Rapid Ion Mobility Separations of Bile Acid Isomers Using Cyclodextrin Adducts and Structures for Lossless Ion Manipulations

Abstract

Bile acids (BAs) constitute an important class of steroid metabolites often displaying changes associated with disease states and other health conditions. Current analyses for these structurally similar compounds are limited by a lack of sensitivity and long separation times with often poor isomeric resolution. To overcome these challenges and provide rapid analyses for the BA isomers, we utilized cyclodextrin adducts in conjunction with novel ion mobility (IM) separation capabilities provided by structures for lossless ion manipulations (SLIM). Cyclodextrin was found to interact with both the tauro- and glyco-conjugated BA isomers studied, forming rigid noncovalent host-guest inclusion complexes. Without the use of cyclodextrin adducts, the BA isomers were found to be nearly identical in their respective mobilities and thus unable to be baseline resolved. Each separation of the cyclodextrin-bile acid host-guest inclusion complex was performed in less than 1 s, providing a much more rapid alternative to current liquid chromatography-based separations. SLIM provided capabilities for the accumulation of larger ion populations and IM peak compression that resulted in much higher resolution separations and increased signal intensities for the BA isomers studied.

Figure 1.

SLIM SUPER IM multipass long ion path length module design used for separations (top) and a depiction of the SLIM separation compression ratio ion mobility programming (CRIMP) process (bottom).

Figure 2.

Chemical structures of the tauro-BAs studied. SLIM SUPER IM–MS analyses were performed using a 85.5 m path length, and all four tauro-BA isomers were observed as [M+K]⁺ and [M–H]⁻ ions. Traveling wave IM separations were optimized for best ion transmission in both polarities; however, the four isomers could not be baseline separated in either mode.

Figure 3.

BA isomer SLIM SUPER IM separations with and without a CRIMP step. (A) 18 m separation of the four isomers without (top) and with compression (bottom). (B) 72 m separation of the four isomers without (top) and with compression (bottom). All species shown are the [M+α+H+K]²⁺ ions, where M is the BA and α is the cyclodextrin. IM peak assignments for the BA isomers from Figure 3 are based on BA species run individually (see the Supporting Information). Ions were accumulated for 1 s in-SLIM prior to separation.

Figure 4.

Glyco-BA isomer separations. SLIM SUPER IM separation of the three glyco-BA isomers using the same parameters as the tauro-BA separations. 72 m SLIM SUPER IM separation as [M+α+H+K]²⁺ ions (left) and 85.5 m SLIM SUPER IM separation as [M+K]⁺ ions (right), where M is the BA and α is the cyclodextrin. IM peak assignments are based on BA species run individually (see the Supporting Information). Ions were accumulated for 1 s in-SLIM prior to separation.