Purification of major glucosinolates from Brassicaceae seeds and preparation of isothiocyanate and amine metabolites

Consumption of Brassica vegetables containing glucosinolates is associated with decreased risk of cancer at several tissue sites. Studies of the relative potency and metabolism of the major dietary isothiocyanates have been hindered by limited availability of high-purity isothiocyanates and their glucosinolate precursors. We describe a simple procedure for the preparation of glucosinolates sinigrin, gluconapin, progoitrin, glucoiberin, glucoraphanin, glucoalyssin and gluconasturtiin from vegetable seeds. Glucosinolates were extracted from the seeds of mustard, oilseed, broccoli, cauliflower and winter cress. Purification by preparative anion exchange and reversed-phase column chromatography gave the pure glucosinolates. Good separation was achieved without using ion-pair reagents. Related isothiocyanates and amine degradation products were prepared by myrosinase-catalysed hydrolysis of the corresponding glucosinolate and by organic synthesis, respectively. The compounds were characterized by analytical HPLC, 1H NMR, 13C NMR, elemental analysis and mass spectrometry. Optimized conditions for detection and quantitation of these glucosinolates and metabolites by LC-MS/MS are also given. Large-scale glucosinolate preparation could be performed, giving amounts suitable for pharmacological evaluation in vivo. These methods will facilitate studies of the pharmacological activity and metabolism of dietary glucosinolates.