Introduction

Bile acids (BAs) are components of the gastrointestinal (GI) tract that enable absorption of lipids, cholesterol, and fat-soluble vitamins, ultimately regulating the GI tract [1, 2]. BAs were first proposed as potential tumor-promoting agents in 1939, due to their high concentrations in certain areas of the body [3]. In the last two decades, studies analyzing BA circulation during cholesterol metabolism (Fig. 1) have led to new treatments for hypercholesterolemia [4, 5], and shown that BAs can act as nuclear receptor ligands, detecting and controlling their own concentrations within the body, but also be strongly cytotoxic [2, 6]. These properties have made it apparent that BAs are not only important in hepatic, biliary, and intestinal diseases, but many other diseases [7,8,9,10,11]. Thus, gaining a better understanding of BA metabolic pathways is essential for determining their role in each process, especially in individuals that have disrupted BA metabolisms due to genetic predisposition, diet, and/or cancer.

Fig. 1
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 are illustrated to show their similarities

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Since all BAs are originally derived from cholesterol as shown in Fig. 1, they share a similar four-ring steroid structure connected to a carbon side chain [12]. This unique amphipathic structure makes BAs structurally appropriate for absorbing nutrients in the digestive system [13]. However, understanding and measuring the full repertoire of BAs can be exceptionally challenging due to their structural similarities. BAs are categorized into primary and secondary groups based on their processing in the liver and intestines [6, 7, 14]. The primary BAs, cholic acid (CA) and chenodeoxycholic acid (CDCA), are the end-products of cholesterol metabolism in the liver which can be conjugated (mainly with glycine or taurine, depending on the animal species) to form taurocholic acid (TCA), glycocholic acid (GCA), taurochenodeoxycholic acid (TCDCA), and glycocheno-deoxycholic (GCDCA) acid as shown in Fig. 2. This conjugation creates BAs that are impermeable to cell membranes, resulting in their high concentrations in bile and intestinal content. Conjugation also plays a pivotal role in fat digestion and absorption when the BAs reach the colon via the gall bladder, bile duct, and duodenum.

Fig. 2
figure 2

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

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In the colon, deconjugation and dehydroxylation occur through bacterial enzymes, leading to the formation of dozens of distinct but structurally similar BAs [2]. For example, the enzymatic action of the bacterial microbiota converts CA and CDCA into secondary BAs by removing the hydroxyl group from the seventh carbon atom on the molecule forming deoxycholic acid (DCA) from CA, and lithocholic acid (LCA) from CDCA. The newly formed secondary BAs then pass into the portal vein and reach the liver, where they join new primary BAs and are reconjugated with glycine or taurine and stored in the gallbladder. However, other changes can occur to the BAs. For example, some BAs such as LCA, the most toxic substance produced in the body and a known carcinogen, enter the liver where they are sulfated or esterified to glucuronic acid and excreted. This recycling of BAs is known as enterohepatic circulation and can occur 10 times every day, forming additional BAs. The numerous resulting structurally similar BAs cause great difficulties in fully understanding BA metabolic pathways, and even though they have almost identical structures, they have quite different functions [6, 15]. Therefore, new analyses are needed to fully evaluate BAs responsible for pathogenic conditions.

While BAs can be analyzed directly with mass spectrometry (MS)-based techniques, often in negative ionization mode due to their many hydroxyl groups, the BA structural similarities and numerous isomers require LC separation times often between 10 and 45 min [16,17,18,19,20,21,22,23]. LC gradients between 4 and 7 min have also been utilized but these are often for just a subset of the BAs [24], which makes new higher throughput and great coverage approaches greatly desirable. Ion mobility spectrometry coupled with mass spectrometry (IMS-MS) is an attractive approach due to its ability to separate isomeric structures on a millisecond time scale [25, 26]. In this work, we utilized drift tube IMS (DTIMS) coupled with MS (DTIMS-MS) to characterize the sodiated and deprotonated BA structures occurring in positive and negative mode electrospray ionization (ESI). Copper and zinc metal ions were also added to the BA isomers that were difficult to separate with DTIMS to evaluate if the metal bound complexes could be distinguished. The results of these analyses are detailed in this manuscript, showing the utility of DTIMS-MS for rapid BA analyses.

Materials and methods

Chemicals, solvents, and sample preparations

Seventy-two BA standards (56 unlabeled and 16 deuterated) were characterized in this manuscript. The 56 unlabeled BA standards were purchased from Sigma-Aldrich (St. Louis, MO) and Steraloids (Newport, RI) and the 16 deuterium-labeled BAs were purchased from C/D/N Isotopes Inc. (Point-Claire, Quebec, Canada). Each BA standard was dissolved in high-purity methanol purchased from Thermo Fisher Scientific (Waltham, MA) to a concentration of 5 mg/mL. Prior to analysis, each BA standard was further diluted with methanol to a final concentration of 0.5 μM.

