Bile acids (BA), crucial for lipid digestion, are synthesized from cholesterol in the liver
and act as biomarkers and signaling molecules, influencing disease states through
complex interactions with gut microbiota. A metabolomics LC-MS workflow:
Simultaneous Quantitation and Discovery (SQUAD), was implemented for fecal BA
and BA conjugates analysis. The workflow incorporates Real-Time Library Search
(RTLS), for enhanced identification confidence of relevant unknowns through spectral
similarity measures during method execution.
Human stool material and BA standards from NIST and CIL were utilized to build the
library needed for SQUAD. The feces were spiked with labeled standards and
extracted with 80% methanol. Fresh fecal samples were also obtained from a dietary
intervention involving mice fed one of three diets, each comprising 15% of kcal from
fat. The diets varied in fat source: standard (soybean oil), SFA-rich (cocoa butter and
soybean oil), and MUFA-rich (olive and soybean oils). Following 29 days under each
dietary condition, fecal samples were collected, and metabolites were extracted using
80% methanol. Subsequently, metabolites were separated using a reversed-phase
column. The tribrid mass spectrometer enabled sensitive PRM-quantitation on the
linear ion trap and HRAM Orbitrap MS1 scanning for higher annotation rates.
The labeled and unlabeled BA standards were used to construct calibration curves for
absolute quantitation. The quantitation provided a wide dynamic range with metrics of
10 fmol lower limit of quantification and a 0.25 fmol lower limit of detection for most
targets. MS n-based quantitation enabled selective detection of co-eluting isomers and
isobars, enhancing discrimination between analyte signals and matrix interferences.
This study enables the assessment of bile acids and other metabolic profile differences
in the mice fecal samples across various groups due to improved annotation
capabilities of the HRAM data. Finally, confidence in the targeted and untargeted
MS-based workflow's quality was obtained by regularly monitoring the spiked IS.