HPLC Quantification of Steviol Glycosides

SGs (steviol glycosides stevioside and rebaudioside A) were separated and quantified using high-performance liquid chromatography (HPLC) by the modified method [53 (link)]. An Agilent 1200 series HPLC system (Agilent Technologies Inc., Santa Clara, CA, USA) with a diode array detector was used. Samples were filtered through a syringe filter with a PVDF membrane (pore diameter, 0.22 µm) and separated on a reversed phase column (Purospher STAR RP-18e 5 µm Hibar 2 × 250 mm, Merck, Germany) with a precolumn. Injection volume was 10 µL at 70 °C column temperature. Isocratic elution with a mobile phase consisting of 70% deionized water acidified with HCl to pH 2.75 and 30% acetonitrile was used for separation, with an additional washing step with 50% acetonitrile at a flow rate of 0.25 mL min⁻¹. Stevioside and rebaudioside A were detected at the wavelength of 210 nm. Identification of stevioside and rebaudioside A in the samples was done by means of retention time and UV spectra. Calibration was done by plotting the peak area responses against the concentration values in the concentration range from 1 to 1000 µg mL with linear dependence for both analytes. Each analysis was repeated three times, and the mean value was used. Working under an isocratic mode, less than 10 min was required to separate the components of interest without affecting resolution.