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Sheath (N2), ion sweep, and auxiliary gas (N2) pressure were, respectively, set at 10, 2.0, and 5?psi. In MS2 (MS/MS) experiments, the normalized collision energy was optimized for each of the compounds. The particular transitions, the collision energy, and the tube lens settings were specific for each analyte and obtained using the TSQ Tune Master software buy JQ1 in the optimization MS + MS/MS mode. These were shown in Table 1. Table 1 Time-scheduled SIM conditions, m/z ions and corresponding structures used in the LC-ESI-MS/MS analysis of CEs. 3. Results and Discussion 3.1. Comparison of Retention Time, Intensity, and Ion Fragmentation of CEs between ESI and APCI Mass Spectra ESI is one of the primary ionization techniques for the coupling of LC to MS, while APCI is a supplementary technique to electrospray and suitable for thermally stale polar and nonpolar compounds due to no generation of charged ions. ESI is particularly suited for polar organic compounds and is sensitive to matrix effects. High molecular weight compounds can be observed as multicharged molecular ions in ESI. In contrast to ESI, the APCI technique is used to analyze smaller molecular compared to ESI technique [16, 19, 20]. Owing to different ionization mechanism and characteristics of CEs, E-64 we hypothesized that ESI is more suitable for isolation and identification of CEs than APCI; firstly, polarity of CEs is due to the ester group. Secondly, the least molecular weight of CEs is 428.7, which is combined cholesterol with acetic acid (C2:0). The retention time (RT) of CEs by using both ESI and APCI process is expressed in Table 1. Overall, RT of CEs on total ion chromatogram (TIC) was affected by a number of carbon chains and double bonds. The Chl-lignocerate (C24:0) appeared at 28.49?min of RT, while Chl-myristate was separated at 16.06?min of RT. RT increased with longer carbon chain lengths. CEs containing the same number of carbons with different number of double bonds such as Chl-linoleate (C18:2), Chl-oleate R428 datasheet (C18:1), and Chl-stearate (C18:0) appeared at 15.55, 18.13, and 21.31?min of RT, respectively. The present double bonds reduced RT. These findings are similar to a previous study in which fatty acid's chain length and double bond influenced the RT in the analysis fatty acid [21, 22]. Under ESI technique, both full and product ion scan of CEs standards were demonstrated to generate protonated molecular ions such as [M+Na]+ and [M+NH4]+ (Table 1).