Spiral-coil countercurrent chromatographic separation of betanin and betanidin ethyl-esters monitored by continuous ESI-MS/MS injection and LC-ESI-MS/MS

Betalains present in Beta vulgaris L. (Chenopodiaceae) have a high potential as natural pigments for food applications. They have been used as a substitute of synthetic dyes in processing of gelatine, strawberry yogurts, ice creams, fruits cocktails, candies and biscuits. The stability of betalains is strongly influenced by numerous factors – sugar content, and impact of light, oxygen, water activity, pH and temperature. A mixture of betacyanin mono-, di- and tri-ethyl-esters was separated by a large-volume spiral-coil countercurrent chromatography (spCCC) prototype which yielded fractions continuously off-line injected to an ESI-MS/MS device. This method yielded a reconstituted pigment profile and finally enabled the MS-target-guided isolation procedure for the whole spCCC-experiment. The individual fractions were analyzed by LC/ESI-MS/MS. Betanin and betanidin mono-, di- and tri-ethyl-esters as well as other decarboxylated derivatives were detected in the chromatograms. The ethyl-esters of betanin were only partially fractionated, but clearly fractionated from the ethyl-esters of betanidin which will be of great value for biological evaluations.