The desirability plots (Figures 5, S8-10) visualize the possibility of maximizing carboxylic-BNMA responses (scoring 0 to 1) when varying parameters. As shown in Figure , low concentrations of EDC and 4-BNMA with short coupling times at 60 ᵒC should ideally maximize the reaction efficiency in ACN. The EDC activation time -t1, should be considered carefully; mixing EDC simultaneously with 4-BNMA resulted in a low yield of acid derivatives. The result is in accord with Quincy et al. study indicating that the derivatization of carboxylic acids by EDC and amine could be efficient at low molecular ratios between reagents and total -COOH. However, these findings are contrary to that of Bryce et al., who used excessive amounts of EDC and 4-BNMA without considering coupling times. Therefore, we suggest the optimal condition for EDC/4-BNMA coupling of carboxylic acid using ACN solvent, with molar ratios of EDC: -COOH 2:1 and 4-BNMA: -COOH 10:1, t1 = 0.5 hours, t2 = 1 hour at 60 o C. In previous studies, 70% alcohol aqueous solution and sodium hydroxide have been applied to extract carboxylic acids from crude oil. Barrow et al. dissolved crude oil in acetonitrile at 100 µg mL -1 with 1% ammonium hydroxide addition for direct infusion to nano-ESI FT-ICR MS. In this study, acetonitrile and methanol were selected as solvents for LLE of crude oil acids, with the relative polarity of 0.460 and 0.762, respectively. Ammonium hydroxide was selected to assist the deprotonation of carboxylic acids and promote their solubility in the aqueous-solvent solution, improving extraction efficiency. Because of the significant influences of aprotic and protic solvents on derivatization, the labeling conditions were adjusted based on DOE1 to achieve equivalent responses in DOE2. For experiments with ACN, the adjustment of t1 was to normalize the difference between ACN and MeOH based on the desirability plots at a score of 0.4 -maximum score for MeOH (Figures and).