HPLC

Hernandez, Karel Vives, Moreno-Romero, Jordi, Hern, Martha, Torre, andez de la, P, Claudia, Manríquez, erez, Leal, Darcy Ríos & Martínez-Garcia, Jaime F., 2022, Effect of light intensity on steviol glycosides production in leaves of Stevia rebaudiana plants, Phytochemistry (113027) 194, pp. 1-10 : 7

4.3. SGs analysis by HPLC

To quantify the levels of SGs, high performance liquid chromatography (Agilent 1200 series HPLC ) was used under isocratic conditions in a Varian System (Darmstadt, Germany), consisting of a Pro Star 230 pump, a 335-diode array detector set to a wavelength of 210 nm. To detect stevioside and rebaudioside A, a Luna HILIC analytical column (250 × 4.6 mm, 5 μm particle size, Phenomenex, Aschaffenburg, Germany) was used. The mobile phase consisted of acetonitrile: water (85:15, v: v) adjusted to a flow rate of 1.0 ml⋅min 1 maintained at 25 ◦ C.

Steviol glycoside standards (stevioside and rebaudioside A, obtained from Sigma Aldrich, Darmstadt, Germany) were prepared by diluting them in acetonitrile and ethanol (1:1) at a concentration of 1 mg ⋅ml 1 and determined under the conditions described above ( Fig. 6 ).

For the extraction of SGs, the procedure of W¨olwer-Rieck et al. (2010) was used. After harvesting, S. rebaudiana leaves were freeze-dried at all experimental stages. Samples were ground using a mortar and pestle and liquid nitrogen to produce a fine powder. 12 mg were weighed and collected in a 2 ml Eppendorf tubes. Subsequently, the extraction process was carried out with a volume of 1.5 ml of ethanol and acetonitrile (1:1; v: v) in a heating block at 102 ◦ C for 30 min. The supernatant was recovered. Plant material was re-extracted using the same procedure three additional times, to then reach a final volume of 10 ml. Each extract was cooled to room temperature and centrifuged for 15 min at 12,000 rpm The SGs content was expressed in μg⋅mg 1 of dry weight.

Figures

Fig. 6. Chromatograms of stevioside and rebaudioside A standard solutions used for the quantification of S. rebaudiana leaves extracts. Intensity of absorbance in milli-Absorbance Units (mAU).