COMPREHENSIVE CHROMATOGRAPHIC AND SPECTROSCOPIC METHODS FOR THE SEPARATION AND IDENTIFICATION OF INTACT GLUCOSINOLATES

Much effort has been devoted to developing methods for the efficient i solation and identification of glucosinolates. Existing methods for se paration involve ion exchange, GLC, and HPLC (mostly after chemical mo dification by enzymatic sulfate removal and/or silylation). We demonst rate a simple and direct strategy for analyzing the glucosinolate cont ent of plant extracts, made possible by a new combination of widely av ailable techniques: (a) reverse-phase paired-ion chromatography (PIG) of plant extracts, (b) hydrolysis of glucosinolates by myrosinase and quantitation of resulting isothiocyanates by cyclocondensation with 1, 2-benzenedithiol; (c) a novel method for replacing the PIC counterions by ammonium ions, permitting direct bioassay, mass, and H-1 NMR spect rometry; (d) mass spectrometric analysis of ammonium salts by negative -ion fast atom bombardment (FAB) to determine mit of the [M-H](-) ion, and by chemical ionization (CI) in ammonia to obtain accurate masses of characteristic fragment ions, principally [R-CN:NH4](+), [R-CH=NOH: H](+) and [R-CH=NOH:NH4](+); and (e) high-resolution H-1 NMR spectrosc opy of intact glucosinolates. FAB and CI mass spectra, as well as high -resolution H-1 NMR spectra were obtained for a variety of glucosinola te standards. The results provide guidance for the isolation and chara cterization of unknown glucosinolates from plants. These combined proc edures were applied to a sample of broccoli (cultivar SAG;A), in order to resolve and identify its major glucosinolates: 4-methylsulfinylbut yl glucosinolate (glucoraphanin) and 4-methylthiobutyl glucosinolate ( glucoerucin). Thus, this analytical strategy provides a powerful techn ique for identifying and quantitating glucosinolates in plant extracts without resorting to derivatization. (C) 1996 Academic Press, Inc.