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.

• HILIC-MS/MS method covers free CoA and acyl-CoAs (short to long-chain) in one run. • The method employing a zwitterionic HILIC column was characterized. • Limit of detection is in the range of (1.3–12.4) pmol mL−1 in the targeted method. • Application in HepG2 cells shows high acyl-CoAs and low free CoA in starved state. Acyl-CoAs play a significant role in numerous physiological and metabolic processes making it important to assess their concentration levels for evaluating metabolic health. Considering the important role of acyl-CoAs, it is crucial to develop an analytical method that can analyze these compounds. Due to the structural variations of acyl-CoAs, multiple analytical methods are often required for comprehensive analysis of these compounds, which increases complexity and the analysis time. In this study, we have developed a method using a zwitterionic HILIC column that enables the coverage of free CoA and short- to long-chain acyl-CoA species in one analytical run. Initially, we developed the method using an LC-QTOF instrument for the identification of acyl-CoA species and optimizing their chromatography. Later, a targeted HILIC-MS/MS method was created in scheduled multiple reaction monitoring mode using a QTRAP MS detector. The performance of the method was evaluated based on various parameters such as linearity, precision, recovery and matrix effect. This method was applied to identify the difference in acyl-CoA profiles in HepG2 cells cultured in different conditions. Our findings revealed an increase in levels of acetyl-CoA, medium- and long-chain acyl-CoA while a decrease in the profiles of free CoA in the starved state, indicating a clear alteration in the fatty acid oxidation process. [ABSTRACT FROM AUTHOR]