HILIC–MS/MS Using Zwitterionic Column Simultaneously Quantifies Free CoA and Short- to Long-Chain Acyl-CoA Compounds


A variety of chromatographic techniques have been used to quantify the full range of acyl-CoAs, formed when a fatty acid bonds with coenzyme A, but added complexity and analysis time adversely affect the level of throughput.

A recent study in the Journal of Chromatography A describes the development of a zwitterionic hydrophilic-interaction chromatography (HILIC) column, meaning one that is intended to be electrically neutral, for the purposes of simultaneously determining free coenzyme A (CoA) and short- to long-chain acyl-CoA compounds, which are formed when CoAs bond with fatty acids (FAs) (1). The method of analysis was a targeted method that combined HILIC with tandem mass spectrometry (MS/MS).

3d illustration of human body liver | Image Credit: © PIC4U - stock.adobe.com

3d illustration of human body liver | Image Credit: © PIC4U - stock.adobe.com

In their research, this team of six authors, affiliated with Leiden University and University Medical Centre Groningen in The Netherlands, said an efficient analytical method to identify acyl-CoAs is needed because of their involvement in physiological and pathophysiological pathways—particularly the metabolization of FAs through beta oxidation (1). That process breaks down FAs within the liver, producing adenosine triphosphate (ATP) in low-glucose conditions; the authors explained that acyl-CoAs undergo the fatty acid beta oxidation (FAO) process inside the mitochondria after CoAs form thioester (when sulfur atoms replace oxygen atoms) bonds with FAs. Fatty acid oxidation disorders (FAODs) may occur, causing acyl-CoA ester accumulation, when enzymes or other proteins in the pathway exhibit deficient activity.

The authors acknowledged that previous research had already established liquid chromatography coupled to mass spectrometry (LC–MS) as the most effective approach for analyzing the full range of CoAs. However, challenges do exist, including carbon chain length, degree of saturation, instability in aqueous solutions, and resultant difficulties in sample preparation (1). Reversed-phase liquid chromatography (RPLC) combined with HILIC, or in some cases two-dimensional (2D)-LC–MS have helped with some problems including low signal intensity and poor recovery but have made the overall process more complex and time-consuming, with efforts to mitigate either of those factors having the potential of impacting throughput capability.

The practical application of the HILIC–MS/MS platform in this study was demonstrated in cells of the Hep G2 human liver cancer cell line, which for this experiment were cultured in both supplemental and starved states (1). This did not require the use of an ion-pairing reagent, which can contaminate the MS instrumentation. In the latter of the two cultured Hep G2 states, the researchers’ findings indicated an increase in FAO relative to other downstream metabolic processes. Overall, the research team evaluated their chosen method’s performance as a success: “sensitive, linear and repeatable.”

Extrapolating their results to further chromatographic research and analysis, the researchers concluded by saying that the simplicity and robustness of HILIC–MS/MS for determining the full range of acyl-CoAs in a single run suggest that the approach can be implemented in clinical settings, however suggesting that further examination and possible use of ion mobility-mass spectrometry (IM-MS) may be needed in the future (1).


(1) Singh, M.; Kiyuna, L. A.; Odendaal, C.; Bakker, B. M.; Harms, A. C.; Hankemeier, T. Development of Targeted Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry Method for Acyl-Coenzyme A Covering Short- to Long-Chain Species in a Single Analytical Run. J. Chromatogr. A 2024, 1714, 464524. DOI: 10.1016/j.chroma.2023.464524

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