Factors affecting separation and detection of bile acids by liquid chromatography coupled with mass spectrometry in negative mode

Abstract

Bile acids (BAs) are cholesterol metabolites with important biological functions. They undergo extensive host-gut microbial co-metabolisms during the enterohepatic circulation, creating a vast structural diversity and resulting in great challenges to separate and detect them. Based on the bioanalytical reports in the past decade, this work developed three chromatographic gradient methods to separate a total of 48 BA standards on an ethylene-bridged hybrid (BEH) C18 column and high-strength silica (HSS) T3 column and accordingly unraveled the factors affecting the separation and detection of them by liquid chromatography coupled with mass spectrometry (LC-MS). It was shown that both the acidity and ammonium levels in mobile phases reduced the electrospray ionization (ESI) of BAs as anions of [M-H]^-, especially for those unconjugated ones without 12-hydroxylation. It was also found that the retention of taurine conjugates on the BEH C18 column was sensitive to the strength of formic acid and ammonium in mobile phases. By using the volatile buffers with an equivalent ammonium level as mobile phases, we comprehensively demonstrated the effects of the elution pH value on the retention behaviors of BAs on both the BEH C18 column and HSS T3 column. Based on the retention data acquired on a C18 column, we presented the ionization constants (pK _a) of various BAs with the widest coverage beyond those of previous reports. When we made attempts to establish the structure-retention relationships (SRRs) of BAs, the lack of discriminative structural descriptors for BA stereoisomers emerged as the bottleneck problem. The methods and results presented in this work are especially useful for the development of reliable, sensitive, high-throughput, and robust LC-MS bioanalytical protocols for the quantitative metabolomic studies. Graphical Abstract Nonlinear curve fitting of capacity factors and elution pH value for the separation of common unconjugated bile acids.

Keywords: Bile acid; Electrospray ionization; High-performance liquid chromatography; Ionization constant; Mass spectrometry; Structure-retention relationship.

Fig. 1.

Chemical structure of C24 bile acids. The mostly known structural diversity appears at (1) A/B ring fusion stereochemistry (trans/5α-H or cis/5β-H); (2) sites of hydroxylation at C3, C6, C7 and/or C12; (3) dehydrogenation and epimerization of the hydroxyl groups; and (4) conjugation of glycine or taurine at the C24-carboxyl group and/or conjugation of glucuronide or sulfate at hydroxyl/ C24-carboxyl groups.

Fig. 2.

Retention time of unconjugated BAs eluted by the key intermediate gradient programs during optimization of the Gradient-II on an ACQUITY BEH C18 column. Mobile phase A: 0.01% formic acid (v/v) in water; Mobile phase B: acetonitrile; Blue square: monohydroxyl-BAs (isoLCA/ alloLCA/LCA, blue square); Green diamond: dihydroxyl-BAs (muroCA/βUDCA/UDCA/HDCA/ CDCA/DCA/isoDCA); Red circle: trihydroxyl-BAs (βUCA/UCA/ωMCA/βCA/αMCA/βMCA/HCA/ ACA/ CA).

Fig. 3.

Effects of formic acid in mobile phases on the chromatographic retention (a) and negative ionization (b) of BAs. All tests were performed by using the Gradient-I. The ionization efficacy was indicated by peak heights. The peak height data of those BAs detected in the same channel but not fully separated was not available, such as the data of ωMCA/βCA and some data of TUDCA/THDCA.

Fig. 4.

Effects of ammonium in mobile phases on the negative ionization of unconjugated BAs. All tests were performed under the Gradient-II by switching the aqueous phases as 0.008% formic acid, 0.01% formic acid and 0.012% formic acid and the ammonium formate buffers (2 mM) at pH 3.5, 4.0 and 4.5. The ionization efficacy was indicated by peak areas.

Fig. 5.

Ion chromatograms of unconjugated BAs on an ACQUITY BEH C18 column eluted by the Gradient-II with various buffers (from bottom to top: 0.01% formic acid, 2 mM ammonium formate at pH 3.5, 2 mM ammonium acetate at pH 4.5, 2 mM ammonium acetate at pH 5.5, 2 mM ammonium bicarbonate at pH 6.5 and 2 mM ammonium bicarbonate at pH 7.5) and acetonitrile as the aqueous phase and the organic phase respectively.

Fig. 6.

Ion chromatograms of conjugated BAs on an ACQUITY BEH C18 column eluted by the Gradient-I with various buffers (from bottom to top: 0.01% formic acid, 2 mM ammonium formate at pH 3.5, 2 mM ammonium acetate at pH 4.5, 2 mM ammonium acetate at pH 5.5 and 2 mM ammonium bicarbonate at pH 6.5) and acetonitrile as the aqueous phase and the organic phase respectively.

Fig. 7.

Retention variations with the pH value of mobile phases for the unconjugated BAs (A, C) and the conjugated BAs (B, D) on BEH C18 column (A, B) and HSS T3 column (C, D).

Fig. 8.

Plot of capacity factors against the pH of the mobile phase on ACQUITY BEH C18 column for the unconjugated BAs (A), the glycine-conjugated BAs (B), taurine-conjugated BAs (C) and the other unconjugated and conjugated BAs (D). The data of conjugated BAs and unconjugated BAs was collected under the Gradient-I and Gradient-II, respectively.

Fig. 9.

Correlation between the predicted LogP and the retention time acquired by the Gradient-I for a total of 35 BAs with the cis A/B ring juncture (5β) and without β-hydroxyl groups.