Extensive evaluation of plasma metabolic sample preparation process based on liquid chromatography-mass spectrometry and its application in the in vivo metabolism of Shuang-Huang-Lian powder injection

Abstract

Shuang-Huang-Lian powder injection (SHLPI) is a natural drug injection made of honeysuckle, scutellaria baicalensis and forsythia suspensa. It has the characteristics of complex chemical composition and difficult metabolism research in vivo. LC-MS platform has been proven to be an important analytical technology in plasma metabolomics. Unfortunately, the lack of an effective sample preparation strategy before analysis often significantly impacts experimental results. In this work, twenty-one extraction protocols including eight protein precipitation (PPT), eight liquid-liquid extractions (LLE), four solid-phase extractions (SPE), and one ultrafiltration (U) were simultaneously evaluated using plasma metabolism of SHLPI in vivo. In addition, a strategy of "feature ion extraction of the multi-component metabolic platform of traditional Chinese medicine" (FMM strategy) was proposed for the in-depth characterization of metabolites after intravenous injection of SHLPI in rats. The results showed that the LLE-3 protocol (Pentanol:Tetrahydrofuran:H₂O, 1:4:35, v:v:v) was the most effective strategy in the in vivo metabolic detection of SHLPI. Furthermore, we used the FMM strategy to elaborate the in vivo metabolic pathways of six representative substances in SHLPI components. This research was completed by ion migration quadrupole time of flight mass spectrometer combined with ultra high performance liquid chromatography (UPLC/Vion™-IMS-QTof-MS) and UNIFI™ metabolic platform. The results showed that 114 metabolites were identified or preliminarily identified in rat plasma. This work provides relevant data and information for further research on the pharmacodynamic substances and in vivo mechanisms of SHLPI. Meanwhile, it also proves that LLE-3 and FMM strategies could achieve the in-depth characterization of complex natural drug metabolites related to Shuang-Huang-Lian in vivo.

Keywords: Sample pretreatment method; Shuang-Huang-Lian powder injection; Supramolecular solvent; UNIFI™ metabolism platform; UPLC/Vion™-IMS-QTof-MS.