The optimized chromatographic analysis was carried out using a Waters Alliance 2695 liquid chromatography system (Waters, Milford, CT, USA) equipped with 2996 photodiode array detector (Waters, Milford, CT, USA) and ACE C18 (150 × 4.6 mm, 3 μm) column (ACT, Aberdeen, UK). Since all triterpenoids cannot be separated in a single chromatographic run, different chromatographic conditions were used.
For the analysis of triterpenoid acids (maslinic, corosolic, betulinic, oleanolic, ursolic acids) and neutral triterpenoids with chromophores (betulin, erythrodiol, uvaol), the mobile phase consisted of acetonitrile and water (89:11, v/v), delivered at a flow rate of 0.7 mL/min in the isocratic mode. The column temperature was set at 20 °C with an injection volume of 10 μL. Whereas the isocratic elution system for the analysis of neutral triterpenoids, which lacks chromophores (lupeol, β-amyrin, α-amyrin, friedelin) and phytosterol (β-sitosterol), consisted of acetonitrile and methanol (10:90, v/v). The column temperature was set at 35 °C, the flow rate was 1 mL/min, and the sample injection volume was 10 μL.
Detection of all triterpenoids was performed at a wavelength of 205 nm corresponding to the maximum absorption, and peaks were identified with retention times as compared with standards.
For the analysis of triterpenoid acids (maslinic, corosolic, betulinic, oleanolic, ursolic acids) and neutral triterpenoids with chromophores (betulin, erythrodiol, uvaol), the mobile phase consisted of acetonitrile and water (89:11, v/v), delivered at a flow rate of 0.7 mL/min in the isocratic mode. The column temperature was set at 20 °C with an injection volume of 10 μL. Whereas the isocratic elution system for the analysis of neutral triterpenoids, which lacks chromophores (lupeol, β-amyrin, α-amyrin, friedelin) and phytosterol (β-sitosterol), consisted of acetonitrile and methanol (10:90, v/v). The column temperature was set at 35 °C, the flow rate was 1 mL/min, and the sample injection volume was 10 μL.
Detection of all triterpenoids was performed at a wavelength of 205 nm corresponding to the maximum absorption, and peaks were identified with retention times as compared with standards.
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