An HPLC method for simultaneous quantitation of individual isothiocyanates and oxazolidinethione in myrosinase digests of rapeseed meal

A simple, rapid and precise method for simultaneous quantitation of individual isothiocyanates and oxazolidinethione inmyrosinase digests of rapeseed meal has been developed. The method consists of inactivation of native myrosinase activity present in the seedmeal, followed by digestion with mustard myrosinase (thioglucoside glucohydrolase, EC 3.2.3.1) to hydrolyze rapeseed glucosinolates quantitatively to isothiocyanates and oxazolidinethione. These hydrolytic products are extracted in methylene chloride as soon as they are formed and finally resolved by a reverse phase high pressure liquid chromatography (HPLC) technique on a μ Bondapak C₁₈ column using aqueous acetonitrile as solvent and an ultraviolet (UV) absorbance detector set at 254 nm. The lower limits of quantitation by this method in a single aliquot applied to the column were 0.2 μg for the isothiocyanates and 0.01 μg for the oxazolidinethione. Recoveries of allyl isothiocyanate, oxazolidinethione and sinigrin added toB. juncea, prior to digestion, were quantitative and averaged at 94.5, 93.0 and 91.2 percent with standard deviations of 1.5, 3.3 and 2.8 percent, respectively. The butenyl and pentenyl isothiocyanates and oxazolidinethione in Tower (B. napus) and Candle (B. campestris) rapeseeds, and allyl isothiocyanate inB. juncea were the major hydrolytic products of glucosinolates. The identity of peaks corresponding to these compounds on a HPLC chromatogram was confirmed by mass spectroscopy.