High-performance liquid-phase separation of glycosides analytical and micropreparative HPLC combined with spectroscopic and enzymatic methods for generating a glucosinolate library

Analytical and micropreparative high-performance liquid chromatographic (HP LC) methods assisted by spectroscopic and enzymatic tools were developed fo r generating a glucosinolate library from white cabbage. This involved the study of the chromatographic behavior of glucosinolates by reversed-phase c hromatography (RPC) using octadecyl-silica (ODS) stationary phases and ion- exchange chromatography with strong anion-exchange (SAX) columns, The gluco sinolate extract from white cabbage was fractionated on a micropreparative Zorbax ODS column of 250 x 6.2 mm id. The collected fractions from the ODS column effluent were further purified by rechromatographing the early eluti ng fractions on a SAX column and the late-eluting peaks on the same ODS col umn under different elution conditions. These studies demonstrated the bene fits of using more than one HPLC method in the isolation of pure glucosinol ates. These chromatographic separation schemes combined with efficient samp le cleanup provided pure glucosinolate fractions that were readily identifi ed by high-field nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (MS), namely negative-ion liquid secondary ionization mass spe ctrometry (LSIMS). The structural determination of a given glucosinolate by 1- and 2-dimensional NMR was further confirmed by LSIMS, which gives the m ass of the molecular anion of glucosinolates. In addition, the structure of a given glucosinolate fraction was readily assessed by performing RPC of t he myrosinase digest of the particular glucosinolate and that of the standa rd degradation product that has a structure that is reflective of the paren t glucosinolate. Also, through the myrosinase degradation products (e.g., i sothiocyanates) under controlled conditions, the quantitative determination by RPC of the corresponding glucosinolates in the white cabbage extract wa s reliably achieved. By using the ability of myrosinase to hydrolyze the gl ucosinolates, it was possible to profile the glucosinolates in the cabbage extract quite rapidly. For the glucosinolates present at very low amounts i n white cabbage, and also for the HPLC fractions containing more than one g lucosinolate, the isothiocyanate degradation products resulting from the hy drolyzing effect of myrosinase were analyzed by gas chromatography/MS, and their structures were determined. This in turn allowed the determination of the structure of the parent glucosinolates. In summary, white cabbage seem s to contain at least 8 glucosinolates.