Figure shows the two-step derivatization of carboxylic acid, i.e., carbamide activation with EDC and, subsequently, substitution with 4-BNMA. The carboxyl (-COOH) activation with EDC prevents acid-base reaction with 4-BNMA, making the acid susceptible to nucleophilic substitution. The derivatization was optimized with respect to EDC and 4-BNMA concentrations (corresponding to reagent: -COOH molar ratios in the range of 1 to 100), EDC and 4-BNMA coupling times (t1 and t2), and solvent (DCM, ACN, EtOH, MeOH). Seven acid standards were used with molecular structures and reactions scheme shown in Figures and. Briefly, a 980 µL mixture of acid standards (each at a concentration of 20 µM) was placed in a 2 mL vial. To this solution, 5 -15 µL of EDC stock solution (in DCM, ACN, EtOH or MeOH) was added and the mixture was kept at 60 ᵒC for t1 hours. The reaction was continued by adding 5 -15 µL of 4-BNMA stock solution held at 60 ᵒC for t2 hours. Finally, the solution was cooled to room temperature, dried under a low nitrogen gas flow for 10 minutes, and reconstituted in 1 mL ACN-H2O (1:1, v/v). Before analysis, the sample was filtered using a syringe filter (0.2 µm, polytetrafluoroethylene).