Isomeric differentiation of bile acids using three‐dimensional MS2 spectrum

Abstract Bile acids (BAs) currently occupy the research hotspot because of their unreplaceable physiological functions as well as their unique abilities to reflect the physiopathological status. It is always challenging to characterize BAs‐submetabolome using LC–MS/MS, primarily attributing to the high‐level structural diversity. The key obstructing confirmative identification is isomeric identification because of the inherent “isomer‐blind” disadvantage of MS/MS. The isomerism styles of BAs are broadly divided into: type‐I, 5 α ‐H and 5 β ‐H epimers; type‐II, α ‐OH and β ‐OH epimers; type‐III, C‐OH positional isomers; and type‐IV, hybrid isomers bearing two or more isomerism fashions. Herein, we aim to comprehensively pursue isomer‐selective clues for BAs through constructing three‐dimensional MS 2 (3D‐MS 2 ) spectrum that was accomplished by fortifying energy‐resolved MS (ER‐MS) program as the new dimension. The breakdown graphs of primary MS 2 spectral signals composed of 3D‐MS 2 spectrum namely full collision energy ramp (FCER)‐MS 2 spectrum for each BA species after appropriate normalization, and seven isomeric BAs covering all isomerism styles were utilized as representative cases. After recording MS 2 spectra with progressive CE levels, differences were observed for their FCER‐MS 2 spectra. Diagnostic fragment ions (DFIs) occurred within type‐I, type‐II, type‐III or type‐IV isomers, and noteworthily, 2.02 Da neutral loss discriminated BAs containing cis ‐6,7‐diol and trans ‐6,7‐diol functional groups. Notably, the features such as the maximal relative ion intensity (RII max ) and optimal CE (OCE) enabled isomeric differentiation for all types, even type‐II and type‐III isomers. Above all, it is feasible to acquire in‐depth isomer‐selective MS/MS clues for BAs using FCER‐MS 2 spectrum, regardless of the isomerism manner.