Identification and Simultaneous Determination of Twelve Active Components in the Methanol Extract of Traditional Medicine Weichang'an Pill by HPLC-DAD-ESI-MS/MS

Abstract

Weichang'an (WCA) pill, a traditional Chinese patent medicine consisting of ten Chinese medicinal herbs, has been used to treat irritable bowel syndrome and functional dyspepsia for several decades. In this study, twelve bioactive constituents in the methanol extract of WCA were accurately identified since MS/MS fragmentation behavior of the references and the standards by using HPLC-DAD-ESI-MS/MS analysis and a reliable and accurate method for the simultaneous determination was developed. Twelve active components including costunolide and dehydrodehydrocostus lactone from the principal herb Radix Aucklandiae; naringin, hesperidin and neohesperidin from Fructus Aurantii; magnolol and honokiol from the ministerial herbs Cortex Magnoliae officinalis; aloe-emodin, rhein, emodin, chrysophanol and physcion from adjunctive and messenger herb Radix et Rhizoma Rhei were analyzed in the samples. The chromatographic separation was performed on a Kromasil C18 column with gradient elution of acetonitrile-methanol and 1.0% acetic acid water. In this condition, linearity, inter- and intra-day precision and accuracy were within acceptable ranges. The developed method showed satisfactory precision and accuracy with overall intra- and inter-day variations of 0.68-1.33% and 0.67-2.05% respectively, and the overall recoveries of 97.54-102.69% for twelve compounds. The proposed approach was successfully applied as a powerful tool for the quality control of WCA pill.

Keywords: HPLC-DAD-ESI-MS/MS; Quantitative analysis; Traditional medicine; Weichang’an Pill..

Figure 1

Chemical structures of the twelve bioactive compounds to be determined in WCA

Figure 2

Chromatograms of WCA by HPLC-MS (A) TIC chromatogram in ESI positive mode.(B) TIC chromatogram in negative ESI mode. (C) TIC chromatogram of MSⁿ in ESI positive mode. (D) TIC chromatogram of MSⁿ in ESI positive mode.

Figure 3

Typical chromatograms of the standard mixture (A) and WCA methanol extract (B) at 230, 254, 280 nm. (1) naringin; (2) hesperedin; (3) neohesperedin; (4) aloe-emodin; (5) rhein; (6) emodin; (7) honokiol; (8) dehydrodehydrocostus lactone; (9) costunolide; (10) magnolol; (11) chrysophanol; (12) physcion.