MSACL 2025 Abstract

Simultaneous Quantitation and Discovery (Squad) of Fecal Bile Acids and Conjugates in Adults With Autism Spectrum Disorder

Bashar Amer (1), Alexey Melnik (2), James B Adams (3), Rosa Krajmalnik-Brown (3), Khemlal Nirmalkar (3), Brandon Bills (1), Rafael Melani (1), and Susan S. Bird (1)
(1) Thermo Fisher Scientific, San Jose, California, United States; (2) Bileomix Inc., Farmington, CT, United States; (3) Biodesign Center for Health Through Microbiomes, Arizona State University, Tempe, Arizona, United States

Ema Ruzic (Presenter) Thermo Fisher Scientific

Presenter Bio: Experienced scientist and marketing professional with a strong foundation in serology, DNA analysis, and molecular biology. Currently serving as a Vertical Marketing Manager at Thermo Fisher Scientific, leading the development of impactful content, customer engagement strategies, and strategic event planning. Known for analytical strength, independent leadership, and adaptability across roles. Previous experience as a Market Development Specialist included product launch support, market analysis, and customer relationship building. Combines scientific expertise in biology and chemistry with cross-functional marketing and project management skills to drive results in dynamic environments.

Relevant Financial Disclosures (within past 24 months, reported on Jul 15, 2025) No relevant financial relationship(s) to disclose. Conflict mitigation NOT REQUIRED.

INTRODUCTION:
Bile acids (BA) are synthesized from cholesterol in the liver and play a crucial role in lipid digestion and absorption. Fecal bile acids serve as biomarkers and signaling molecules due to their intricate interplay with gut microbiota. Disordered microbiomes can alter the composition and size of the bile acid pool, producing a variety of conjugated bile acids and structurally similar metabolites. These changes may indirectly confer disease states. Here, a metabolomics workflow was developed for the simultaneous quantitation and discovery (SQUAD) of fecal BA and BA conjugates on the Thermo Scientific™ Orbitrap Ascend™ Tribrid™ mass spectrometer. The workflow utilizes Real-Time Library Search (RTLS) for spectral similarity measures and heightened identification confidence for molecular species during method execution.

METHODS:
Human stool materials were obtained from adults with and without autism spectrum disorder (ASD). BA and microbially conjugated bile acid (MCBAs) standards were purchased from Bileomix Inc. Analytes were separated on a Thermo Scientific™ Hypersil GOLD™ column within a Thermo Scientific™ Vanquish™ Horizon system. Data were acquired on the Thermo Scientific™ Orbitrap Ascend™ Tribrid™ mass spectrometer, which facilitates sensitive and highly dynamic PRM-quantitation utilizing the linear ion trap. It also allows Orbitrap MS1 scanning for higher annotation rates. AcquireX™ and RTLS workflows were used to maximize the number of relevant compounds interrogated by MS2 and MS3 for confident annotation. Thermo Scientific™ TraceFinder™ 5.2 and Compound Discoverer™ 3.4 software were used for data processing, analyte quantitation, and unknown annotation.

RESULTS:
The BA and BA conjugate standards were used to evaluate a SQUAD metabolomics workflow for BA analysis in the collected fecal samples on an Ascend MS. The standards were used to generate calibration curves for absolute quantitation and to build an MS2 spectral library for the annotation of relevant unknown BA and BA conjugates via RTLS. This workflow facilitates reliable ion trap quantitation over a wide dynamic range (i.e., 5 – 6 orders of magnitude) of the targeted BA in feces. A lower limit of quantification (LLOQ) of 10 femtomoles and a lower limit of detection (LLOD) of 0.25 femtomoles was observed for most of the targets. Moreover, MSn-based quantitation of the mass analyzer enabled the selectivity required to detect and accurately quantify co-eluting isomers and isobars. It also leads to improved discrimination between signals derived from analytes and those resulting from matrix interferences.

Simultaneously, the HRAM Orbitrap data and the increased percentage of fragmented compounds using the advanced deep scan AcquireX workflow resulted in improved annotation capability compared to traditional DDA on a wider dynamic range of fecal compounds. Additionally, Real-Time Library Search provided spectral similarity measures and identification confidence scores for BA species upon which acquisition decision-making can be based during method execution.

DISCUSSION:
In our analysis we identified several microbially conjugated bile acids that were previously determined to be associated with gut conditions including IBD, in particular, we found in high abundance leucine/isoleucine, tyrosine, and phenylalanine conjugated bile acids. It’s important to note that neither chromatography nor MS2-level fragments were able to distinguish Isoleucine from leucine-conjugated bile acids. MS3 level on the Ascend system was required to detect specific fragments and quantify levels of Ile/leu conjugates.