Establishment Of The High Mass Resolution Spectrometry Method For Detecting Fecal Bile Acids Profiling In Mice And Its Application

Objective1.To establish a method for detecting fecal bile acids profiling in mice by ultra performance liquid chromatography-triple quadrupole time-of-flight mass spectroscopy(UPLC-Triple TOF-MS/MS).2.To analyze the bile acids profiling of fecal and small intestinal contents in low fat diet(LFD),high fat diet-induced obesity resistance(DIR)and high fat diet-induced obesity(DIO)groups by UPLC-Triple TOF-MS/MS,to find the differences of the bile acids profiling in fecal and small intestinal contents among three groups,and explore the association between the individual bile acids and obesity.Methods1.The fecal and small intestine contents analytes were extracted by solid phase extraction,and then detected by the UPLC-Triple TOF-MS/MS method.The analytes were separated by gradient elution on a Kinetex XB-C18 reverse phase chromatography column.The mobile phase conditions:15 mmol/L ammonium acetate(mobile phase A)and acetonitrile(mobile phase B).The electrospray ionization(ESI)was operated by negative mode.The accurate MS and MS/MS data of bile acids were obtained by full scan and product ion scan modes which were used for qualitative and quantitative analysis.2.The fecal and small intestinal bile acids profiling in LFD,DIR and DIO groups mice were analyzed by the target and non-target strategy.3.Nonparametric tests,Spearman correlation analysis and other statistical methods were adopted to explore the correlation between the individual bile acids and obesity.Results1.Forty kinds of known bile acids were quantified and 22 kinds of possible bile acids were identified and semi-quantified by UPLC-Triple TOF-MS/MS method.2.The fecal bile acids were linear in the range of 0.125-250 nmol/mg with a good correlation(R~2>0.9900),and the detection limits ranged among 0.005-0.030 nmol/mg.The intra-day precisions were less than16.03%,and the inter-day precisions were less than 20.84%.The average recoveries for fecal BAs were in the range of 80.64%to 121.46%.3.There were some differences in the fecal and small intestinal bile acids profiling among LFD,DIR and DIO groups.Compared with DIO group,the concentrations of total bile acids(total BAs),cholic acids(CAs),deoxycholic acids(DCAs)and chenodeoxycholic acids(CDCAs)in DIR and LFD groups were significantly lower(P<0.017).The small intestinal bile acids profiling of the three groups were significantly different.Compared with LFD group,the concentrations of total BAs and taurine-conjugated bile acids(T-BAs),CAs and muricholic acids(MCAs)in DIR and DIO groups were significantly higher(P<0.017),and the concentrations of these bile acids in DIR group were significantly higher than those in DIO group(P<0.017).4.Compared with DIO group,the concentration of intestinal tauro-?-MCA(T-?-MCA)in DIR group was significantly increased(P<0.017),and the concentration of fecal chenodeoxycholic acid(CDCA)in DIR group was significantly reduced(P<0.017).Intestinal T-?-MCA was negatively related to obesity(r_s=-0.788,P=0.000),and fecal CDCA was positively related to obesity(r_s=0.536,P=0.000).Conclusion1.This study established an UPLC-Triple TOF-MS/MS method for fecal bile acids profiling analysis with good repeatability and high accuracy.2.There were differences in the intestinal and fecal bile acids profiling among LFD,DIR and DIO groups.Intestinal T-?-MCA was negatively related to obesity,fecal CDCA was positively related to obesity.It indicates that the development of DIR may be related to the alterations of bile acids metabolism and gut microbiota.