Evaluating the structural complexity of isomeric bile acids with ion mobility spectrometry

Abstract

Bile acids (BAs) play an integral role in digestion through the absorption of nutrients, emulsification of fats and fat-soluble vitamins, and maintenance of cholesterol levels. Metabolic disruption, diabetes, colorectal cancer, and numerous other diseases have been linked with BA disruption, making improved BA analyses essential. To date, most BA measurements are performed using liquid chromatography separations in conjunction with mass spectrometry measurements (LC-MS). However, 10-40 min LC gradients are often used for BA analyses and these may not even be sufficient for distinguishing all the important isomers present in the human body. Ion mobility spectrometry (IMS) is a promising tool for BA evaluations due to its ability to quickly separate isomeric molecules with subtle structural differences. In this study, we utilized drift tube IMS (DTIMS) coupled with MS to characterize 56 different unlabeled BA standards and 16 deuterated versions. In the DTIMS-MS analyses of 12 isomer groups, BAs with smaller m/z values were easily separated in either their deprotonated or sodiated forms (or both). However, as the BAs grew in m/z value, they became more difficult to separate with two isomer groups being inseparable. Metal ions such as copper and zinc were then added to the overlapping BAs, and due to different binding sites, the resulting complexes were separable. Thus, the rapid structural measurements possible with DTIMS-MS show great potential for BAs measurements with and without prior LC separations.

Keywords: Bile acids; Collisional cross sections; Ion mobility spectrometry.

Figure 1.

A schematic of the metabolic pathway that transforms cholesterol into primary and secondary bile acids through processing in the liver and the intestines. Structures of primary and secondary bile acids to illustrated to show their similarities.

Figure 2.

A schematic of the primary BA structures (left) and examples of their glycine and taurine conjugated forms (right).

Figure 3.

The m/z versus CCS trend line for the 56 unlabeled BAs in their A) deprotonated [M-H]⁻ and B) sodiated [M+Na]⁺ forms. The unconjugated, glycine conjugated and taurine conjugated BAs are noted with blue, orange and grey colors. The CCS measured in DTIMS with nitrogen gas are noted as ^DTCCS_N2, with the unit of Ų.

Figure 4.

Arrival time distributions for the sodiated and deprotonated forms of lithocholenic acid (LCLA). Three peaks were observed for the sodiated LCLA complex, but the dominate conformer was much smaller than the deprotonated form.

Figure 5.

Isomeric arrival time distribution comparisons for A) 3-ketocholanic acid (black) and lithocholenic acid (red) and B) 3a-hydroxy-7,12-diketocholanic acid (black) and 3a-hydroxy-6,7-diketocholanic acid (red). The deprotonated spectra for each pair are shown on the left, while the sodiated spectra are shown on the left. Opposite separation trends were observed for the two different isomer pairs.

Figure 6.

Small deprotonated BAs illustrated greater IMS separations than larger BAs. The arrival time distributions for two representative isomer groups are shown in their deprotonated forms for A) m/z = 387.2541 and B) m/z = 514.2844. The group at m/z = 387.2541 represents one of the smaller isomer groups analyzed, while the group at m/z = 514.2844 was the largest isomer group characterized in this work.

Figure 7.

Metal ions enable better separations for the different BAs, but also induce multiple conformations. The IMS arrival time distributions for the BA isomers (GCDCA, GDCA, GUDCA and GHDCA) with the exact mass of 449.3141 are shown in their A) deprotonated, B) sodiated, C) copper complexed, and D) zinc complexed forms. The multiple peaks observed for the copper and zinc complexed forms illustrate the multiple binding locations for each cation on the BAs.