B-178 Sensitive and selective quantitation of bile acids in children with autism spectrum disorder using targeted MS2/MS3 on the Stellar MS

Abstract Background Bile acids (BA), synthesized from cholesterol in the liver, are crucial for lipid digestion and absorption. In the gastrointestinal tract, primary bile acids are converted by host microbiota into various secondary and conjugated bile acids. Disordered microbiomes can alter the composition and concentration of the bile acid pool, impacting human health. Normally, blood levels of bile acids are low, but they can increase due to disorders such as liver disease. Consequently, identifying and quantifying these compounds has become a significant research focus. We present a highly selective targeted MS2 and MS3 approach for the quantitation of bile acids and their conjugates in serum using the Thermo Scientific™ Stellar™ mass spectrometer. Methods Unlabeled and stable-isotope-labeled bile acid standards were purchased from Cambridge Isotope Laboratories. These standards were used to prepare dilution series solutions in 50% methanol and generate calibration curves. Bile acids were extracted from the serum of adults with and without autism spectrum disorder (ASD) using 80% methanol, separated on a reverse-phase C18 column, and analyzed using the Thermo Scientific™ Stellar™ mass spectrometer. A scheduled method combining targeted-MS2 and targeted-MS3 scan modes with two activation types, CID and HCD, was employed to quantify these molecules. Data was processed using TraceFinder™ 5.2 software. Results The BA standards were utilized to generate calibration curves for absolute quantitation, facilitating reliable ion trap quantitation over a wide dynamic range of 5 to 6 orders of magnitude for the targeted BA. The MSn-based quantitation of the mass analyzer provided the selectivity needed to detect and accurately quantify co-eluting isomers and isobars, while also improving discrimination between analyte signals and matrix interferences. The new Stellar mass spectrometer enhances quantitative methods by combining different activation types and integrating targeted MS2 and MS3 acquisition modes in a single run. Bile acid standard curves (0.1 to 1000 nM) demonstrated a dynamic range over 5 orders of magnitude and excellent sensitivity in the femtomole range. Enhanced selectivity was achieved with MS3 acquisition, allowing accurate quantitation of co-eluting isomers that MS2 could not separate. This platform also provides valuable structural insights, potentially eliminating the need for extended chromatographic separation. The developed method was successfully employed to quantify metabolites in fecal samples. Conclusion High specificity and sensitivity with combined MS2 and MS3 levels for the quantitation of bile acids in blood