4.5. Simultaneous Determination of Betulin and Betulinic Acid by Reverse-Phase HPLC

Betulin and betulinic acid were dissolved in methanol at 2 mg/mL and 1 mg/mL, respectively, in order to prepare the stock solutions. A series of dilutions according to the required concentrations were carried out to prepare the standard solutions for RP-HPLC assay. Standard curves of betulin and betulinic acid were prepared with five different concentrations each. The sample solution was filtered through a 0.22 μm membrane filter, and then an aliquot (10 μL) of the clean solution was injected into the RP-HPLC system. The assaying system used is described in the literature by Liu et al. [4]. In brief, two Waters 510 pumps (Waters, Milford, MA, USA), a sample injector (Rheodyne, Cotati, CA, USA) with a 20 μL loop, and a Waters 996 photodiode array detector made up the RP-HPLC system. A reversed-phase Symmetry C18 (250 mm × 4.6 mm i.d., 4 μL; Waters) column was used with acetonitrile:water in the ratio 91:9 (v/v) as the mobile phase at a flow rate of 1.0 mL/min under 30 °C. The detection wavelength was set at 210 nm.