Primary bile acids are cholesterol-derived molecules that are synthesized in the liver and are collected and pre-concentrated in the bile contained in the gall bladder. During gastric emptying, bile is secreted into the duodenum via the common bile duct, where bile acids play a crucial role in emulsifying and absorbing dietary fats (8,9). The two primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA) are bioconverted into chemically distinct, bacterially produced secondary bile acids and an expanding list of microbially-conjugated bile acids (MCBAs) by host microbiota. Of considerable recent interest is the potential role of these bacterial-derived secondary bile acids in human physiology and their contributions to pathologies such as inflammatory bowel disease and cancer. Consequently, there is great interest in identifying and quantifying these compounds in diverse samples.
Analysis of bile acids by nominal mass instruments, such as triple-quadrupole MS (TQMS) systems, is challenging because of the high chemical background found in several precursor ions to precursor ion-based multiple-reaction monitoring (MRM) transitions used in current state-of-the-art assays. High-resolution mass spectrometry (HRMS) generates a full product ion spectrum for each targeted bile acid and extracting fragment ions with a narrow mass-to-charge (m/z) window can reduce background chemical interferences and improve the signal-to-noise (S/N) of the assay. The detection of individual bile acid isomers currently depends on chromatographic resolution; collision-induced dissociation (CID)-based fragmentation cannot distinguish these isomeric metabolites. Electron-activated dissociation (EAD) is a complementary fragmentation mode to chromatographic separation. The ZenoTOF 7600 system has an EAD cell that provides complimentary fragmentation that can be activated and scheduled within a typical quantitative bioanalytical method to provide structural characterization of bile acids. To demonstrate the power of EAD for structural characterization, the DCA sub-class was structurally characterized to identify EAD-specific fragments for each isomer.