Z spray

A Waters Acquity Ultra-high Performance Liquid Chromatography™ I-Class system (Waters Corporation, Milford, CT, USA) was used for the determination of seven bisphenols and four parabens. A Waters Xevo^® TQ-XS (Waters Corporation, Milford, CT, USA) with an orthogonal Z-Spray™ electrospray ionization (ESI) source (Waters Corporation, Milford, CT, USA) was used for the spectrometric measurements. The column was a Waters UPLC^® BEH C₁₈ (2.1 mm × 50 mm, 1.7 μm particle size). A ScanVac CoolSafe™ lyophilizer (Lynge, Denmark) was used for lyophilization of food samples. Other laboratory equipment was a vortex-mixer (IKA, Staufen, Germany), a GX400 laboratory balance (Mettler-Toledo, Columbus, OH, USA), a Universal 32 centrifuge (Hettich, Tuttlingen, Germany), a Spectrafuge™ 24D centrifuge (Labnet International, Inc., Edison, NJ, USA) and a SBHC0NC sample concentrator (Stuart, Staffordshire, UK), and an Ultrasons-HD series ultrasonic bath (Selecta, Barcelona, Spain). For the treatment and analysis of data and for equipment control, MassLynx 4.1 software (Waters Corporation, Milford, CT, USA) from Waters was used.

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.