Rapid separation and determination of betulinic acid from a complex matrix using combination of TLC and RP-HPLC

Hitherto, only a few studies are reported about using the combination of TLC and RP-HPLC for the separation and determination of analyte(s) from a complex matrix. The present study is aimed to develop a simple and rapid method for the separation and determination of betulinic acid from a complex matrix, extracts of Orthosiphon stamineus, using a combination of the two techniques. The samples having higher contents of the analyte and fewer interfering species were prepared using TLC. The samples were then eluted through C18 column using isocratic solvent system comprising acetonitrile, methanol and acetic acid acidified water of pH 2.8 in a ratio of 70 : 20 : 10 (v/v/v), respectively, and detection was carried out at 210 nm. The method was validated and applied successfully to quantify betulinic acid in various types of extracts of the plant. The limit of detection (LOD) and limit of quantification (LOQ) were found to be 0.0005 and 0.0050 μg/ml, respectively. The method exhibited linearity in a concentration range of 0.005-100.00 μg/ml (R2= 0.9999). The recovery was found to be 97.10 - 97.60 % (RSD 5%), whereas, intra-day and inter-days accuracy values were 97.13 - 98.67% (RSD 5%) and 96.45 - 98.00% (RSD 5%), respectively. The results of the present study indicate that the developed method is simple, rapid, sensitive and accurate, and may be of a value to natural product industry and researchers for the standardization of extracts containing betulinic acid in a lesser time and consuming fewer solvents.