DOE2 with a two-level full factorial design (FFD) was used to define the optimal extraction condition of acidic fractions with LLE. The model oil consisted of seven carboxylic acid standards was used. Three factors were examined, including ammonium hydroxide concentration (corresponding to pH 6.8 -10.5 at 25 ᵒC), water content, and solvent types (Table ). For extractions, 200 µL model oil was extracted two times with 600 µL designed solutions by shaking at 1000 rpm for 2 hours. The extractants were isolated by centrifugation at 4400 rpm for 15 mins and collected into 2 mL amber vials. In the case of using ammonium hydroxide for LLE, 0.3% hydrochloric acid solution was used to adjust the extractant's pH to 6.5 before EDC -4-BNMA labeling. The optimal labeling condition obtained from DOE1 were applied for DOE2: 320 µM EDC (molar ratio EDC: -COOH 2:1), 1600 µM 4-BNMA (molar ratio 4-BNMA: -COOH 10:1), t1 = 0.5 hours, t2 = 0.5 hours, and T = 60 ᵒC. For experiments with ACN, t1 was increased to 12.5 hours. Samples were cooled to room temperature, dried with nitrogen gas, and reconstituted in 1 mL ACN-H2O (1:1, v/v), followed by filtering with 0.2 µm syringe membrane before analysis.