The stevioside molecule 1 ( Fig. 1) is comprised of a glycone (sugars) attached to the steviol moiety. The class of Stevia-related sweeteners has been indicated to benefit the glucose metabolism ( Jeppesen, Gregersen, Alstrup, & Hermansen, 2002) and renal function ( Hsieh et al., 2003). Despite its natural origin and possible benefits, there have been serious concerns Fasudil manufacturer about its safety; hence, the toxicity, carcinogenicity

and genotoxicity of stevioside have been investigated. These studies have been conducted mainly in Japan, where Stevia is approved as a food additive ( Aze et al., 1991, Matsui et al., 1996b, Matsui et al., 1996a, Pezzuto et al., 1985, Toskulkao et al., 1997 and Xili et al., 1992). The results have often suggested that stevioside has no serious toxicity to mammals. Recently, however, an in vitro study of the metabolism of several glycosidic sweeteners showed that Stevia-related compounds are degraded to steviol 2 ( Fig. 1) by human faecal homogenates, and no apparent inter-species differences in the intestinal metabolism between rats and humans

PD98059 concentration of Stevia-related compounds were observed ( Koyama et al., 2003). Since steviol is highly lipophilic, it has been postulated that it will be absorbed into the systemic circulation ( Wingard et al., 1980). Steviol has also been known to be mutagenic after metabolic activation in the mutation assay using S. typhi TM677 ( Pezzuto et al., 1985), and a possible decrease of the fertility of male rats was also suggested ( Melis, 1999). This apparent toxicity led Australia and Canada, for instance, to approve Stevia only as a food supplement, but not as a food additive. These studies provide therefore conflicting conclusions and insufficient toxicological information about the safety of steviol. Therefore, the concerns about the safety use of the natural stevioside sweetener still remain ( WHO, 1999). Lack of critical scientific reports on stevioside and their discrepancies about the toxicological

effects of its aglycone steviol led the European Commission in 2000 to refuse to accept Stevia as a food or drug additive ( FAO/WHO, 2004). Normally, stevioside and steviol have been analysed by HPLC with ultraviolet Myosin detectors (Hutapea et al., 1999 and Koyama et al., 2003). Herein we applied direct infusion ESI(+)-MS for the on-line monitoring of stevioside hydrolysis. ESI(+)-MS has been used as an interesting “ion-fishing” technique, since it is able to gently transfer with high speed and sensitivity either positive or negative ions (even transient species) directly from solutions to the gas phase (de la Mora et al., 2000). Due to these outstanding features, ESI-MS (and its tandem version ESI-MS/MS) is rapidly becoming a major tool in chemistry and biochemistry for the fast screening of reaction intermediates in solution (Santos, 2008, Santos, 2010 and Santos et al.