BAFinder: A Software for Unknown Bile Acid Identification Using Accurate Mass LC-MS/MS in Positive and Negative Modes

Most
LC-MS based bile acid analyses target common bile acids. The
identification of unknown bile acids remains challenging in untargeted
experiments. Here, a software named BAFinder was developed to improve
the identification of unknown bile acids from accurate mass LC-MS/MS
data in both the positive and negative ESI modes. A wide variety of
bile acid structures were covered in BAFinder, including oxidized
bile acids and sugar conjugates that were often ignored. The annotation
of unknown bile acids was based on a thorough investigation of MS/MS
fragmentation patterns of 84 bile acid reference standards in both
modes. Specifically, BAFinder took the peak alignment result and MS/MS
spectra, grouped candidate features in positive and negative modes,
searched their representative MS/MS spectra against a MS/MS library,
and used characteristic product ions and neutral losses to annotate
bile acids not covered in the library. Finally, the number of hydroxyl
groups and double bonds, conjugation, and isomer information of bile
acids were reported with four different levels of annotation confidence.
The use of BAFinder was demonstrated through successful application
to the analysis of human plasma and urine samples, in which a total
of 112 and 244 bile acids were annotated and 75 and 111 of them were
confirmed with standards or synthesized compounds, respectively. The
software is freely available at https://bafinder.github.io/.