Triterpene purification by HPLC

We utilized a bead milling process to purify raw rubber from T. koksaghyz roots⁶² and extracted therein-comprised lipids by acetone for 7 days at room temperature. Semi-preparative HPLC of the lipid extract was carried out using a Shimadzu LC20A HPLC system (Shimadzu, Duisburg, Germany) coupled to a UV detector (SPD-M20A) and a fraction collector (FRC-10A). The triterpenes were separated using an Ultra C18 column (250 × 21.2 mm, particle size: 5 µm, Restek GmbH, Bad Homburg, Germany) and methanol as solvent with a flow rate of 10 ml min⁻¹. The column oven temperature was set to 40 °C. Detection was carried out at 205 nm and the triterpene fractions were collected, dried using Rocket evaporator system (Thermo Fisher Scientific), dissolved in acetone and analysed by GC-MS. In a second purification step, an Ultra Biphenyl column was used as a stationary phase (250 × 21.2 mm, particle size: 5 µm, Restek GmbH, Bad Homburg, Germany). The column oven temperature was set to 40 °C and the triterpenes were separated with a gradient of methanol (A) and water (B) at a flow rate of 8 ml min⁻¹ using the following elution profile: 0–25 min, isocratic 90% A; 25–71 min, linear from 90% to 100% A; 71–75 min, isocratic 100% A; followed by column re-equilibration: 75–76 min, linear from 100% to 90% A; 76–85 min, isocratic 90% A. Triterpenes were identified by GC-MS as previously described⁷ using standard compounds (β-amyrin, α-amyrin, lupeol and lupenone were purchased from Extrasynthese, Genay, France; Taraxerol and β-amyrone from Sigma-Aldrich, Taufkirchen, Germany). For quantification, the fractions of one HPLC-run (C18-column) were collected, dried, dissolved in 1 ml of acetone and analysed by GC-MS. Peak areas of total ion counts (TICs) were used for calculating the percentage amount of the single triterpenes.