Determination of ascorbigens in autolysates of various Brassica species using supercritical fluid chromatography

A new method of analysis based on normal phase supercritical fluid chromatography (SFC) has been developed for investigation of ascorbigens [2-C-(indol-3-ylmethyl)-beta-L-xylo-3-hexulofuranosonic acid gamma-lactone derivatives]. This method has been adapted to preparative isolation and quantitative determinations of individual ascorbigens comprising ascorbigen, neoascorbigen, and 4-methoxy-ascorbigen. The structures of these compounds have been revealed from 1D (H-1, C-13) and 2D (COSY, KMQC, HMBC) NMR experiments. The developed SFC method had an acceptable linearity for the ascorbigens with correlation coefficients (R-2) > 0.9995 (n = 10) in the range of 0.13-4.9 nmol injected, detection limits were below 13 pmol, retention time stabilities were excellent, and relative response factors have been determined. The SFC method has been used for determination of ascorbigens produced during autolysis of indol-3-ylmethylglucosinolates in various Brassica vegetables and rapeseed seedlings. Generally 30-60% of the indol-3-ylmethylglucosinolates in the plants were transformed into ascorbigens, with the concentration in autolysates varying from 0.51 +/- 0.002 to 3.72 +/- 0.21 mu mol/g of dry weight (DW) for ascorbigen, from 0.05 +/- 0.01 to 2.42 +/- 0.23 mu mol/g of DW for neoascorbigen, and from 0.03 +/- 0.0002 to 0.84 +/- 0.07 mu mol/g of DW for 4-methoxyascorbigen.