MSACL 2026 Abstract

Exploring Single-Point External Calibration Using Spiked Matrices for Bile Acids Quantification by LC-HRMS

J. Will Thompson (1), James Campbell (1), Bashar Amer (2), Cynthia M. Grim (2), Susan S. Bird (2), Whitney Stutts (3), Crystal L. Pace (3) and J. Scott Mellors (1)
(1) Move Analytical LLC, Carrboro, NC, (2) Thermo Scientific, San Jose, CA, (3) University of North Carolina at Chapel Hill, Chapel Hill, NC

Will Thompson, PhD (Presenter) Move Analytical

Presenter Bio: Cofounder, Move Analytical LLC in 2025. Principal Scientist and Director of Life Science Business Development at 908 Devices Inc, from 2021-2025. Assistant Director of the Proteomics and Metabolomics Shared Resource at Duke School of Medicine from 2007 to 2021.

Relevant Financial Disclosures (within past 24 months, reported on Apr 22, 2026) Ownership Interest Move Analytical LLC Discussing Products? YES, presenter will present educational content related to products/services of one or more ineligible company(ies) listed above. Using Generic Terms? YES Conflict mitigation IN PROCESS.

INTRODUCTION:
A major barrier to clinical and translational metabolomics is the need for complex (multi-analyte) and costly calibration strategies, limiting scalability and inter-laboratory reproducibility. Single-point external calibration (SPEC) has shown promise in proteomics(1) and newborn screening(2) but remains underutilized in metabolomics. Here, we evaluate SPEC for bile acid quantification and develop a commercially available spiked biological matrix to enable robust, transferable quantification of bile acids from multiple matrices and across laboratories using high-resolution mass spectrometry (HRMS).

METHODS:
32 bile acids (labeled and unlabeled) were purchased from Cambridge Isotope Laboratories, and used to create a bile acids panel which was analyzed in multiple biological matrices (serum, plasma, urine, feces). Samples were extracted with methanol containing stable isotope internal standards and analyzed by LC-HRMS (Orbitrap platforms; 10-minute gradient). Quantification was performed using MS1 data in Skyline, with automated experimental design, system suitability, and reporting in MoveApp™, and downstream statistical analysis in Python.

RESULTS:
Evaluation of calibration strategies in surrogate matrices demonstrated that, with sufficient signal-to-noise, a single-point calibration with regression through zero accurately quantified bile acids across a 10–5000 nM range. Stable-isotope dilution experiments revealed that SPEC quantification using spiked biological matrices had improved recovery relative to traditional calibration curves formulated in water or water/methanol.

A spiked matrix calibration material (~250 nM), along with low (~15 nM) and high (~2500 nM) QC materials was generated and value-assigned using stable isotope dilution. Inter-laboratory validation across multiple sites demonstrated strong reproducibility: in pooled serum, 21 bile acids were detected in all labs with 20 showing <30% RSD; in pooled urine, 22 bile acids were detected with 13 <30% CV; and in a single-donor fecal sample, 8 bile acids were consistently detected with 4 <30% CV. 29 and 32 of the 32 quantitative bile acids demonstrated interlaboratory precision less than 25% CV for QC Low and QC High, respectively. Additional bile acid conjugates, including glucuronides and amino acid conjugates, were reproducibly detected across laboratories. Data collection from additional laboratories and sample types is ongoing.

DISCUSSION:
We demonstrate that single-point external calibration using spiked biological matrices enables accurate, cross-laboratory quantification of bile acids without the need for full calibration curves. The SPEC approach simplifies quantitative workflows and could be used to help overcome the perceived irreproducibility of discovery metabolomics, assisting in translational adoption of HRMS-based metabolomics. Additional quantitative figures of merit as well as opportunities for expansion to other method types will be presented.

References:
1. Pino LK et al Anal Chem. 2018 Nov 6;90(21):13112-13117.
2. Pickens CA, Int. J. Neonatal Screen. 2020, 6(3), 75.