[en] The official ISO protocol (similar to U.E. recommendations established in 1987 by six european laboratories) for determination of individual glucosinolate content in rapeseed using HPLC was published for the first time in 1992 (ISO 9167-1). Numerous laboratories, all over the world, use now this method, especially in order to control the 00 varieties. The goals of our research were to considerably reduce the analysis time without loosing precision. A single extraction is now suggested (200 mg of ground seeds stirred in 10 ml of 75°C methanol/water 70/30 with an internal standard for 10 min). Then 1-6 ml of the crude extract is directly put on DEAE A-25 resin prepared according to the ISO method. Glucosinolates are desulfated by addition of 100 µl of concentrated Helix pomatia sulfatase (H1, EC 3.1.6.1) quickly prepared by ethanol precipitation. The desulfatation process can be reduced to 1 hour without any problems with all kind of glucosinolate (alcenyl, benzyl, indolyl, methylthio, methylsulfinyl, methylsulfonyl...). Elution of the desulfo-glucosinolates is realised with 4 x 0.5 ml of distilled water. Desulfo-glucosinolates are separated by HPLC using an Inertsil 3 ODS-3 column (100 x 3 mm, 3 µm) with a water/acetonitrile gradient (from 2 to 25% in 35 min). Resolution is very nice and the limited flow (0.4 ml/min) reduce the solvent costs and the elimination of wastes. The suggested fast method has been compared with the official ISO method analysing the three certified reference materials prepared by BCR (now IRMM) and recommended by U.E. and ISO (CRMs 366, 190 and 367). The recovery of indolyl desulfo-glucosinolates, specially 4-OH glucobrassicin, is higher with the quicker method. Results obtained with the two methods are very close for other desulfo-glucosinolates.
