A fast and reliable LC-QqQ-MS/MS analytical approach for the quantification of key bile acids in feces, serum, and liver: application to an intergenerational study

Bile acids (BAs) are key metabolites of the crosstalk between the microbiota and the host that have been associated with health and disease states. Several methods have been developed for their analysis, mainly using liquid chromatography coupled to tandem mass spectrometry with a triple-quadrupole mass analyzer (LC-QqQ-MS/MS). However, there is a lack of fast and reliable methods to routinely measure the most described BAs in different biological matrices. Here, we present a novel analytical approach enabling the quantification of the 36 most common endogenous BAs in humans and rodents in three biological matrices – human feces, human serum, and rat liver tissue – using a LC-QqQ-MS/MS platform. We used the method to characterize BA composition of these three matrices, and validated it for feces and serum in terms of linearity, accuracy, precision, matrix effect, limit of detection, and limit of quantitation. We demonstrated its applicability in an intergenerational study analyzing fecal samples from 58 healthy individuals – including infants (3–8 months old) and their mothers and grandmothers. 24 BAs were quantified, compared, and correlated with 15 bacterial functions of BA pathways measured by shotgun metagenomics. Primary BAs were increased in Infants, whereas secondary BAs were increased in elders. These secondary BAs had significant positive correlations with the bacterial enzymes of these pathways. This novel methodology uses 8 internal standards for quality assurance and quantitation, and independent external calibration curves with matrix addition to assess the selection and quantitation of the BAs. This methodology will be useful for further studies needing to quantify BAs.