We started the chromatographic separation with a 20 min gradient as reported in section Table . In order to achieve the quantification of all acyl-CoA species in a single analytical run, we employed a ZIC-cHILIC column which contains a phosphorylcholine group (Figure ) that consists of a negatively charged inner moiety and a positively charged outer moiety . We chose to use this ZIC-cHILIC column for the chromatography optimization of acyl-CoA as it was reported to effectively separate various compounds containing phosphate groups like ATP, ADP, NAD, sugar phosphates, etc. .The presence of a zwitterionic stationary phase requires lower concentrations of buffer compared to other types of stationary phases, as the zwitterionic stationary phase contains both positive and negative charges. This causes weak electrostatic interactions with the analytes and hence only a low concentration of buffer is needed .The peak tailing observed in the acyl-CoA species can be attributed to the presence of the phosphate group on CoA moeity . This phenomenon occurs due to the strong interaction between the phosphate group and specific sites within the column. Additionally, these phosphate groups have a tendency to adhere to the stainless parts of the LC-MS instrumentation, further contributing to the challenges in analysis .The buffer salts such as ammonium acetate and ammonium formate etc., are known to maintain the ionization of analytes and decrease the interaction between stationary phase and analytes . We have used ammonium acetate buffer as this is one of the most commonly used buffers for metabolomics studies employing HILIC chromatography . In this study, we investigated the impact of different concentrations of ammonium acetate on the peak shape, separation and retention of acyl-CoAs. Mobile phases were prepared with concentrations of 2.5 mM, 5 mM, and 10 mM ammonium acetate and the buffer concentration was kept same in both mobile phases to maintain consistent ionic strength throughout the gradient elution. Figure displays the peaks of the acyl-CoA standards for each buffer concentration and peak asymmetry factors were calculated and summarized in Table . Notably, as the concentration of ammonium acetate increased, the peak tailing of the acyl-CoAs decreased. After evaluating different concentrations of ammonium acetate, a final concentration of 5 mM was chosen, as it resulted in satisfactory peak shapes. Additionally, maintaining a lower salt concentration helps prevent excessive salt precipitation within the instrument, ensuring its proper functionality and longevity. Furthermore, we assessed the influence of pH variation and flow rate on peak tailing but did not observe any significant effects (data not shown). As a result, a flow rate of 0.25 mL/min was chosen for chromatographic separation with the presence of 5 mM ammonium acetate in the mobile phases.