization.74 75 Evaporative light scattering (ELSD) is also a promising detection method.76 Flow-injection analysis... 

Reaction with phenylisothiocyanate (PITC) in alkaline conditions produces stable phenylthiocarbamyl (PTC) adducts which can be detected either in the ultraviolet below 250 nm or electrochemically. However, this method involves a complex derivatization procedure and offers poorer sensitivity than the alternatives available for individual amino acids. It is useful, however, in conjunction with the automated analysis of peptides when single derivatized residues can be cleaved and analysed after conversion in acidic conditions to phenylthiohydantoins. 

Determination of iodo amino acids by HPLC with inductively coupled plasma (ICP)-MS detection had LOD 35-130 pg of I, which is about one order of magnitude lower than with UVD usually applied for these compounds175. Amino acids and peptides containing sulfur, such as cysteine, cystine, methionine and glutathione, can be determined after HPLC separation by pulsed electrochemical detection, using gold electrodes176. 

Aromatic and sulfur-containing amino acids were separated by HPLC, and subjected to post-column UV irradiation before electrochemical detection with GCE vs AgCl/Ag electrodes. The analytes showed different behavior during lamp off and on periods. Thus, for example, tyrosine (46) and tryptophan (47) showed inherent electrochemical response at +0.80 V, but none at +0.60 V however, on turning on the UV lamp they showed sensitive response at both potentials126. 

Depending on the derivatizing agent used, spectrophotometric or fluorometric detectors are usually employed. Electrochemical detection of underivatized amino acids is limited to amino acids possessing aromatic or sulfur-containing side-chain, even if derivatization procedmes to attach electrochemical active moieties to the amino acids can be employed, as well as other approaches... 

Fig. 6.2.2a-bc High-performance liquid chromatography (HPLC) with electrochemical (EC) detection of neurotransmitter metabolites, a standard mixture b cerebrospinal fluid (CSF) sample - control c CSF sample - aromatic amino acid decarboxylase (AADC) deficiency. Peak identification 1 = 5HIAA (7.7 min), 2 = 3-MD (9.6 min), 3 = HVA (11.7 min)... 

The detection step involves electrochemical oxidation at a nickel electrode. This electrode has been applied to measurements of glucose (4), ethanol (5), amines, and amino acids (6,7). The reaction mechanism involves a catalytic higher oxide of nickel. The electrolyte solution consists of 0.1 M sodium hydroxide containing 10-4 M nickel as suspended nickel hydroxide to ensure stability of the electrode process. The flow-injection technique offers the advantages of convenience and speed in solution handling and ready maintenance of the active electrode surface. 

The ubiquitous electrochemical behavior of ferrocene and its relative chemical stability have made this organometallic complex a useful group for the preparation of redox-active devices. The incorporation of ferrocene-modified amino acids into larger polypeptide structures can therefore lead to electrochemically active de novo designed proteins. In addition, the attachment of ferrocene derivatives to peptides make them electroactive and eligible to electrochemical detection. Hence, it is not surprising that the first synthesis of a ferrocene-modified amino acid dates back to the 1950s. 

Another approach to dealing with the nonelectrochemically active nature of most amino acids is to generate, in situ, chemical reactions at the electrode surfaces to produce electrochem-ically active products for detection. Related to this concept, is the online use of immobilized enzymes (142) to react with amino acids. A by-product of this reaction is hydrogen peroxide, which is then quantified by amperometric detection. 

L Dou, J Mazzeo, IS Krull. Determination of amino acids, peptides and proteins by HPLC combined with electrochemical detection. BioChromatography 5(2) 74-96, 1990. 

S. J. Setford, S.F. White and J.A. Bolbot, Measurement of protein using an electrochemical bi-enzyme sensor, Biosens. Bioelectron., 17 (2002) 79-86. P. Sarkar and A.P.F. Turner, Application of dual-step potential on single screen-printed modified carbon paste electrodes for detection of amino acids and proteins, Fresenius J. Anal. Chem., 364 (1999) 154-159. 

J. Wang, M.P. Chatrathi, A. Ibanez and A. Escarpa, Micromachined separation chips with post-column enzymatic reactions of class enzymes and end-column electrochemical detection assays of amino acids, Electroanalysis, 14 (2002) 400-404. 

J. Wang, G. Chen and M. Pumera, Microchip separation and electrochemical detection of amino acids and peptides following precolumn de-rivatization with naphthalene-2,3-dicarboxyaldehyde, Electroanalysis, 15 (2003) 862-865. 

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