2. Academic Validation
  3. Quantifying endogenous and tracer-derived ketone bodies using a dual-label UHPLC-MS/MS method

Quantifying endogenous and tracer-derived ketone bodies using a dual-label UHPLC-MS/MS method

  • Talanta. 2025 Sep 30;298(Pt A):128919. doi: 10.1016/j.talanta.2025.128919.
Sandra Adámez-Rodríguez 1,  Eric D Queathem 2,  Abdirahman Hayir 3,  María Luisa Marina 1,  María Castro-Puyana 1,  Patrycja Puchalska 4
Affiliations

Affiliations

  • 1 Department of Analytical Chemistry, Faculty of Chemistry, University of Alcalá, Ctra. Madrid-Barcelona, km. 33.600, Alcalá de Henares, E-28871, Madrid, Spain.
  • 2 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota Medical School, Minneapolis, MN, USA; Department of Integrative Biology and Physiology, University of Minnesota Medical School, Minneapolis, MN, USA; Division of Molecular Medicine, Department of Medicine, University of Minnesota Medical School, Minneapolis, MN, USA.
  • 3 Division of Molecular Medicine, Department of Medicine, University of Minnesota Medical School, Minneapolis, MN, USA.
  • 4 Division of Molecular Medicine, Department of Medicine, University of Minnesota Medical School, Minneapolis, MN, USA. Electronic address: ppuchals@umn.edu.
PMID: 41043268 DOI: 10.1016/j.talanta.2025.128919
Abstract

Acetoacetate (AcAc) and β-hydroxybutyrate (βOHB) are ketone bodies involved in energy metabolism, particularly during physiological states of glucose scarcity, such as fasting, exercise, and the implementation of a ketogenic diet. The production (ketogenesis) and utilization (ketolysis) of ketone bodies are dynamic processes that can be quantified using stable isotope-labeled tracers in metabolic tracing studies, necessitating precise and sensitive analytical methods for accurately measuring both labeled and unlabeled pools. Although UHPLC-MS/MS has recently emerged as a reliable tool for quantifying ketone bodies, its dependence on 13C-labeled internal standards limits its utility in 13C-based tracer studies. AcAc, in particular, poses challenges due to its chemical instability and the scarcity of authentic, stable, isotopically labeled internal standards. While the chemical reduction of AcAc to βOHB provides a solution, this necessitates a cumbersome desalting step. To overcome these limitations, we developed a novel approach using deuterated AcAc (d3-AcAc) and [3,4,4,4-d4]βOHB as internal standards for the simultaneous quantification of 13C-labeled and unlabeled ketone bodies in biological samples. We optimized the synthesis of AcAc from ethyl-AcAc via base-catalyzed hydrolysis, achieving 99.2 ± 0.2 % purity at 60 °C for 3 h, as confirmed by 1H NMR. Stability assessments in the extraction buffer and post-extraction serum samples confirmed the robustness of newly synthesized d3-AcAc for at least 5 h. A comparative analysis against the labor-intensive conventional method demonstrated superior precision, accuracy, and ease of application, enabling high-throughput metabolic and clinical studies. The optimized UHPLC-MS/MS method substantially improves metabolic tracing capabilities, enabling rapid and accurate investigation of ketone body tracing studies across various physiological and pathological conditions.

Keywords

Acetoacetate; Beta-hydroxybutyrate; Flux modeling; Ketone bodies; Quantification; Stable isotopes.

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