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Assay of free and glycine- and taurine-conjugated bile acids in serum by high-pressure liquid chromatography by using post-column reaction after group separation.

ABSTRACT: An accurate and sensitive method that involves the group separations of serum bile acids (i.e. free and glycine- and taurine-conjugated bile acid fractions) by ion-exchange chromatography on piperidinohydroxypropyl-Sephadex LH-20 is described. Each group was then analysed by high-pressure liquid chromatography by using the post-column reaction technique with immobilized 3 alpha-hydroxy steroid dehydrogenase. The bile acid patterns in the umbilical venous serum samples were analysed by this method. Taurochenodeoxycholate predominated in the umbilical blood.

SUBMITTER: Onishi S

PROVIDER: S-EPMC1158324 | biostudies-other | 1982 Apr

REPOSITORIES: biostudies-other

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Project description: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.

Dataset Information

Assay of free and glycine- and taurine-conjugated bile acids in serum by high-pressure liquid chromatography by using post-column reaction after group separation.

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