Developing chromatographic methods for acyl-CoAs is challenging due to their structural complexity. These compounds exhibit significant variations in their physicochemical properties by factors such as carbon chain length, degree of saturation, and the presence of functional groups . Additionally, acyl-CoAs have low endogenous levels and are highly unstable in aqueous solutions. Due to these reasons they are susceptible to hydrolysis, making sample preparation challenging and resulting in poor recovery and low signal intensity . The quantification of acyl-CoAs has previously been accomplished using a variety of analytical techniques, such as gas chromatography, capillary electrophoresis, and reversed phase liquid chromatography (RP-LC) coupled to UV or fluorescence detection . However, liquid chromatography coupled to mass spectrometry (LC-MS), is the most widely used technique for acyl-CoA analysis due to its higher sensitivity and selectivity. On the other hand, severe peak tailing, signal deterioration, and poor detection limits are common obstacles associated with this approach . Various efforts have been made to cover the full range of acyl-CoA in a single analytical run. One such method is to use ion-pairing reagents such as triethylamine or dimethylbutyl amine . However, ion-pairing reagents are reported to decrease mass spectrometry signal intensity and frequent cleaning of detectors is required. Another approach is to use two-dimensional(2D)LC-MS , but the complexity and analysis time are increased by inclusion of a second dimension of separation, which has an impact on throughput. Furthermore, a RP-LC-MS/MS technique based on phosphate methylation after acyl-CoA derivatization has been reported . Nonetheless, derivatization complicates sample preparation and requires investigation for the evaluation of complete chemical conversion.