Preparation of standards
Steviolbioside was prepared according to Wood, Allerton, Diehl, and Fletcher (1955), by refluxing 4 g stevioside (purity > 98%) in 10% potassium hydroxide for 1 h. After cooling, the solution was acidified to pH 5 using glacial acetic acid. The white precipitate was isolated by centrifugation and crystallized from hot methanol to a purity of >99%. Identity and purity were checked by detailed HPLC, LC/MS-ELSD and NMR analyses (Geuns, 2010 and Struyf et al., 2008). A similar procedure was applied to 4 g rebaudioside A (purity > 98%) to obtain rebaudioside B. Isosteviol was prepared by acid hydrolysis of stevioside according to Mosettig et al. (1963). A solution of 1 g stevioside (purity > 98%) in 15 mL 48% aqueous hydrobromic acid was stirred at room temperature for 20 h. The resulting precipitate was collected by centrifugation, washed with water several times and crystallized from hot methanol to give hexagonal crystals of isosteviol.