Anthocyanin Purification by Solid Phase Extraction. Solid
phase extraction (SPE) was used for anthocyanin purification (both
before and after hydrolysis) prior to instrumental analysis. Because the
purple tomato contains a relatively low content of anthocyanins, SPE can
also serve as a means of concentration. The purification of anthocyanins
was accomplished using Strata-X 33 μm Polymeric Reversed-Phase
cartridges (Phenomenex, USA).30 The cartridge was activated by
passing 2 mL of MeOH solution acidified with formic acid (5%) and
equilibrated with 2 mL of waterformic acid (95:5). The crude extract
was concentrated to ca. 1/5 of its original volume by a rotary evaporator,
then 5 mL of this concentrated solution was carefully loaded on to the
cartridge, which was then washed with 10 mL of waterformic acid
(95:5) to remove sugars, protein, and substances that could interfere
with the analysis of anthocyanins. The elution of the anthocyanins was
performed by the addition of 2 mL of methanolformic acid (95:5).
The eluate was dried under nitrogen and redissolved in waterformic
acid (95:5) to a final volume of 500 μL.