The extraction protocol was based on the conditions previously optimized by Rostagno et al. [24]. In short, 0.5 g of each coffee sample were extracted with 15 ml of 50% methanol/water, followed by 15 ml of 75% methanol/water, and finally with 15 ml of 100% methanol. Extraction was performed on a multi-frequency ultrasound bath [10], [25] and [26], operating at 25 kHz for 20 min at 60 °C. After each extraction step, the sample is centrifuged for 10 min at 10 °C and a velocity of 4000 rpm using a universal centrifuge. The supernatant was collected and the solid residue was subjected to the next extraction step. After the final extraction, the supernatants were combined, and water was added to obtain a final volume of 100 ml. 20 μl of the obtained extract was filtered and injected into the HPLC apparatus. Analyses were performed in triplicate.