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Composition of tomatoes and tomato products in antioxidants (WG1) page 94
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of the aqeous extract after removal of the organic solvents. Fractionation of phenolics can also
be applied on silica and polyamide minicolumns.
Hydrolysis of glycosides may be applied prior the analysis. Three types of hydrolytic
treatments are used for this purpose, acidic, enzymatic and alkaline. The rate of acid-base
hydrolysis of glycosides depends on acid-base strength, the nature of the sugar and the
position of attachement to the flavonoid nucleus.
HPLC analysis
HPLC combines the advantages of simultaneous separation and quantification without
the need for preliminary derivatization in most cases. Normal-phase chromatography has been
used for the quantification of flavonoids in skin of ripe tomatoes. Non polar components were
removed from the plant material by extraction, following which the aqueous phase was
suitable to clean-up on a polyamide column. Flavonoids were eluted with methanol prior to
acetylation. The recovered acetates were separated isocraticallyd on Lichrosorb Si60 using
benzene-cetonitrile, benzene-ethanol or octane-ethanol-acetonitrile solvent systems and
detection either 312 or 270nm. As an alternative to HPLC bare adsorbent, supercritical fluid
chromatography allows excellent separation of polymethoxylated flavones. Carbon dioxide
modified with methanol gave rapid elution of these compounds as sharp, well resolved peaks.
For these normal-phase systems, there is the concern of highly polar materials that may be
retained irreversibly on column, with the result that the separation characteristics may be
gradually altered. Thus reversed-phase chromatography has invariably been the method of
choice.
The separation of flavonoids usually eluted on C8 or C18 columns with aqueous
mobile phase with methanol, acetonitrile or tetrahydrofuran as organic modifiers. Under the
usual reversed-pase conditions, diglycosides precede monoglycosides which precede
aglycones. The elution pattern for flavonoids containing equivalent substitution patterns is
flavanone followed by flavonol and flavone. Hence, Hertog et al. (1993) published a method
to quantify flavonols (quercetin, kaempferol, myricetin) and flavones (apigenin, luteolin) on
tomato juice. Glycosides were hydrolyzed under acidic conditions and aglycones were
extracted. This products were isocratically eluted on a Nova-Pak C18 with acetonitrilephosphate
buffer as mobile phase and detected at 370 nm.