Isomers-oriented separation of forty-five plasma bile acids with liquid chromatography-tandem mass spectrometry

Some bile acids (BAs) were considered as biomarkers or have therapeutical effect on metabolic diseases. However, due to the existence of isomers and limitations in sensitivity, simultaneous quantification of multiple BAs remains a challenge. The aim of this study is to establish an accurate and sensitive method for the determination of multiple BAs with similar polarity. A LC-MS/MS analytical method capable of quantifying forty-five BAs simultaneously using nine stable isotope internal standards was developed and fully validated based on key isomers-oriented separation strategy. The method was further applied to analyze plasma samples to describe the dynamic profile of BAs after high glucose intake. The chromatography and mass spectrum conditions were optimized to enable the accurate quantification of forty-five BAs, while ensuring the lower limit of quantification between 0.05-10 ng/mL. The results of system suitability, linearity, dilution integrity, accuracy and precision demonstrated the good quantitative capacity and robustness of the method. A total of thirty-five BAs were quantified in plasma samples from twelve healthy Chinese individuals. The established method featured superior sensitivity and better separation efficiency compared to previous studies. Meanwhile, BAs exhibited correlations with glucose and insulin, suggesting their potential as biomarkers for metabolic disorders.