Determination of ketone bodies in biological samples via rapid UPLC-MS/MS

Patrycja Puchalska, Alisa B. Nelson, David B. Stagg, Peter A. Crawford

Research output: Contribution to journalArticlepeer-review

22 Scopus citations


Efforts to enhance wellness and ameliorate disease via nutritional, chronobiological, and pharmacological interventions have markedly intensified interest in ketone body metabolism. The two ketone body redox partners, acetoacetate (AcAc) and D-β-hydroxybutyrate (D-βOHB) serve distinct metabolic and signaling roles in biological systems. A highly efficient, specific, and reliable approach to simultaneously quantify AcAc and D-βOHB in biological specimens is lacking, due to challenges of separating the structural isomers and enantiomers of βOHB, and to the chemical instability of AcAc. Here we present a single UPLC-MS/MS method that simultaneously quantifies both AcAc and βOHB using independent stable isotope internal standards for both ketones. This method incorporates one sample preparation step requiring only 7 min of analysis per sample. The output is linear over three orders of magnitude, shows very low limits of detection and quantification, is highly specific, and shows favorable recovery yields from mammalian serum and tissue samples. Tandem MS discriminates D-βOHB from structural isomers 2- or 4-hydroxybutyrate as well as 3-hydroxyisobutyrate (3-HIB). Finally, a simple derivatization distinguishes D- and L-enantiomers of βOHB, 3-HIB, and 2-OHB, using the same rapid chromatographic platform. Together, this simple, efficient, reproducible, scalable, and all-encompassing method will support basic and clinical research laboratories interrogating ketone metabolism and redox biochemistry.

Original languageEnglish (US)
Article number122048
StatePublished - Apr 1 2021

Bibliographical note

Funding Information:
The authors are grateful for support from NIH ( DK091538 , AG069781 ). The authors thank Yi-Cheng Sin and Laura Kyro for technical and graphics support, respectively. BioRender supported the construction of cartoon images.

Publisher Copyright:
© 2020


  • Acetoacetate instability
  • Acetoacetate internal standard
  • D-beta-hydroxybutyrate structural and stereo-isomers
  • Quantification of ketone bodies


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