Previous EDC/N-methylated amine coupling methods require mixing the sample with EDC and amine simultaneously at concentrations above 2.4×10 6 and 4.7×10 folds of total carboxyl concentration (-COOH) for incubation at 60 ᵒC, 1 hour. There reported methods have had several drawbacks, including (i) greatly excessed reagents without removal, (ii) the coupling times were not thoroughly investigated, and (iii) high water content caused EDC hydrolysis. Herein, we optimized the EDC/4-BNMA derivatization and LLE steps using DOE techniques. The supporting information describes the full DOE setup and analysis details, validation of DOE and plot information. Initially, individual FFD sets with four quantitative factors (1 -4) were carried out to find optimal reaction conditions in a specific solvent -ACN or EtOH, as they are favourable for LLE of acids in crude oil. Because of the potential of labeling approach in different solvents involving other extraction techniques, DOE1 was setup with inclusion of factor 5. The GSD allows (i) combining individual DOEs to add a complete design of all possible factor compositions -sequential experimentation, and (ii) reducing experiments for a mix of quantitative and multilevel qualitative factors.