UHPLC Q-Orbitrap Mass Spectrometry-Based Molecular Networking for Identification of Chemical Constituents in the Multi-Herb Formula Runyan Mixture

Abstract

Traditional Chinese medicine (TCM) in-hospital preparations are approved for use only in the hospital where they are prepared. They are widely used in China because of their efficacy and affordable price. However, few researchers focused on their quality controls and treatment mechanisms, for which a key consideration is the elucidation of their chemical composition. Runyan mixture (RY) is a typical in-hospital TCM preparation comprising a formula of eight herbal drugs used for adjuvant therapy of upper respiratory tract infections. The chemical constituents of formulated RY have not yet been elucidated. In the present work, RY was analyzed by a ultrahigh-performance liquid chromatography system equipped with high-resolution orbitrap mass spectrometry (MS). The acquired MS data were processed by MZmine and a feature-based molecular networking was constructed to identify the metabolites of RY. 165 compounds including 41 flavonoid O-glycosides, 11 flavonoid C-glycosides, 18 quinic acids, 54 coumaric acids, 11 iridoids, and 30 others were identified. This study demonstrates an efficient method to identify compounds in complex herbal drug mixtures using high-resolution MS and molecular networking tools which will support future research into quality controls and treatment mechanisms of in-hospital TCM preparations.

Figure 1

UPLC-UV chromatogram at 280 nm (A) and base peak ion chromatograms (BPC) in negative model (B) of RY.

Figure 2

MN and annotation of flavonoid C-glycosides in RY. (A) MN of flavonoid C-glycosides in RY. (B) The MS/MS spectrum of vicenin 2. (C) The proposed fragmentation pathway of vicenin 2. (D) The MS/MS spectrum of FC10 of proposed structure 6‴-(3-hydroxy-3-methylglutaryl) violanthin.

Figure 3

MN and annotation of flavonoid O-glycosides in RY. (A) MN of flavonoid O-glycosides in RY. (B) The MS/MS spectrum of FO7 and FO8. (C) The flavonoid aglycones tentatively identified in RY.

Figure 4

MN of coumaric acids. Five kinds of coumaric acids are indicated with different colors. (A) Sub-MN consisting of 4-hydroxybenzoyl, 3,4-dihydrobenzoyl, coumaroyl, and caffeoyl compounds. (B) Sub-MN consisting of feruloyl compounds.