Response surface modeling of betulinic acid pre-concentration from medicinal plant samples by miniaturized homogenous liquid–liquid extraction and its determination by high performance liquid chromatography

This work describes a miniaturized homogenous liquid–liquid extraction (HLLE), combined with high performance liquid chromatography (HPLC) procedure to determine betulinic acid in the medicinal plant samples. The method was based on the rapid extraction of betulinic acid from a methanol solution of sample into 67μL chloroform, as an extraction solvent. After addition of water into the mixture, the extracting solvent phase immediately formed a distinct water-immiscible phase under the vial, which could easily be separated, evaporated and re-dissolved in 0.5mL of acetonitrile for further HPLC analysis. The effects of various experimental parameters in the extraction step were also studied using response surface methodology. Three independent variables were volume of extracting solvent (A: 30–90μL), time (B: 1–10min) and volume of water (C: 1–10mL). The statistical analysis showed that the independent variable A, the quadratic term (A2) and the interaction between B and C have significant effects on the peak area of betulinic acid (p