LC-ESI-MS/MS Method for Simultaneous Determination of Triterpenoid Glycosides and Aglycones in Centella asiatica L.

Abstract

A new and rapid method for simultaneous identification and estimation of bioactive triterpenoid glycosides [asiaticoside (AS) and madecassoside (MS)] and their aglycones [asiatic acid (AA) and madecassic acid (MA)] in Centella asiatica was developed by using high-performance liquid chromatography (HPLC) coupled with triple-quadrupole mass spectrometry (MS/MS). Estimation was based on multiple reaction monitoring (MRM) using the precursor → product ion combination for determination of four analytes using Alltima C18 column (50 × 4.6 mm, 3 µm). An electrospray ionization (ESI) tandem interface in positive mode was employed prior to mass-spectrometric detection. The method was subjected to a thorough validation procedure in terms of linearity, limit of detection (LOD) and quantification (LOQ), accuracy, and precision. Six-point calibration curves were linear in the range of 50–500 ng mL−1 for AS and MS, and 25–250 ng mL−1 for AA and MA, with excellent linearity (R 2 > 0.98). With the optimized conditions, the four analytes were detected accurately within 10 min. LOD and LOQ ranged from 2.5 to 5 and 10 to 15 ng mL−1, respectively. Method accuracy in terms of average recoveries of all four analytes ranged between 98.61 and 102.85 % at three spiking levels with intra- and interday precision relative standard deviation (RSD, %) of 1.01–4.62 and 1.13–4.16, respectively. The new method was successfully applied to estimate the concentration of these four bioactive compounds in extracts of C. asiatica prepared by nonpolar-to-polar solvents.