| Objective:(1)To develop an off-line two-dimensional mixed-mode liquid chromatography × reversed-phase liquid chromatography system.(2)Comprehensive ingredients profiling of Sanguisorba officinalis L.(S.officinalis)(3)Construction a strategy for comprehensively profiling the metabolites and the metabolic rules of S.officinalis in vivo.Methods:(1)The chromatographic columns with different selectivity were screened to develop the off-line two-dimensional liquid chromatography(2D-LC)system and the orthogonality of the system was evaluated.(2)The 2D-LC system tandem mass spectrum was used to separate and characterize S.officinalis based on molecular ions and fragment ions under the guidance of molecular network.(3)An ultra-high performance liquid chromatography tandom highresolution mass spectrum and molecular network were utilized to comprehensively profile the metabolic rules of S.officinalis in vivo.Results:(1)An off-line 2D-LC system which exhibits a great separation selectivity and orthogonality,which is composed of a newly prepared stationary phase(M-PVS)and a traditional C18 phase.The retention time(R2 = 0.0582)and m/z value(R2 = 0.0121)detected in the second dimension did not correlate with the first dimension fraction number.The number of detected compounds and separated peaks were significantly increased while ion suppression was reduced when onedimensional liquid chromatography upgraded to 2D-LC.(2)In addition,270 compounds of S.officinalis(127 of which were tentatively characterized as new compounds,and further confirmation is needed),including ellagic acids,flavonoids,glycosides,and saponins,were characterized.(3)A total of 1678 exogenous metabolites were detected,of which 913,601,364,and 160 were present in urine,feces,plasma,and bone marrow,respectively.Furthermore,104 metabolites were tentatively characterized,including 31 metabolites from phase I and 57 metabolites from phase II.Metabolites of S.officinalis in rats were mainly excreted through the urine and feces.Conclusion:(1)The 2D-LC system has the ability to provide higher peak capacity and different selectivity,which is an important method for the separation of complex samples.(2)The 2D-LC system tandem HRMS with powerful characterization ability is suitable for the separation and analysis of Traditional Chinese Medicine(TCM).(3)The established metabolic analysis method in vivo has the ability to intuitively profile the metabolite information,such as metabolites characterization,tissue distribution and metabolic pathways,which is a powerful method for the metabolite profiling of TCM. |
