Online coupling of the Ussing chamber, solid-phase extraction and high-performance liquid chromatography for screening and analysis of active constituents of traditional Chinese medicines

Abstract

A semi-automated online platform was established successfully for preliminary screening of potential active flavonoids of traditional Chinese medicines (TCMs) in multicomponent system. Online coupling of the in vitro intestinal absorption model, solid phase extraction (SPE) and high-performance liquid chromatography (HPLC) was actualized at the first time. The Ussing chamber model was selected to absorb the constituents of TCMs. A mini chromatographic column filled with C18 was used as a SPE column for online enrichment of flavonoids. HPLC was applied to analyze the constituents screened by platform. With the use of rutin as a model flavonoid, the specifications of SPE column, eluting solvent, elution time and flow rate of eluent were systematically investigated to optimize online system. Under the optimal conditions, the linear range of rutin was 0.125-368 µg/mL with the correlation coefficient (R²) greater than 0.9947. The limit of detection (LOD) was as low as 0.0500 µg/mL and the limit of quantification (LOQ) was 0.125 µg/mL. The intra-day relative standard deviation (RSD) and inter-day RSD was 2.5% and 3.8%, respectively. The recoveries of rutin in the intestinal absorption samples ranged from 93.2% to 94.0%. Finally, the online system was applied to screen the potential active flavonoids of Scutellaria baicalensis Georgi (Huangqin, HQ) and Polygoni Cuspidati Rhizoma et Radix (Huzhang, HZ). A total of 14 flavonoids of these two TCMs were identified by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS), and 12 flavonoids of them were screened as the potential active components by online Ussing chamber-SPE-HPLC. In comparison with offline method and gavage in rats, the online system can screen the active constituents from TCMs more accurately and completely. The results demonstrated that the online system was reliable and sufficiently accurate for screening and determination of the potential active flavonoids of TCMs in multicomponent system.

Keywords: Active constituents; Flavonoid; Intestinal absorption; Online screening; Traditional Chinese medicines; Ussing chamber-SPE–HPLC.