Flavonoids analytical methods

Swain, T., Analytical methods for flavonoids, in Chemistry and Biochemistry of Plant Pigments, Goodwin, T.W., Ed., Academic Press, London, 1965, chap. 19. 

Knowledge of the flavonoid content of plant-based foods is paramount to understanding their role in plant physiology and human health. Analytical methods are also important to identify adulteration of beverages, for example. And flavonoids are indispensable markers for chemotaxonomic purposes. 

Various analytical methods exist for flavonoids. These range from TLC to CE. With the introduction of hyphenated HPLC techniques, the analytical potential has been dramatically extended. Gas chromatography (GC) is generally impractical, due to the low volatility of many flavonoid compounds and the necessity of preparing derivatives. However, Schmidt et al. ° have reported the separation of flavones, flavonols, flavanones, and chalcones (with frequent substitution by methyl groups) by GC. 

Stalikas, C.D. 2008. Phenolic acids and flavonoids occurrence and analytical methods. In Free Radicals and Antioxidant Protocols, Methods in Molecular Biology, 2nd edn. (Uppu, R.M. Murthy, S.N. Pryor, W.A. Parinandi, N.L., Eds.). Humana Press, New York, pp. 65-90. 

Flavonols and flavones are present in many food products and medicinal plants and show relevant antioxidant activity in vitro. In this chapter, classical analytical methods sueh as thin layer ehromatography and two-dimensional paper chromatography together with modem methodologies such as HPLC-MS-MS are reported. Preparative ehromatography methods are also reviewed as well as spectroseopie methods used for flavonoid characterization and identification, including UV spectrophotometry and MS spectrometry. Chemical and enzymatic methods used in flavonoid identification are also reviewed. 

Despite several shortcomings, paper chromatography was clearly advantageous to qualitative analytical methods. One of the major advantages was its application in the quantitative analysis of flavonoids. This point is manifested by several reports using paper chromatography... 

Wade, R. BCirksey, S. Lee, K. Smith, J. 1992. New analytical methods in the USA for deteeting fruit juice adulteration. Fluessiges Obst. 59 62-72 (English Version). Walle, T. 2004. Absorption and metabolism of flavonoids. Free Radic. Biol. Med. 36 829-837. 

Given the various biological effects ascribed to flavonoids, questions arise on bioavailabihty. Therefore, there is a need for analytical methods for the analysis of flavonoid aglycones and glycosides in human biofluids. In addition, the useMness of flavonoid as a biomarker for the intake of fruit and vegetables must be evaluated. MS methods for the determination of flavonoids in biological samples were reviewed by Prasain et al. [68]. 

Extensive reviews of analytical methods for anthocyanins (Francis, 1982 Jackman et al., 1987b Strack and Wray, 1994) and other flavonoids (Williams and Harbome, 1994) as well as phenolic acids (Herrmann, 1989) have been published. In these reviews, extraction procedures, methods for fractionation of groups of polyphenols and the identification and quantification of individual components are presented. Here, a brief presentation of more recently published methods for grape and berry polyphenolic analyses is given with respect to their relationship to antioxidant activity and health benefits. 

The chromatographic profile should feature the fundamental attributions of sameness and differences of the constituents of the investigated extract and define their quality in order to get reproducible biological data in terms of safety and efficacy. The analytical methods should be so precise that different batches of the extract in question will have the same efficacy. As described above, it has now become routine to define a ginkgo extract in terms of the content of the two groups, the flavonoid glycosides and... 

Hyphenated analytical methods used in studies of flavonoids fall into several categories, the most important of which involve mass spectrometry (MS, usually as LC-UV-MS see... 

The direct analysis of flavonoids is not possible due to the high complexity of the sample matrices. Sample pretreatment is a requirement in all flavonoid analysis to remove possible interferences, to increase the concentration of the analyte and hence the sensitivity of the analytical method, to adapt the analyte to the detection method, and to provide a more reproducible and robust analysis method. The selection of the proper sample treatment procedure depends on the type of flavonoids and the sample matrix (plant, food, or liquid sample such as biological fluids and drinks). Solid samples are normally lyophilized and homogenized before extraction liquid samples are normally filtered and/or centrifuged before the isolation or extractive procedure. 

The electroactive property of flavonoids was used to optimize a FIA coupled to an electrochemical detection (FIA-EC) method to determine quercetin and rutin natural plant extracts and pharmaceutical preparations [65]. In this method, flavonoids were determined at normal (unheated) and hot platinum microelectrodes using cyclic voltammetry. The use of the FIA system causes an increase of the analytical signal by more than 6 times because of the increase of the temperature to about 76°C in a small zone close to... 

Estimation of the daily intake of flavonoids is not a straightforward matter because of the mentioned variation in the content and composition of flavonoids in foods. Moreover, the variability and the lack of standardized analytical methods for their estimation further hinder the creation of compositional databases necessary to calculate flavonoids consumption. Intakes wUl also be affected by seasonal and regional availability of plant foods, as weU as by dietary habits dependant of cultural traditions and individual preferences. 

Analytical methods for the isolation, separation, and characterization of anthocyanins have been described (Markham, 1982 Jackman et al., 1987 Harbome, 1998 Rivas-Gonzalo, 2003 Andersen and Francis, 2004). A comprehensive and highly recommended source for anyone involved in anthocyanin analysis is that of Strack and Wray (1989). Further details of flavonoid chemistry can be found in volumes of The Flavonoids series (Harbome et al., 1975 Harbome and Mabry, 1982 Harbome, 1988, 1994). Recent advances in flavonoid research are thoroughly described in the book Flavonoids Chemistry, Biochemistry and Applications, edited by Andersen and Markham (2006). Extensive information on the occurrence of anthocyanins in various natural products reported after 1992 was presented by Andersen and Jordheim (2006). One of the most useful sources of current protocols of anthocyanin analysis is the Handbook of Food Analytical Chemistry, Pigments, Colorants, Flavors, Texture, and Bioactive Food Components, edited by Wrolstad et al. (2004). This book is a practical how to manual that contains detailed in-stmctions on the following topics (1) extraction, isolation, and purification of anthocyanins, (2) characterization and measurement of anthocyanins by UV-Vis... 

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