The following optimal conditions were applied for the analysis of crude oils: 0.5 mL crude oil was extracted two times with 1.5 mL of 10% v/v aqueous acetonitrile solution with 0.01% ammonium hydroxide. A total of 3 mL extractant was collected into a 4 mL amber vial by centrifugation at 4400 rpm for 15 mins. To the extractant, 10 µL EDC stock solution was added and held at 60 ᵒC for 0.5 hours before adding 10 µL 4-BNMA stock solution and keeping it at the same temperature for 1 hour. For LC-Orbitrap HRMS analysis, 3 mL extractant was dried under the nitrogen stream and reconstituted in 1 mL ACN-H2O (1:1, v/v). Notably, the optimal concentrations of reagents were held at EDC: -COOH and 4-BNMA: -COOH molar ratios of 2:1 and 10:1, respectively. The carboxylic acid concentration in six crude oil samples was estimated based on their reported TAN, assuming that only monocarboxylic acids engender acidity. 3 Results and discussions