Preparative fractionation of 5′-O-caffeoylquinic acid oxidation products using centrifugal partition chromatography and their investigation by mass spectrometry

Abstract

When apples are processed into juices or ciders, a great variety of neoformed molecules are generated by enzymatic oxidation of polyphenols. These phenolic oxidation products could be responsible of specific organoleptic properties in apple juices and ciders. 5′-O-Caffeoylquinic acid (CQA) is the major hydroxycinnamic acid in apple and the preferential substrate of apple polyphenoloxidase (PPO). Its main oxidation products were synthetized and purified at the multi-milligrams scale to decipher their structures using mass spectrometry.CQA oxidation products were first synthetized in model solution by oxidizing CQA, in the presence of oxygen and PPO. Then, a specific method involving centrifugal partition chromatography (CPC) was developed to fractionate the main oxidation products corresponding to CQA dehydrodimers (MW 706 Da). For this purpose, CPC was performed in Elution-Extrusion Countercurrent Chromatography (EECCC) mode using a two-phase solvent system precisely selected according to the partition coefficient of the targeted compounds.After a last purification step using semi-preparative reversed phase HPLC, ten CQA dehydrodimers resulting from oxidative coupling were successfully purified, with a UV 280 nm chromatographic purity superior to 85%. Hypothetical structures were formulated for all CQA dehydrodimers based on their UV–vis, MS and MSn spectra.According to this study, centrifugal partition chromatography in combinaison with semi-preparative HPLC was a promising tool to fractionate or purify phenolic oxidation products.