The red wine extract was centrifuged at 3000g for 10 min at
4 C, filtered through a 0.22-lm PTFE membrane filter and directly
injected into the HPLC. The ZF homogenate was also centrifuged at
3000g for 10 min at 4 C, filtered through a 0.22-lm PTFE membrane
filter and directly injected into the HPLC.
An LTQ Orbitrap Velos mass spectrometer (Thermo Scientific,
Hemel Hempstead, UK) equipped with an ESI source in negative
mode was used to acquire mass spectra in profile mode with a
resolution of 30,000 at m/z 400. Operation parameters were as follows:
source voltage, 4 kV; sheath gas, 20 (arbitrary units); auxiliary
gas, 10 (arbitrary units); sweep gas, 2 (arbitrary units); and
capillary temperature, 275 C. Default values were used for most
other acquisition parameters (FT Automatic gain control (AGC) target,
5 105 for MS mode and 5 104 for MSn mode). The LTQ
delivers single-stage mass analysis providing molecular mass
information, two-stage mass analysis (MS/MS) and multi-stage
mass analysis (MSn) which gives structural information. Exact
mass measurements are accomplished using Orbitrap-MS. Red
wine extract and ZF were first analysed in full MS mode with the
resolution set at 30,000 at m/z 400. The successive analyses were
done in MSn mode with the resolution set at 15,000 at m/z 400.
An isolation width of 100 amu was used and precursors were fragmented
by high-energy C-trap dissociation (HCD) with normalised
collision energy of 2% and an activation time of 10 ms. The maximum
injection time was set to 100 ms with two micro scans for
MS mode and to 1000 ms with one micro scan for MSn mode.
The mass range was from m/z 100 to 1000. The data analyses were
performed using XCalibur software. The liquid chromatograph was