The Study Of A Novel Strategy For The In Vivo Metabolite Identification Of Licorice Using UHPLC-MS/MS

Traditional Chinese medicines(TCMs)have been used for the treatment of various diseases with a long history,and have aroused worldwide interests due to the significant efficacy in treating chronic and systemic diseases.The comprehensive study of the in vivo metabolism is an essential part of pharmaceutical research of TCMs,which can facilitate understanding the potential therapeutic material basis in vivo.However,the in vivo metabolite identification of TCMs is quite difficult because of the complicated chemical compositions,endogenous interferences,trace concentrations and unpredictable metabolic pathways.Here,we proposed a sensitive,reliable and economical strategy using ultra-high performance liquid chromatographytriple quadrupole-linear ion trap mass spectrometry(UHPLC-QTRAP-MS/MS)to comprehensively and rapidly identify the in vivo metabolites of TCMs at an oral clinical dosage.Licorice,the roots and rhizomes of Glycyrrhiza uralensis,Glycyrrhiza inflata,and Glycyrrhiza glabra,was selected as the model herb because it is widely used in most traditional Chinese medicine prescriptions.First,the metabolism of six representative licorice compounds in rat liver microsomes was studied to obtain their fragmentation behaviors and metabolic pathways.Multiple ion monitoring-enhanced product ion(EPI)and neutral loss-EPI scan were used to detect the untargeted metabolites in vitro.Subsequently,a sensitive multiple reaction monitoring(MRM)-EPI method was established according to the in vitro results and predicted metabolites.Finally,the in vivo metabolites of licorice were detected and identified using the QTRAP-based MRM-EPI method.The number of licorice metabolites identified by UHPLC-QTOFMS/MS and UHPLC-QTRAP-MS/MS were compared to investigate this strategy.After oral administration of licorice water extract at a normal dosage,a total of 45 metabolites in urine,21 metabolites in feces and 35 metabolites in plasma were detected using this new strategy.However,only 21 metabolites in urine,10 metabolites in feces and 14 metabolites in plasma were tentatively characterized when using conventional UHPLC-QTOF-MS/MS method.It is obvious that UHPLC-QTRAP-MS/MS method has advantages in identification of the in vivo metabolites of TCMs.As a result,18 metabolites had not been previously reported and 6 metabolites were first detected in vivo.Other metabolites were consistent with literature reports.These results suggested that this new strategy is sensitive and reliable,and is especially suitable for the in vivo metabolite identification of TCMs after oral administration at normal clinical dosage.It could be widely used as a reliable tool for comprehensive detection and characterization of in vivo metabolites of TCMs.