Vitamin electrochemical detection

Chromatographic methods including thin-layer, hplc, and gc methods have been developed. In addition to developments ia the types of columns and eluents for hplc appHcations, a significant amount of work has been done ia the kiads of detectioa methods for the vitamin. These detectioa methods iaclude direct detectioa by uv, fluoresceace after post-column reduction of the quiaone to the hydroquinone, and electrochemical detection. Quantitative gc methods have been developed for the vitamin but have found limited appHcations. However, gc methods coupled with highly sensitive detection methods such as gc/ms do represent a powerful analytical tool (20). 

Vitamin D2 and D3 exhibit identical UV absorption spectra and they do not possess fluorescence. Electrochemical detection is limited and only few methods are applied in food analysis [530,533], MS detection has been applied achieving satisfactory detection limit (10 mol/mL) [534,535],... 

Sanchez-Perez, A., Delgado-Zamarreno, M.M., Bustamante-Rangel, M., and Hernandez-Men-dez, J. 2000. Automated analysis of vitamin E isomers in vegetable oils by continuous membrane extraction and liquid chromatography-electrochemical detection. J. Chromatogr. A 881 229-241. 

All of the fat-soluble vitamins, including provitamin carotenoids, exhibit some form of electrochemical activity. Both amperometry and coulometry have been applied to electrochemical detection. In amperometric detectors, only a small proportion (usually <20%) of the electroactive solute is reduced or oxidized at the surface of a glassy carbon or similar nonporous electrode in coulometric detectors, the solute is completely reduced or oxidized within the pores of a graphite electrode. The operation of an electrochemical detector requires a semiaqueous or alcoholic mobile phase to support the electrolyte needed to conduct a current. This restricts its use to reverse-phase HPLC (but not NARP) unless the electrolyte is added postcolumn. Electrochemical detection is incompatible with NARP chromatography, because the mobile phase is insufficiently polar to dissolve the electrolyte. A stringent requirement for electrochemical detection is that the solvent delivery system be virtually pulse-free. 

Schneiderman et al. (90) extracted retinyl palmitate from commercial breakfast cereals using supercritical C02 and determined the vitamin by means of reversed-phase HPLC and electrochemical detection. Chromatograms of an unfortified wheat sample and a fortified bran-based cereal product are shown in Fig. 10. 

MA Schneiderman, AK Sharma, KRR Mahanama, DC Locke. Determination of vitamin K, in powdered infant formulas, using supercritical fluid extraction and liquid chromatography with electrochemical detection. J Assoc Off Anal Chem 71 815-817, 1988. 

H Hasegawa. Vitamin D determination using high-performance liquid chromatography with internal standard-redox mode electrochemical detection and its application to medical nutritional products. J Chromat 605 215-220, 1992. 

MM Delgado Zamarreno, A Sanchez Perez, MC Gomez Perez, J Hernandez Mendez. Automatic determination of liposoluble vitamins in butter and margarine using Triton X-100 aqueous micellar solution by liquid chromatography with electrochemical detection. Anal Chim Acta 315 201-208, 1995. 

Because the vitamins occur in food in trace quantities, detection sensitivity is often an issue. Ultraviolet absorbance is the most common detection method. Fluorescence and electrochemical detection are used in specific cases where physicochemical properties permit and where increased sensitivity and selectivity are desired. Refractive index is seldom used, due to its lack of specificity and sensitivity. 

The use of ELISA is broad and it finds applications in many biological laboratories over the last 30 years many tests have been developed and vahdated in different domains such as clinical diagnostics, pharmaceutical research, industrial control or food and feed analytics for instance. Our work has been to redesign the standard ELISA test to fit in a microfluidic system with disposable electrochemical chips. Many applications are foreseen since the biochemical reagents are directly amenable from a conventional microtitre plate to our microfluidic system. For instance, in the last 5 years, we have reported previous works with this concept of microchannel ELISA for the detection of thromboembolic event marker (D-Dimer) [4], hormones (TSH) [18], or vitamin (folic acid) [24], It is expected that similar technical developments in the future may broaden the use of electroanalytical chemistry in the field of clinical tests as has been the case for glucose monitoring. This work also contributes to the novel analytical trend to reduce the volume and time consumption in analytical labs using lab-on-a-chip devices. Not only can an electrophoretic-driven system benefit from the miniaturisation but also affinity assays and in particularly immunoassays with electrochemical detection. 

Delgado Zamarreno MM, Sanchez Perez A, Bustamante Rangel M, and Hernandez Mendez J. Automated analysis for vitamin E in butter by coupling sample treatment-continuous membrane extraction—liquid chromatography with electrochemical detection. Anal. Chim. Acta 1999 386 99-106. 

With the major constituents in foods the choice of LC detector is often the most important issue. Compounds such as vitamins, carbohydrates etc. may not have a strong ultraviolet (UV) chromophore. Therefore refractive index (RI) detection and, increasingly, electrochemical detection are often used. As discussed later, the choice of detector is even more important when determining the concentration of components in the foodstuff rather than the bulk constituent. 

Hart, J.P. Sheares, M.J. McCarthy, P.T. Enhanced sensitivity for the determination of endogenous phylloquinone (vitamin Kl) in plasma using high-performance liquid chromatography with dual-electrode electrochemical detection. Analyst 1985,110, 1181-1184. 

Fluorescence (FL) detection is another selective and sensitive technique. Vitamin E, a-, /3-, S-, and y-to-copherols have been analyzed by RP-HPLC using FL as well as UV detection. In addition, a newer technique, electrochemical detection, has been reported in the analysis of phenolic compounds of olive oil [7]. 

