Characterization of Bile Acid Isomers and the Implementation of High-Resolution Demultiplexing with Ion Mobility-Mass Spectrometry

Bile acids (BAs) are a complex suite of clinically relevantmetabolitesthat include many isomers. Liquid chromatography coupled to mass spectrometry(LC-MS) is an increasingly popular technique due to its high specificityand sensitivity; nonetheless, acquisition times are generally 10-20min, and isomers are not always resolved. In this study, the applicationof ion mobility (IM) spectrometry coupled to MS was investigated toseparate, characterize, and measure BAs. A subset of 16 BAs was studied,including three groups of isomers belonging to unconjugated, glycine-conjugated,and taurine-conjugated BA classes. A variety of strategies were exploredto increase BA isomer separation such as changing the drift gas, measuringdifferent ionic species (i.e., multimers and cationized species),and enhancing the instrumental resolving power. In general, Ar, N-2, and CO2 provided the best peak shape, resolvingpower (R (p)), and separation, especiallyCO(2); He and SF6 were less preferable. Furthermore,measuring dimers versus monomers improved isomer separation due toenhanced gas-phase structural differences. A variety of cation adductsother than sodium were characterized. Mobility arrival times and isomerseparation were affected by the choice of adduct, which was shownto be used to target certain BAs. Finally, a novel workflow that involveshigh-resolution demultiplexing in combination with dipivaloylmethaneion-neutral clusters was implemented to improve R (p) dramatically. A maximum R (p) increase was observed with lower IM field strengths to obtain longerdrift times, increasing R (p) from 52 to187. A combination of these separation enhancement strategies demonstratesgreat potential for rapid BA analysis.