Drift tube IMS analysis and collision cross section measurements

An Agilent 6560 IMS-QTOF MS (Agilent Technologies, Santa Clara) equipped with the gas kit upgrade was utilized for all of the drift tube IMS (DTIMS) measurements in this study [27, 28]. The gas kit contains a flow controller to automatically regulate the drift tube pressure and baratrons so drift gases other than nitrogen can be used. BAs were ionized using ESI in both positive and negative mode. For the DTIMS measurements, the ions were passed through the inlet glass capillary, focused by a high-pressure ion funnel, and accumulated in an ion funnel trap. Ions were then pulsed into the 78.24-cm-long IMS drift tube filled with ~ 3.95 Torr of nitrogen gas, where they traveled under the influence of a weak electric field (10–20 V/cm). Ions exiting the drift tube were refocused by a rear ion funnel prior to QTOF MS detection and their arrival times (tA) were recorded and collision cross section (CCS) values calculated [29]. All CCS values were measured using seven stepped electric field voltages to obtain the most accurate values and each sample was analyzed in both positive and negative ionization modes. CCS values are listed in Table S1 in the Electronic Supplementary Material (ESM). The detailed instrumental settings are given in the ESM, Tables S2–S4, and follow those previously published in an interlaboratory study [30] and a drift tube IMS CCS database study [31]. Each sample was measured in triplicate and relative standard deviations (RSD) were obtained for all of the analytes. The Agilent IM-MS Browser software was utilized for data processing and all stepped field CCS calculations.

Results and discussion

To evaluate the structural complexity of BA isomers, 72 different BA standards were evaluated with DTIMS-MS. The standards included primary and secondary BAs, 12 isomer groups, and unlabeled and deuterated versions. To determine when and if separation was possible with DTIMS, both positive and negative ionization were utilized and the resulting sodiated and deprotonated forms of each standard were studied. All triplicate measured CCS values are noted in Table S1 (see ESM) along with CAS numbers for all molecules so the exact structure of each BA can be looked up in PubChem (https://pubchem.ncbi.nlm.nih.gov/) if desired. In our analyses, initially, the results for the unlabeled and deuterated versions were compared. Of the 72 standards studied here, 56 BAs were unlabeled, while 16 BAs were in a deuterated form having either four or five deuterium substitutions. As expected, similar CCS values (< 0.3% difference) were observed for the unlabeled and labeled forms in both positive and negative modes. Since < 0.3% difference is the experimental error of the instrument used in this study, no structural changes were observed upon deuteration of the BAs. This information is important to understand since deuterated BAs can be spiked into complex mixtures in metabolomics studies for absolute quantitation of endogenous BAs. The similar CCS values of the labeled and unlabeled BAs therefore add confidence to BA identifications in complex mixtures.

The role of conjugation is also of interest in the structural evaluation of BAs. Of the 56 unlabeled BAs analyzed in this study, 35 were unconjugated, while 9 where conjugated with glycine and 12 with taurine. To understand whether conjugation affects BA structure, the DTIMS CCS and m/z trends were evaluated for the whole collection of unlabeled BA standards. As shown in Fig. 3, significant differences were observed in the positive and negative ion modes. In negative ion mode, the deprotonated forms of the unconjugated and conjugated BAs had very similar CCS values ranging from ~ 198 to 215 Å2 (Fig. 3a). While the unconjugated BAs displayed a wide CCS distribution that spanned the whole CCS range, the glycine and taurine conjugates each had very narrow ranges with glycine conjugates only spanning 3 Å2 and taurine conjugates occurring over 6 Å2. However, in positive ion mode (Fig. 3b), the range of CCS values for the sodiated complexes increased for all forms from ~ 188 to 222 Å2. Additionally, the range for each different type of BA also increased: unconjugated forms extended over 25 Å2, glycine conjugates spanned 8 Å2, and taurine conjugates occurred over 10 Å2. The extended CCS distribution illustrated the effect of both the sodium binding location and BA structural flexibility as the hydroxyl groups moved to coordinate with the sodium ion. These structural alterations thus resulted in either compaction or extension of the BAs as shown by the CCS values change. For example, the sodiated form of litocholenic acid (LCLA) was much smaller than its deprotonated form (Fig. 4). Interestingly, sodium binding also induced multiple conformers in the BAs as shown by the three peaks for the sodiated form of LCLA. However, a single conformation often dominated the sodiated complexes (> 80% of all forms), so only the dominate CCS conformer values are noted in Table S1 (see ESM).

