HPLC Analysis of Steviol Glycosides in Botanical Extracts

For analysis, 250 mg of dry biomass was extracted with 2 ml of methanol in an ultrasonic bath for 1 h at 30°C. Obtained extracts were filtered through a PTFE, 0.22 μm Millipore filter. Reversed-phase (RP)-HPLC analysis was conducted as described by Bayraktar et al. [29 ], with our modification, on a Merck-Hitachi liquid chromatograph (LaChrom Elite) equipped with a DAD detector L-2455 and Purospher RP-18e column. Analysis was carried out at 40°C with a mobile phase consisting of A—water and B—acetonitrile. The gradient was as follows: 10–20% B for 0–2 min; 20–35% B for 2–6 min; 35–40% B for 6–9 min; 40–50% B for 9–10 min; 50–80% B for 10–15 min; 80% B for 15–20 min; 80–10% B for 20–40 min; 10% B for 40–45 min at a flow rate 1 ml min^-1. Detection was performed at λ = 210 nm, and the UV spectra of all samples were scanned between 190 nm and 360 nm. Quantification was defined by measuring the peak area with reference to the standard curve derived from five concentrations (from 0.0625 mg ml^-1 to 1 mg ml^-1) of authentic reference compounds: stevioside (50956; Sigma-Aldrich) and Reb A (38462; Sigma-Aldrich).