HPLC Techniques with Electrochemical Detection

Manuel Chicharro Santamarta, Monica Moreno Barambio and Alberto Sdnchez Arribas Departamento de Quimica AnaUtica y Andlisis Instrumental, Universidad Autonoma de Madrid, C/Francisco Tomas y Valiente, 7, 28049, Madrid, Spain 

In this scene, HPLC is a paramount technique in the modern age of food analysis with hundreds of applications [3], some of which include the detection of aflatoxins, amino acid analysis, vitamins separation, profiling of various food components, analysis of colorants and their residues, or the determination of the sugars content. 

The detector is a fundamental component in the chromatographic system. These devices allow the visualization of the analytes once separated in the chromatographic column thus, both the sensitivity and the efiiciency that can be attained with the system are dependent 

Edited by Alberto Escarpa, Marta Cristina Gonzdlez and Miguel Angel Lopez. 2015 John Wiley Sons, Ltd. Published 2015 by John Wiley Sons, Ltd. 

Other important detector types are fluorescence detector (FL), light scattering detector (LS), and the refractive index detector (RI). The FL detection offers the higher sensitivity and the lower detection limits. Nevertheless, these properties are frequently counteracted by the limited number of compounds with native FL therefore, additional derivatization reactions of analytes are usually needed, and the cost of acquisition and maintenance. In addition, when FL is used in HPLC systems, the linear dynamic range is often small for many analytes (even when the dynamic range is relatively large) and care should be taken 

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HPLC Techniques with Electrochemical Detection 95 Table 4.3 HPLC-ED analysis of amino acids... 

HPLC Techniques with Electrochemical Detection 99 Table 4.5 HPLC-ED analysis of natural phenolics... 

HPLC Techniques with Electrochemical Detection 101 Table 4.7 HPLC-ED analysis ofmycotoxins... 

UV detection, diode-array detector (DAD) and fluorescence have been the detection techniques used, coupled to HPLC for the analysis of OTC. UV detection is set at 355 nm [49-51], 350 nm [40], or at 353 nm [52], Using the diode array detector [49] offers advantages that the target peak can be identified by its retention time and absorption spectrum. Compared to UV detection, fluorescence detection is generally more specific and is less interfered by other compounds in the sample matrix [51]. A HPLC method with electrochemical detection has also been suggested recently. Zhao et al. [53] described HPLC with a coulometric electrode array system for the analysis of OTC, TC, CTC, DC, and methacycline (MC) in ovine milk. An amper-ometric detection coupled with HPLC was developed by Kazemifard and Moore [54] for the determination of tetracyclines in pharmaceutical formulations. 

The second exampleinvolved a similar procedure however this time the electrochemical and chromatographic analyses were used to monitor cerebral spinal fluid (CSF) in the lateral ventricles of a living rat. Electrical stimulations and drug administrations were clearly followed via the electrochemical techniques while HPLC coupled with electrochemical detection identified the transmitters and metabolites that were involved. 

GC, for their direct detection of carbohydrates, with high sensitivity and specificity and without the need for sample derivatization. Specifically, the technique of high-performance anion-exchange chromatography with pulsed electrochemical detection (HPAEC-PED) is fast becoming the technique of choice for this. HPLC techniques with R1 detection are greatly limited in their ability to analyze complex carbohydrates in foods. 

The variety of detection modes available for HPLC analysis that provide additional information about the eluent as it exits the column greatly facilitates unknown characterization. The majority of analytical methods for phenolic compounds includes HPLC with spectrophotometric-based detection techniques (UV absorption, fluorescence, photo diode array—PDA) as well as HPLC with electrochemical detection. 

The suprahypothalamic neurotransmitter level can be assessed by a determination of catecholamines in circumscribed brain areas, the technique requires preparation of frozen tissue and isolation of specific nuclei by the micropunch technique. The catecholamines and indolamines can be measured by a radio-enzymatic methods and by a high-pressure liquid chromatography (HPLC) with electrochemical detection. These mechanistic investigations are mostly initiated due to questions arising from the receptor interaction profile of the drug candidate, they may be required to prove that such receptor interactions truly change the functional state of neurotransmitters (functional expression). Mostly, however, the peripheral effects of such neurotransmitter mechanisms (for instance prolactin secretion) are sufficiently distinct. 

Since L-tyrosine is the main precursor of melanins and a substrate of tyrosinase, the L-DOPA/L-tyrosine ratio could more accurately reflect tyrosinase activity than l-DOPA alone. In 1997, we developed two reversed-phase HPLC techniques, one with electrochemical detection to measure simultaneously l-DOPA, norepinephrine (NE), epinephrine (E), dopamine (DA), and DOPAC (3,4-dihydroxyphenyl acetic acid) (all compounds easily oxidizable between +0.15 and +0.50 V), and one with fluorimetric detection to measure L-tyrosine (and phenylalanine) on the same blood sample. 

Many analytical techniques such as, radiochemistry, gas chromatography, and liquid chromatography have been developed for the determination of biogenic amines, their precursors, and metabolites. HPLC with electrochemical detection is considered to be one of the most popular methods for determining biogenic amines, owing to its simplicity, versatility, sensitivity, and specificity. 

Very few biological materials have been analyzed for the presence of MBOCA or its metabolites. MBOCA and its metabolites have been measured in urine of exposed humans and experimental animals. Hemoglobin adducts have also been measured in the blood of exposed animals. The most frequently used techniques are gas chromatography (GC) with electron capture detection (ECD) and high-performance liquid chromatography (HPLC) with electrochemical detection (ED). Detailed methodologies from selected studies are presented in Table 6-1. 

The latter technique of electrochemical detection focused on its utility (coupled with HPLC) for the determination of halogenated anilines in environmental and biological samples. 

Absolute qualitative identification can be assured only if samples are removed and analyzed. Two examples of such a procedure have been reported. The first was an attempt to determine if direct electrical stimulation of the caudate nucleus resulted in the release of dopamine as well as ascorbic acid from that tissue. Micro voltammetric and stimulating electrodes were micromanipulated into excised caudate tissue which was flushed with warmed, oxygenated buffer. Reference and auxiliary electrodes were nearby. Quantitative information was taken, stored, manipulated, and displayed by a minicomputer. Simultaneously a push-pull cannula device sampled the caudate and delivered the perfusate to an iced vial. Changes in the electrochemical signal that followed stimulation were correlated with changes in the dopamine and ascorbic acid content of the perfusate as determined via HPLC with electrochemical detection. It was found that little if any ascorbic acid was released as a result of electrical stimulation in these experiments. Although there is some question concerning the stability of ascorbate in an iced vial, the above example does illustrate this coincident analytical technique. 

Hybrid techniques such as HPLC-EC or electrochemical detection in combination with flow injection analysis (FIA) will foreseeably play a much more important role in the environmental analysis. 

The degree of purity of a sugar sample can be ascertained or the identification and assay of mixtures of sugars can normally be achieved by any one or a combination of chromatographic methods, of which GC of volatile derivatives or HPLC with electrochemical detection of the sugars or derivatives prepared in order to confer suitable spectroscopic properties, are comparable in their efficacy. Supercritical fluid chromatography (SFC) is also a flexible, rapid, and efficient technique comparable with HPLC but by no means as widely used. 

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