ANALYSIS Phenolic Acids in Fruits and Vegetables

II. ANALYSIS

Soluble HBA or HCA derivatives are frequently extracted from fruits and vegetables with ethanol or methanol-water solutions (80/20, v/v), using low temperatures and adding an antioxidant to prevent oxidation during the extraction procedure. Chemical or enzymatic hydrolysis of the plant material is necessary when phenolic acids are linked to cell wall constituents to give insoluble forms [6]. Apolar solvents or supercritical carbon dioxide may be useful to extract phenolic lipids [7,8]. In the case of acylated flavonoids, solvents must be adapted to the characteristics of the flavonoid itself, e.g., acidic methanol for fruit anthocyanins, although some artefacts may appear under these conditions.

Purification of the raw extract is essential. This may be performed in a first stage by removing chlorophylls and carotenoids and in a second stage by extracting phenolic acids with ethyl acetate from the depigmented aqueous extract, using a method previously described for fruits [2]. A preliminary analysis on a polyamide column has the advantage of separating the two groups of HCA derivatives: glucose derivatives on the one hand and quinic, tartaric, malic, or galactaric derivatives on the other [7]. Paper chromatography, classical or high-performance thin-layer chromatography, and column chromatography have been used extensively since the 1960s to separate phenolic acids, both before and after hydrolysis of esters and glycosides. Furthermore, separation of phenolic acid conjugates has greatly progressed thanks to high-performance capillary electrophoresis [9,10] and high-performance liquid chromatography (HPLC), which also allows quantitative determinations. In particular, the development of reversed-phase columns has greatly improved the separation performance of HCA and HBA derivatives [7].

In addition to analytical separations, the identification of phenolic acids has greatly benefited from the development of modern techniques (infrared [IR] and nuclear magnetic resonance [NMR] spectroscopy, mass spectrometry, etc.), that have added to the accurate knowledge of the structure of natural phenolic molecules [7]. New analytical approaches, including Raman spectroscopy, also allow in situ detection of HCA covalenty linked to cell wall constituents [6]. Some early approximate identifications have now been rectified, but there may be others as yet unrecognized [4].

In some unusual cases, spectrophotometric estimation of a major phenolic acid may be performed directly in plant extracts, such as chlorogenic acid in apples, pears, or potatoes [2,11], but this gives approximative information. From a quantitative point of view, HPLC techniques appear to be the most suitable, and they have been widely developed for estimating individual plant phenolic acids in their native forms [7]. Numerous examples concerning fruits and vegetables have already been reported [1,2]. Nevertheless, given the diversity and complexity of the combined forms naturally present, it has often been easier to determine phenolic acids released after hydrolysis of the extract, although some molecules might then be degraded.

A rapid fluorometric determination of p-coumaric, protocatechuic, and gallic acids has also been proposed in persimmon [12], but interference with other phenolic compounds is likely. Moreover, the radical scavenging activities of HBA and HCA may be used for their quantitative determination by chemiluminescence in the presence of hydrogen peroxide [13].