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The use of dietary flavones is becoming increasingly popular for their prevention of cancers,cardiovascular diseases,and other diseases.Despite many pharmacokinetic studies on flavone mixtures,the position(s)of glucuronidation sites on the flavone skeleton in vivo remain(s)uncertain because of the lack of a convenient method to differentiate the isomers in biological samples.Accordingly,this study aimed to develop a new strategy to identify the position of the mono-O-glucuronide of flavones in vivo and to simultaneously determine the parent agent and its major metabolites responsible for complex pharmacokinetic characteristics.The novel strategy involves accurate mass measurements of flavone glucuronides,their [Co(II)(flavone glucuronide-H)(4,7-diphenyl-1,10-phenanthroline)2 ]+complexes generated via the post-column addition of CoBr2 and 4,7-diphenyl-1,10-phenanthroline,and their mass spectrometric fragmentation by UPLC-DAD-Q-TOF and the comparison of retention times with biosynthesized standards of different isomers that were identified by analyzing the shift in UV spectra compared with the spectra of their respective aglycones.We successfully generated a metabolite profiling of flavones in rat plasma after oral administration of a flavone mixture from Dracocephalum moldavica L.,which was used here as the model to demonstrate the strategy.Twelve flavone glucuronides,which were glucuronidated derivatives of acacetin,apigenin,luteolin,diosmetin,chrysoeriol and cirsimaritin,were detected and identified.Glucuronidation of the flavone skeleton at the 3-/7-position was more prevalent,however,luteolin 4-glucuronide levels exceeded luteolin 7-glucuronide levels.Based on the UDP-glucuronosyltransferase(UGT)metabolism profiling of flavones in rat plasma,six main compounds(tilianin,acacetin 7-glucuronide,apigenin 7-glucuronide,luteolin 3-glucuronide,acacetin,and apigenin)were selected as pharmacokinetic markers.Pharmacokinetic results indicated that their maximal concentrations in blood were obtained within 0.4 h,except for the concentration of luteolin 3-glucronide(approximately 9 h).Rat exposure was practically non-linear under the studied dosages(200 to 400 mg/kg).