Guidelines for glucosinolate analysis in green tissues used for biofumigation

Over the last decade glucosinolates contained in different tissues of Brassicaceae and their breakdown products, especially isothiocyanates formed after myrosinase-catalyzed hydrolysis, have been considered not only important antitumoral compounds but also as interesting “biopesticides” for controlling soil borne pathogens. This is the reason why the identification and the quantitative determination of each individual glucosinolate contentsin green tissues (leaves, roots, stem) are of great importance. The aim of this paper is to establish a joint protocol containing the guidelines to determine glucosinolate contents in fresh Brassicaceae tissues by HPLC of desulfo-glucosinolates. Some modifications of the ISO 9167-1 method, initially set up for evaluating rapeseed seeds, are proposed and discussed with the objective of optimizing and standardizing glucosinolate analysis in fresh tissues (leaves, roots or stems) of Brassicaceae. Collection, storage and preparation of fresh samples suitable to be analyzed are important steps during which it is necessary to avoid glucosinolate hydrolysis by the endogenous myrosinase-catalyzed reaction. The techniques of extraction and desulfatation of glucosinolates, the choice and purification of the internal standard, and the HPLC conditions (including the relative response factors) are also presented. The possibility of a convenient and simplified use of an alternative isocratic HPLC method for an accurate qualitative and quantitative determination of glucosinolates is also discussed and evaluated. The identification of desulfo-glucosinolates is performed by comparison of their retention times and UV spectra, and by GC-MS analysis of the compounds resulting from myrosinase-catalyzed hydrolysis of glucosinolates.The experience acquired in the field of the preparation of glucosinolate or desulfo-glucosinolate as chromatographic standards, using free and immobilized sulfatase is also presented.