Fig. 3
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 gray colors. The CCS measured in DTIMS with nitrogen gas are noted as DTCCSN2, with the unit of Å2

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Fig. 4
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

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The effect of different ion types was also investigated for the 12 isomer groups to understand if structural separation was possible and specific to deprotonation or sodiation. In the isomer groups, all but two groups could be distinguished in either positive or negative mode or a combination of both. For example, the isomer pair of 3-ketocholanic acid (3ketoCA) and LCLA with an exact mass of 374.2821 showed very similar arrival time distributions for their sodiated versions in positive mode and were inseparable (Fig. 5a). However, in negative ion mode, the deprotonated form of 3ketoCA displayed a much shorter arrival time than LCLA providing separation at half height of the peak and suggesting differences in the BA conformations. The isomer pair 3a-hydroxy-7,12-diketocholanic acid (3aOH712diketoCA) and 3a-hydroxy-6,7-diketocholanic acid (3aOH67diketoCA) interestingly showed the opposite case (Fig. 5b). In negative mode, the deprotonated structures for these two isomers showed by similar arrival times and were inseparable from each other. However, in positive mode, the sodiated forms displayed very different arrival times and were baseline separated from each other. These opposing trends suggest that isomer separation is specific to the group of BAs being studied and that an overall trend might not be established within the different isomer groups. However, this trend indicates that IMS-MS experiments for the analysis of the 56 BAs in the manuscript would have to be performed in both positive and negative modes unless only a subgroup of isomers was targeted. To eliminate this need, a short LC gradient could be coupled with the IMS-MS analyses if all the BAs could be distinguished either by LC elution time or IMS CCS in a single polarity. The multidimensional LC-IMS-MS evaluations would also provide additional confidence in the BA identifications since the resulting features could be matched to both known LC elution times and IMS CCS values.

Fig. 5
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 right. Opposite separation trends were observed for the two different isomer pairs

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Upon further evaluation of the two isomer groups that could not be distinguished in either positive or negative ion mode or a combination of both, we noticed a trend. Of our 12 isomer groups studied, the 2 groups that could not be distinguished had some of the highest m/z values with exact masses of 449.3141 and 515.2917. In fact, the group with an exact mass of 515.2917 was the largest BA group analyzed in this work. This trend is shown in Fig. 6 where the deprotonated BAs of 387.2541 m/z (Fig. 6a) are compared to those at 514.2844 m/z (Fig. 6b). The group at m/z = 387.2541 represented one of the smaller masses studied and were separable, while those at the largest m/z studied (m/z = 514.2844) showed very similar arrival time distributions and were inseparable. Since we observed significant changes in the BA structure upon sodium binding, we decided to assess whether the addition of metal ions could dramatically change the conformations of the larger BA isomers. Previous studies with other molecule types, such as glycans, have shown improved isomer separations with the use of copper and zinc, so we began our assays with these two metal cations [32,33,34,35,36]. To evaluate the effect of these metals, each standard was injected without and with the metal solutions. As shown in Fig. 7, the deprotonated and sodiated BA isomers with an exact mass of 449.3141 had similar IMS arrival time profiles and were inseparable (Fig. 7, top panel). In contrast, when Cu2+ and Zn2+ bound to the BAs, a reasonable separation of the isomers was attained (Fig. 7, bottom panel). The changes in separation of the BAs indicate that the metal cations bind differently than sodium allowing additional resolution for the four BAs. The multiple peaks in the arrival time distributions however illustrate that Cu2+ and Zn2+ bind to multiple locations on the BAs, making the spectra more complex to analyze. Thus, if specific targeted analyses are desired, this may be one promising way of rapidly separating the BA isomers, but quantitation of the multiple complexed peaks (which may overlap with the other isomers) will be difficult. Another interesting observation in the Cu2+ and Zn2+ arrival time distributions was that GCDCA and GDCA switched conformational sizes in the different metal complexes, indicating that even Cu2+ and Zn2+ are binding in different ways. Therefore, other metal cations could be used with the BAs to possibly produce even better separations, but this study is beyond the scope of this manuscript. We did however try to complex Cu2+ and Zn2+ with the largest BA isomer group in this study (having an exact mass of 515.2917). Unfortunately, no complexes were observed, so the effect of Cu2+ and Zn2+ addition could not be studied for this isomer group.

Fig. 6
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 am/z = 387.2541 and bm/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

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Fig. 7
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

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Conclusions

In this work, we applied DTIMS-MS to characterize BA standards with subtle structural differences. The analyses showed that the structures for BAs can change dramatically from positive to negative ion mode when sodiated and deprotonated ions are formed and detected. In some cases, deprotonation enabled better isomeric separations, but the opposite trend was also observed. Ultimately, evaluation in both ion modes enabled the most confident identification for the entire set of BAs analyzed. Two of the isomer groups we studied were however indistinguishable in either their deprotonated or sodiated forms. In these cases, metal ions such as copper and zinc were added to the overlapping BA isomers and the metal complexation led to the separation of the isomers but also multiple conformations. Our results suggest that IMS-MS is a powerful tool for rapidly identifying BAs and distinguishing them from their isomers. However, no specific IMS structural trends were observed in the study for the different isomer groups such as a certain hydroxyl position resulting in a smaller size or that sodiation would always separate the isomers. Standards will therefore be needed initially to identify the BAs in the assays. Thus, this work detailing the CCS values of 56 unlabeled BAs will serve as a powerful starting point for many IMS-MS-based BA analyses. We also believe that coupling short LC gradients with the IMS-MS assays will provide even more confidence in the BA identifications and may even eliminate the need for both positive and negative mode evaluations if all BAs of interest can be separated in a single polarity either by their LC elution time or their IMS CCS value.