Evaluation of steviol and its glycosides in Stevia rebaudiana leaves and commercial sweetener by ultra-high-performance liquid chromatography-mass spectrometry.

Stevia rebaudiana leaves contain non-cariogenic and non-caloric sweeteners (steviol-glycosides) whose consumption could exert beneficial effects on human health. Steviol-glycosides are considered safe; nonetheless, studies on animals highlighted adverse effects attributed to the aglycone steviol. The aim of the present study was to develop and validate two different ultra-high-performance liquid chromatography methods with electrospray ionization mass spectrometry (UHPLC-MS) to evaluate steviol-glycosides or steviol in Stevia leaves and commercial sweetener (Truvia). Steviol-glycosides identity was preliminarily established by UV spectra comparison, molecular ion and product ions evaluation, while routine analyses were carried out in single ion reaction (SIR) monitoring their negative chloride adducts. Samples were sequentially extracted by methanol, cleaned-up by SPE cartridge and the analytes separated by UHPLC HSS C18 column (150 mm x 2.1 mm I.D., 1.8 microm). The use of CH2Cl2 added to the mobile phase as source of Cl- enhance sensitivity. The LLOD for stevioside, rebaudioside A, steviolbioside and steviol was 15, 50, 10 and 1 ng ml(-1), respectively. Assay validation demonstrated good performances in terms of accuracy (89-103%), precision (<4.3%), repeatability (<5.7%) and linearity (40-180 mg/g). Stevioside (5.8+/-1.3%), rebaudioside A (1.8+/-1.2%) and rebaudioside C (1.3+/-1.4%) were the most abundant steviol-glycosides found in samples of Stevia (n=10) from southern Italy. Rebaudioside A was the main steviol-glycosides found in Truvia (0.84+/-0.03%). The amounts of steviol-glycosides obtained by the UHPLC-MS method matched those given by the traditional LC-NH2-UV method. Steviol was found in all the leaves extract (2.7-13.2 mg kg(-1)) but was not detected in Truvia (<1 microg kg(-1)). The proposed UHPLC-MS methods can be applied for the routine quality control of Stevia leaves and their commercial preparations.