Direct Flavonoid-Focused Chemical Comparison among Three Epimedium Plants by Online Liquid Extraction-High Performance Liquid Chromatography-Tandem Mass Spectrometry

Abstract

It is usually a tedious task to profile the chemical composition of a given herbal medicine (HM) using high performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) due to the time-consuming sample preparation and laborious post-acquisition data processing procedures. Even worse, some labile compounds may face degradation risks when exposed to organic solvents for a relatively long period. As one of the most popular HMs, the promising therapeutic benefits of Epimedii Herba (Chinese name: Yinyanghuo) are well defined; however, the chemical profile, and in particular those flavonoids that have been claimed to be responsible for the efficacy, remains largely unknown. Attempts are devoted here to achieve direct LC-MS measurement and efficient post-acquisition data processing, and chemome comparison among three original sources of Epimedii Herba, such as Epimedium sagittatum (Esa), E. pubescens (Epu), and E. koreanum (Eko) was employed to illustrate the strategy utility. A home-made online liquid extraction (OLE) module was introduced at the front of the analytical column to comprehensively transfer the compounds from raw materials onto the LC-MS instrument. A mass defect filtering approach was programmed to efficiently mine the massive LC-MS dataset after which a miniature database was built involving all chemical information of flavonoids from the genus Epimedium to draw a pentagonal frame to rapidly capture potential quasi-molecular ions (mainly [M-H]^-). A total of 99 flavonoids (66 in Esa, 84 in Eko, and 66 in Epu) were captured, and structurally annotated by summarizing the mass fragmentation pathways from the mass spectrometric data of authentic compounds and an in-house data library as well. Noteworthily, neutral loss of 144 Da was firstly assigned to the neutral cleavage of rhamnosyl residues. Significant species-differences didn't occur among their chemical patterns. The current study proposed a robust strategy enabling rapid chemical profiling of, but not limited to, HMs.

Keywords: Epimedii Herba; flavonoids; mass defect filtering; neutral loss of rhamnosyl residue; online liquid extraction.

Figure 1

Base peak chromatograms of E. sagittatum ESa, (A), E. koreanum EKo, (B) and E. pubescens EPu, (C), in negative ion mode.

Figure 2

Integer parts against decimal parts of the compounds in the in-house chemical library (red solid dots) and the detected MS¹ signals (orange diamonds for Esa, blue solid dots triangles for Eko, and green square for Epu). A five-point frame was drawn to involve all red solid dots, and only those dots in the frame were the potential flavonoids in Epimedium plants.

Figure 3

Proposed mass fragmentation pathways (A) and MS² spectrum (B) of m/z 821 ([M–H]⁻) for epimedin C, a representative isopentenyl flavonoid in Epimedium plants. *: [Y₀–H·]⁻.

Figure 4

Proposed mass cracking pathways (A) and MS² spectrum (B) for oxidized epimedin C that was featured by the 144 Da neutral loss. *: Y₀⁻.

Figure 5

The dried leaves of ESa (A), Eko (B) and EPu (C).

Figure 6

The schematic of OLE–LC–DAD–IT–TOF–MS. Each measurement is divided into two phases, including extraction (0–5 min) and elution (5–42 min) phases via switching the electronic valve from position I to position II.