Identification and quantification of oxo-bile acids in human faeces with liquid chromatography-mass spectrometry: A potent tool for human gut acidic sterolbiome studies

Bile acids (BAs) are endogenous steroids involved in the transport of lipids in bile, acting also as molecular signaling hormones. Primary BAs synthesized in the liver undergo several metabolic pathways in the intestine by gut microbiota to produce secondary BAs. Together with secondary BAs, other metabolites have been recovered from human faeces, including many oxo-BA analogues produced in the colon through oxidation of BA hydroxy groups. However, the complete oxo-BA characterization in biospecimens (particularly intestinal content and faeces) has not been reported yet, hampering the assessment of their potential physiological role. Herein, we have developed and validated a new RP-HPLC-ESI-MS/MS method in negative ionization mode for the simultaneous analysis of 21 oxo-BAs and their 7 metabolic BAs precursors in human faeces. The elution was performed in gradient mode and 28 compounds, including primary, secondary BAs, and their oxo-derivatives, were separated within 50 min at 40 °C column temperature. The method is accurate (bias%