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 67 μL 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 0.5 mL 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: 30–90 μL), time (B: 1–10 min) and volume of water (C: 1–10 mL). 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 < 0.05). The optimized conditions were found to be 67 μL of extracting solvent volume, an extraction time of 4.3 min and 5.2 mL of water volume. Under these conditions, the detection limit (LOD) was obtained as 1.6 ng/g. Furthermore, the relative standard deviation of the 10 replicate was less than 2.7%. The developed procedure was applied to the extraction and determination of betulinic acid in medicinal plant samples.