Pdfoa

Proteinogenic amino acids (i.e., alanine, isoleucine, leucine, phenylalanine, tyrosine, and valine) were quantified in the aqueous extracts of the cocoa beans, in duplicate, by UPLC-MS/MS, using an Acquity UPLC system, equipped with a HSS T3 column and coupled to a Quattro Micro tandem mass spectrometer with a ZSpray electrospray ionization source in the positive ionization mode (Waters). The flow rate of the mobile phase, composed of 5% (v/v) acetonitrile (Thermo Fischer Scientific) with 1 mM formic acid (Merck) and 1 mM pentadecafluorooctanoic acid (PDFOA, Sigma-Aldrich; eluent A) and 90% (v/v) acetonitrile with 1 mM formic acid and 0.5 mM PDFOA (eluent B; Sigma-Aldrich), was 0.23 ml/min. For elution, the following gradient was applied: 0 to 1 min, 99% eluent A and 1% eluent B; 1 to 8 min, 30% eluent A and 70% eluent B; 8 to 10 min, 0% eluent A and 100% eluent B; and 10 to 25 min, 99% eluent A and 1% eluent B. Quantification was performed by external calibration, including an IS solution [8 mg of 2-amino butyric acid (IS) dissolved in 900 ml of ultrapure water (MilliQ) and 1 mM formic acid (Sigma-Aldrich)]. All samples were microcentrifuged (19,400 × g for 15 min at 10°C) and filtered (0.2-μm H-PTFE Millex filters, Merck) before injection (10 μl) into the column.