Acquity uplc pda system

Phenolic acid extracts, including free and bound forms, were analyzed using a Waters Acquity^TM UPLC-PDA System (Waters, Rydalmere, New South Wales, Australia). The compounds were separated on a Waters HSS-T3 column (100 × 2.1 mm i.d; 1.8 μm) maintained at 40 °C, with 0.1% formic acid in Milli-Q-water (v/v) as eluent A and 0.1% formic acid in acetonitrile (v/v) as eluent B. The gradient program is as follows: 3 min, 5% B; 4.3 min, 20% B; 9 min, 45% B; 11 min, 100% B; 14 min, 100% B, and 17 min, 5% B. The flow rate was at 0.4 mL/min. Phenolic acids were quantified at 280 nm using the external calibration curves of phenolic acid standards, including p-hydroxybenzoic acid, vanillic acid, caffeic acid, p-coumaric acid, ferulic acid, and sinapic acid.

Carotenoids were analyzed using a Waters Acquity^TM UPLC-PDA system (Supplementary Table S1). Detected carotenoid compounds were identified using the same UHPLC-MS/MS system as described for the polyphenol analysis (Section 2.3.3) but employing an atmospheric pressure chemical ionization (APCI) operated in positive mode. A full MS scan (m/z 80–1200) was acquired. For the compounds of interest, a MS/MS scan range of m/z 80–650 was selected, with NCE at 20 V. Carotenoids were quantified at 450 nm, using external calibration curves of all-trans beta carotene, lutein and zeaxanthin. Concentrations of carotenoid standards were determined spectrophotometrically (Cintra 303, GBC Scientific Equipment, Braeside, Australia) using the specific molar absorption coefficients of carotenoids in solutions [23 (link)].

Analysis of individual (main) phenolic acids and flavonoids (free and bound) by UPLC-PDA followed the method of Gasperotti et al. [21 (link)], using a Waters Acquity^TM UPLC-PDA System (Waters, Milford, MA, USA). The compounds were separated on a Waters HSS-T3 column (100 × 2.1 mm i.d; 1.8 μm) maintained at 40 °C, with aqueous 0.1% formic acid (eluent A) and 0.1% formic acid in acetonitrile (eluent B). The gradient program (time (min), % B) was: (0.0, 5); (3.0, 5); (4.3, 20); (9.0, 45); (11.0, 0); (14.0, 0) with a flow rate of 0.4 mL/min.
Detected peaks in the feijoa samples were identified by a Thermo high resolution Q Exactive mass spectrometer equipped with a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific Australia Pty Ltd., Melbourne, VIC, Australia). A full scan in negative (ESI) ionization mode was acquired at a resolving power of 70,000 full width half maximum, followed by an MS2 scan range of m/z 100–1200 for the compounds of interest. The Thermo XcaliburTM software (Thermo Fisher Scientific) was used for data acquisition. The detected peeks were identified by matching spectrum, retention time, and MS data obtained from literature. External calibration curves were constructed from the polyphenolic standard solutions (0–2 mg/10 mL in methanol) for quantification, except for dihydroxyflavone, which was quantified as mg of catechin equivalent.