Research Needs. Over the years L-ascorbic acid has been shown to be an essential nutrient for many insects including species of Lepidoptera, Orthoptera, Coleoptera, and Diptera. Others such as cockroaches, houseflies, and mealworms are reared on simple diets without added ascorbic acid. Perhaps those insects require very low levels of vitamin C in their diets. A sensitive analytical method is needed to measure levels of L-ascorbic acid and dehydroascorbic acid in insect tissue and food. Such a method, which is likely to be developed using HPLC with electrochemical detection, could be used to monitor vitamin C levels in feed ingredients as well as in tissues during an insect s life cycle. This information is needed to determine whether ascorbic acid is used to... 

Lurie, I.S. McGuiness, K. The quantitation of heroin and selected basic impur-ties via reversed phase HPLC. II. The analysis of adulterated samples. J.Liq.Chromatogr., 1987, 10, 2189—2204 [also impurities, acetaminophen, acetylcodeine, acetylmorphine, acetylprocaine, aminopyrene, amitriptyline, antipyrene, aspirin, barbital, benztropine, caffeine, cocaine, codeine, diamorphine, diazepam, diphenhydramine, dipyrone, ephedrine, ethylmorphine, lidocaine, meconin, methamphetamine, meth-ap Tilene, methaqualone, monoacetylmorphine, morphine, nalorphine, niacinamide, nicotinamide, noscapine, papaverine, phenacetin, phenmetrazine, phenobarbital, phenolphthalein, procaine, pro-panophenone, propoxyphene, P5rilamine, quinidine, quinine, salic lamide, saUsalicylic acid, secobarbital, strychnine, tetracaine, thebaine, tripelennamine, tropacocaine, vitamin B3, vitamin B5 electrochemical detection]... 

Bryan, P.D. Honigberg, I.L. Meltzer, N.M. Electrochemical detection of retinoids using normal phase HPLC. J.Liq.Chromatogr., 1991, 14, 2287-2295 [LOD 1 ng also acitretin, isotretinoin, tretinoin, vitamin A palmitate]... 

Vitamins, like pharmaceutical substances, can often be determined by UV absorption or electrochemical detection without derivatization. However, vitamins were detennined in a senri-automated manifold by post-column reaction with a coupling reaction with diazotized 5-chloroaniline-2,4-disulphonyl chloride and continuous colorimetric determination of the orange products at 440nm [211]. Post-column derivatization schemes for thiamine and its phosphate esters have also been described [212-214]. 

Vitamin C Vitamin C activity resides in two naturally occurring compounds ascorbic acid and its oxidation product, dehydroascorbic acid. In human tissues ascorbic acid predominates. Ascorbic acid is labile in most samples, oxidizing to dehydroascorbic acid and then degrading to 2,3-diketogluconic acid. Various reagents can be used to prevent this oxidation in plasma or whole blood samples. Extraction with 5% metaphosphoric or trichloroacetic acid is the usual initial preparation. Only ascorbic acid may be detected by UV spectrophotometry at 245-265 nm, the absorption maxima of dehydroascorbic acid being 210 nm. A similar problem exists with electrochemical detection where ascorbic acid oxidizes at +0.7V with carbon electrodes. Fluorescent derivatives may be formed with 2-4-din-itrophenylhydrazine or o-phenyldiamine. These derivatives can be assayed by reversed-phase HPLC. 

Vitamin K HPLC has provided the first assay of the phylloquinones and menaquinones that constitute vitamin K in plasma. Phylloquinone circulates bound to lipoproteins from which it can be extracted with hexane after ethanol protein precipitation. Removal of co-eluted lipids can be achieved with normal-phase cartridge columns. Reversed-phase HPLC is almost universally used for vitamin K measurement. Either UV (270 nm) or electrochemical detection is suitable. Electrochemical detection uses the reductive mode ( —1.3 V) to convert the quinone moiety to hydroquinone the main disadvantage being the need to remove oxygen from the mobile phase. 

UV absorbance detection has been most widely used for vitamin A analysis. However, because retinol and retinyl esters are highly fluorescent, detection limits of one order of magnitude better than in assays with UV detection can be obtained using fluorescence detection. Also, electrochemical detection is a valuable alternative to UV and fluorescence detection provided the eluent contains water to incorporate essential electrolytes. Another detector for LC is the mass spectrometer. The LC-MS approach has also been applied to the analysis of vitamin A and its metabolites. 

The highest sensitivity and selectivity in vitamin E LC assays are obtained by using fluorescence or electrochemical detection. In the former, excitation at the low wavelength (205 nm) leads to improved detection limits but at the expense of selectivity, compared with the use of 295 nm. Electrochemical detection in the oxidation mode (amperometry or coulometry) is another factor 20 times more sensitive. In routine practice, however, most vitamin E assays employ the less sensitive absorbance detection at 292-295 nm (variable wavelength instrument) or 280 nm (fixed wavelength detectors). If retinol and carotenoids are included, a programmable multichannel detector, preferably a diode array instrument, is needed. As noted previously, combined LC assays for vitamins A, E, and carotenoids are now in common use for clinical chemistry and can measure about a dozen components within a 10 min run. The NIST and UK EQAS external quality assurance schemes permit interlaboratory comparisons of performance for these assays. 

Electrochemical detection in LC provides a sensitive assay method for certain vitamins, such as AA, folates, and flavins. AA may be easily detected with femtomolar sensitivity. Sample preparation and matrix interference problems limit the routine applicability of electrochemistry in the analysis of water-soluble vitamins currently to AA. 

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