HPLC Analysis of Steviol Glycosides

Steviol glycosides rebaudioside A (RebA) and stevioside (Stev) were separated and quantified using high-performance liquid chromatography (HPLC) [52 (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 at a flow rate of 0.25 mL min⁻¹ with a mobile phase consisting of 70% deionised water acidified with HCl to pH 2.75 and 30% acetonitrile was used for separation with an additional washing step with 50% acetonitrile. RebA and Stev were detected at the wavelength of 210 nm. Calibration was performed by plotting the peak area responses against the concentration values in the concentration range from 1 to 1000 µg mL⁻¹ with linear dependence for both analytes. Each analysis was repeated three times, and the mean value was used.