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bioRxiv · 10.64898/2026.08.12.744242

Integrated assessment of fatty acid metabolism and cellular energy status using HILIC-MS/MS

Lopes, M.·Roberts, K. D.·Heath, A. E.·Lund, P. J.
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Abstract

Acetyl-CoA and other acyl-CoA thioesters are critical intermediates in the metabolic reactions that cells rely on to produce energy and carry out biosynthesis. Therefore, the analysis of acyl-CoA provides valuable information about the metabolic activity of cells, especially when combined with stable isotope tracing. Acyl-CoA species are routinely monitored by reversed-phase liquid chromatography coupled to tandem mass spectrometry (RPLC-MS/MS). However, drastic differences in the hydrophobicity of short-chain versus long-chain acyl-CoA species have been challenging to accommodate with a single set of RPLC conditions. Here, we describe a convenient method based on hydrophilic interaction liquid chromatography (HILIC-MS/MS) for the concurrent detection of both short-chain and long-chain acyl-CoA and their corresponding acyl-carnitine species. Using this strategy, we tracked the metabolism of isotope-labeled fatty acids in multiple cell lines, which revealed differences in their propensities for fatty acid oxidation and the extent to which isotope incorporation into acyl-CoA mirrored that of acyl-carnitine. We also applied the HILIC-MS/MS workflow to the analysis of NADH and ATP, making it a useful technique for gauging cellular bioenergetics as reflected by the acetyl-CoA/CoA, NADH/NAD+, and ATP/ADP ratios. Altogether, this HILIC-MS/MS platform enables a streamlined analysis of acyl-CoA species and other key intermediates in cell metabolism.

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Lopes, M., Roberts, K. D., Heath, A. E., Lund, P. J.. 2026-08-17. Integrated assessment of fatty acid metabolism and cellular energy status using HILIC-MS/MS. https://doi.org/10.64898/2026.08.12.744242

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