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Total phenolics were extracted according to protocol described
in Tomás-Barberán et al. (2001) using water:methanol (2:8)
containing 2 mM NaF (to inactivate polyphenol oxidase activity
and prevent phenolic degradation) and quantified in duplicated
using the Folin–Ciocalteu reagent and results (mean ± SE) were
expressed as mg gallic acid equivalent 100 g−1 fresh weight. For
individual phenolic compounds,the protocol described by GironésVilaplana
et al. (2012) was followed. Briefly, lyophilised samples
(100 mg) were mixed with 1 mL of methanol/formic acid/water
(25:1:24, v/v/v), vortexed, sonicatedinanultrasonic bathfor 60 min
and centrifuged at 10,500 × g for 5 min The supernatant was filtered
through a 0.45-m PVDF filter (Millex HV13, Millipore, Bedford,
MA, USA) and used for HPLC analysis. The HPLC-DAD-ESI/MSn analyses
were carried out in an Agilent HPLC 1100 series machine
equipped with a photodiode array detector and a mass detector
in series (Agilent Technologies, Waldbronn, Germany). The equipment
consisted of a binary pump (model G1312A), an autosampler(model G1313A), a degasser (model G1322A), and a photodiode
array detector (model G1315B). The HPLC system was controlled by
ChemStation software (Agilent, version 08.03). The mass detector
was an ion trap spectrometer (model G2445A), equipped with an
electrospray ionisation interface and controlled by LCMSD software
(Agilent, version 4.1). The ionisation conditions were 350 ◦C and
4 kV, for capillary temperature and voltage, respectively. The nebuliser
pressure and nitrogen flow rate were 65.0 psi and 11 L/min,
respectively. The full-scan mass covered the range from 100 to
1200 m/z. Collision-induced fragmentation experiments were performed
in the ion trap using helium as the collision gas, with voltage
ramping cycles from 0.3 up to 2V. The mass spectrometry data
were acquired in the positive ionisation mode for anthocyanins
and in the negative ionisation mode for other flavonoids. The MSn
was carried out in the automatic mode on the more abundant
fragment ion in MS (n − 1). The HPLC was equipped with a Luna
C18 column (25 cm × 0.46 cm i.d., 5 m particle size; Phenomenex,
Macclesfield, UK) with a C18 security guard (4.0 mm × 3.0 mm) cartridge
system (Phenomenex, Macclesfield, UK). Water:formic acid
(99:5, v/v) and acetonitrile were used as mobile phases A and B,
respectively, with a flow rate of 1 mL min−1. The linear gradient
started with 8% of solvent B, reaching 15% solvent B at 25 min,
22% at 55, and 40% at 60 min, which was maintained up to 70 min
The injection volume was 20 L. Chromatograms were recorded
at 280, 320, 360, and 520 nm Different phenolics were characterised
by chromatographic comparison with analytical standards
as well as quantified by the absorbance of their corresponding
peaks. Hydroxycinnamic derivatives, p-coumaroylquinic acid and
hydroxybenzoic acid were characterised by chromatographic comparisonaccordingly
toprevious reports basedonretentiontime and
UV–vis spectra at 280 nm (Tomás-Barberán et al., 2001). Anthocyanin
standards (cyanidin 3-glucoside, cyanidin 3-rutinoside and
pelargonidin 3-rutinoside) were purchased from Polyphenols SA
(Sandnes, Norway) and detected at 520 nm Cinnamic acids were
quantified as 5-O-caffeoylquinic acid at 320 nm, and flavonols as
quercetin 3-O-rutinoside at 360 nm and expressed as mg 100 g−1
fresh weight (mean ± SE).
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