MSACL 2025 Abstract

Quantitative and Qualitative Analysis of Bile Acids by High-Resolution Mass Spectrometry Using Electron-Based Fragmentation

Paul RS Baker (1), Santosh Kapil (1), Dilip Kumar R Kandula (1), Robert Proos (1), Maxim D Seferovic (2) and Thomas D. Horvath (3,4,5)
(1) SCIEX, USA, (2) Dept of Obstetrics and Gynecology, Baylor College of Medicine, Houston, TX, (3) Dept of Pathology, Texas Children’s Hospital, Houston, TX, (4) Dept of Pathology & Immunology, Baylor College of Medicine, Houston, TX, (5) Dept of Pharmacy Practice & Translational Research, College of Pharmacy, University of Houston

Thomas Horvath, PhD (Presenter) Baylor College of Medicine / Texas Children's Hospital

Presenter Bio: I am an Assistant Professor in possession of nearly 23 years of academic and FDA/EMEA-regulated pharmaceutical-industry research experience in the field of bioanalytical chemistry. Over the course of my career, I have acquired considerable skill in the development and validation of high-throughput LC-MS/MS-based bioanalytical methods to measure exogenous small-molecule therapeutics (e.g., pharmaceuticals and peptides) or endogenous bio-molecules (e.g., metabolites and lipids) in an assortment of biological matrices and homogenized tissues. I have developed methods which have been implemented in host of projects, including: i) PK/PD assessments of therapeutic small-molecules or enzyme-based drugs; ii) assess the effectiveness or bioequivalence of novel, off-patent formulations; iii) investigate the mechanism of action of new therapeutic compounds; and iv) determine alterations in metabolic pathways based on disease state or therapeutic intervention. My publication record spans a diverse subset of life science research that includes nutritional biochemistry, mosquito metabolism using carbon-13 isotope tracing, and microbiological applications including methods to interrogate the mammalian gut-brain-axis, and pharmaceutical discovery and development. My current research focuses on dissecting microbial derived compounds and their impact on host physiology.

Relevant Financial Disclosures (within past 24 months, reported on Mar 24, 2026) No relevant financial relationship(s) to disclose.

INTRODUCTION:
Analyzing bile acids using nominal mass instruments is challenging due to the high chemical background in several precursor ions, which can interfere with multiple reaction monitoring (MRM) transitions. High-resolution mass spectrometry (HRMS), which generates a full product ion spectrum for each targeted bile acid, can reduce background interferences by extracting fragment ions within a narrow mass-to-charge (m/z) window, improving signal-to-noise (S/N) ratios. While detecting individual bile acid isomers relies on chromatographic resolution when using collision-induced dissociation (CID), electron activated dissociation (EAD) provides structurally diagnostic fragment ions that can differentiate bile acid isomers and reduce isomer resolution time. Here, an HRMS system was used to sensitively quantify bile acids in human plasma with concurrently acquired qualitative data to ensure specificity.

METHODS:
A robust, targeted bile acid analysis method was established using primary reference and deuterated internal standards on an HRMS system. Appropriate MRM transitions were established for both CID- and EAD-based fragmentation. Unique fragments for isomers were identified using EAD-based fragmentation, enabling faster chromatography compared to CID-based fragmentation, where no unique fragments among isomers were generated. Chromatographic separation was performed using a Shimadzu Nexera HPLC system with a Restek Raptor column (C18, 5 µm, 100 x 2.1 mm) over a 17-minute gradient for CID-based fragmentation and a 10-minute gradient for EAD-based fragmentation. Standard metrics were used to assess the assay’s quantitative performance, including lower limit of quantitation (LLOQ), limit of detection (LOD), accuracy and precision.

RESULTS:
Bile acids were detected and quantified in human plasma using an ultra-sensitive HRMS system capable of both CID- and EAD-based fragmentation. Using the longer chromatographic gradient with CID-based fragmentation resulted in a limit of quantitation (LOQ) approximately 10x lower than previously reported with HRMS analysis, which rivals, if not exceeds, the sensitivity of high-end triple quadrupole instruments. In addition, a high-quality MS/MS spectrum was acquired for each targeted bile acid during analysis, which can improve confidence in the assay’s specificity. Using EAD-based fragmentation resulted in identifying unique, diagnostic fragments for some bile acid isomers and when appropriate, this fragmentation mode was used to allow for faster gradients. If a unique fragment was identified, baseline chromatographic separation of those particular isomers was unnecessary. However, EAD-based fragmentation cannot distinguish optical isomers, so some chromatographic separation is still required. Using the 10-minute gradient conditions, bile acids were detected with LOQs approximately 10-fold higher than those acquired with CID-based fragmentation. Despite the higher LOQ for these experiments, they were still below the levels of endogenous bile acids in plasma, making this a suitable approach for bile acid measurement in this sample type. Leveraging EAD-based fragmentation improves specificity and enables faster chromatographic gradients, which can increase sample throughput.

CONCLUSION:
Sensitive, quantitative bile acid analysis with the ability to leverage electron-based fragmentation to improve assay specificity and speed.