Supercritical fluid chromatography as basis for identification and quantitative determination of indol-3-ylmethyl oligomers and ascorbigens.

ABSTRACT

Indol-3-ylmethylglucosinolate (glucobrassicin) occurs in most plants of the Brassicaceae family together with hydroxy and methoxy derivatives of glucobrassicin. These compounds and products produced therefrom have been the subject of considerable research interest due to their potential anticarcinogenic effects, and thereby a need for techniques to work with the individual compounds. A method using normal-phase supercritical fluid chromatography (SFC) with methanol as modifier has been developed for determination and quantification of the various indol-3-ylmethyl derivatives including ascorbigens formed from the glucobrassicin degradation product, indol-3-ylmethanol, under acidic conditions (pH 2-6) with and without the presence of ascorbic acid. The SFC method had detection limits in the 10-100-pmol range. In the absence of ascorbic acid a range of oligomers were formed, whereas the presence of ascorbic acid favoured the formation of ascorbigen and products thereof. Quantitatively important indol-3-ylmethyl oligomers consisting of up to five indol rings have been purified with preparative SFC and identified from MS and 1D and 2D NMR experiments with complete assignment of chemical shifts to all of the atoms. Investigation of the autolysis products of white cabbage showed that ascorbigens were the quantitatively dominating degradation products of indol-3-ylmethylglucosinolates.