Isomeric differentiation of bile acids using three-dimensional MS² spectrum

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 alpha-H and 5 beta-H epimers; type-II, alpha-OH and beta-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 MS2 (3D-MS2) spectrum that was accomplished by fortifying energy-resolved MS (ER-MS) program as the new dimension. The breakdown graphs of primary MS2 spectral signals composed of 3D-MS2 spectrum namely full collision energy ramp (FCER)-MS2 spectrum for each BA species after appropriate normalization, and seven isomeric BAs covering all isomerism styles were utilized as representative cases. After recording MS2 spectra with progressive CE levels, differences were observed for their FCER-MS2 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 (RIImax) 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-MS2 spectrum, regardless of the isomerism manner.