The identification and confirmation of acyl-CoA species were further supported by analyzing their retention time pattern. In HILIC chromatography, the gradient initiates with an organic mobile phase and subsequently transitions to a more aqueous phase. The non-polar nature of a compound increases with a higher number of carbon chains, whereas the polar character is intensified by an increase in the number of double bonds. As a result, acyl-CoA species with longer carbon chains elute first, followed by medium-and short-chain species, as depicted in Figure . Similarly, species with a higher number of double bonds but the same number of carbons elute later compared to species with a lower number of double bonds. For example, C16:0-CoA elutes at 5.56, while C16:1-CoA elutes at 5.59. This distinct retention time pattern is highly valuable for the identification and confirmation of a wide range of acyl-CoA species. . The linear regression coefficients (R 2 ) were above 0.99 for all spiked standards. The LODs and LLOQs were in the range of (1.3-12.4) pmol mL -1 and (3.1-26.6) pmol mL -1 respectively which makes our method sensitive enough to detect the acyl-CoAs in ~1×10 6 HepG2 cells. The intraday and interday precisions were determined at low, medium and high concentration levels.