Comprehensive chromatographic and spectroscopic methods for the separation and identification of intact glucosinolates

Much effort has been devoted to developing methods for the efficient isolation and identification of glucosinolates. Existing methods for separation involve ion exchange, GLC, and HPLC (mostly after chemical modification by enzymatic sulfate removal and/or silylation). We demonstrate a simple and direct strategy for analyzing the glucosinolate content of plant extracts, made possible by a new combination of widely available techniques: (a) reversephase paired-ion chromatography (PIC) 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 ¹H NMR spectrometry; (d) mass spectrometric analysis of ammonium salts by negative-ion fast atom bombardment (FAB) to determine m/z 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 ¹H NMR spectroscopy of intact glucosinolates. FAB and CI mass spectra, as well as high-resolution ¹H NMR spectra were obtained for a variety of glucosinolate standards. The results provide guidance for the isolation and characterization of unknown glucosinolates from plants. These combined procedures were applied to a sample of broccoli (cultivar SAGA), in order to resolve and identify its major glucosinolates: 4-methylsulfinylbutyl glucosinolate (glucoraphanin) and 4-methylthiobutylglucosinolate (glucoerucin). Thus, this analytical strategy provides a powerful technique for identifying and quantitating glucosinolates in plant extracts without resorting to derivatization.