Optimization and Validation of a Fast Liquid Gradient for Determination of Prominent Flavan-3-ols and Flavonols in Fresh Vegetables

Abstract

A fast high-performance liquid chromatography method was used for analysis of prominent flavan-3-ols and flavonols in vegetables. Gradient elution with phosphoric acid-acetonitrile mixtures and phosphoric acid-methanol mixtures allowed fast and complete separation of the studied phenolic compounds within analysis times less than 10 min. The development of two elution gradients using methanol and acetonitrile as modifiers proved to be an excellent approach for the verification of the real polyphenolic composition in vegetables samples because the two optimized methods allowed the separation of the same number of compounds in the same elution order. Diode-array detection was employed for the provisional identification of phenolic compounds that were not available as standards. We preferred methanol as a modifier because it was less toxic and cheaper than acetonitrile. Detection limits ranged between 0.12 and 0.59 μg mL -1 . High recoveries of phenolics from fresh vegetables were measured in all studied cases, independent of the phenolic structure, matrix, and vegetable in question. High levels of procyanidins between 150 and 450 mg kg -1 were found in all studied vegetables. Quantification of quercetin and kaempferol glycosides was only possible in marrow and onion, respectively.