2.9. Identification of Phenolic Compounds by Mass Fragmentation

Phenolic compounds also were identified by LC-ESI-UHR-QqTOF-MS in the pineapple by-products liquid fraction, according to Monforte, et al. [25]. For the characterization of the samples, an UltiMate 300 Dionex UPLC (Thermo Scientific, Waltham, MA, USA), coupled to an ultra-high resolution Qq-time-of-flight (UHR-QqTOF) mass spectrometer with 50,000 full-sensitivity Resolution (FSR) (Impact II, Bruker Daltonics, Bremen, Germany), was used. The identification of the molecules was performed using an Acclaim RSLC 120 C18 column (100 mm × 2.1 mm, 2.2 µm) (Dionex). The injection volume was 1 µL. Two mobile phases were employed—the mobile phase A consisting of water and formic acid (99.9:0.1; % v/v), and mobile phase B of acetonitrile and formic acid (99.9:0.1; % v/v). The gradient started in 5% of mobile phase B until 95% during 7 min, which was maintained constant for 2 min and returned to 5% mobile phase B in 1 min and maintained at 5% for 5 min more at a flow rate of 0.25 mL/min. The parameters were set, as described by Monforte, et al. [25], using positive ionization mode with spectra acquired over a range from m/z 20 to 1000. A 4.5 kV of capillary voltage was applied, and the temperature was 200 °C in the drying gas, drying gas flow was 8 L/min, the pressure was 2 bar in the nebulizing gas, 300 Vpp in collision RF, with a 120 µs of transfer time and pre-pulse storage of 4 µs. A syringe pump delivered post-acquisition internal mass calibration using sodium formate clusters with the sodium formate at the start of each chromatographic analysis. Also, high-resolution mass spectrometry (MS) was used to identify the phenolic compounds present in the fractions. The elemental composition for the compounds was confirmed according to accurate mass and isotope rate calculations designated as mSigma (Bruker Daltonics, Billerica, MA, USA). The accurate mass measurement was within 5 mDa of the assigned elemental composition, and mSigma values of <20 provided confirmation. Identification was carried out using pure standards (1 mg/L) in methanol (LC-MS grade). All the phenolic compounds were identified on their accurate mass [M − H]⁻.