sorption spectra of anthocyanins were recorded
from 200–800 nm in steps of 2 nm using a U-3310
double-beam scanning UV-Visible spectrophotometer
(Hitachi High-technologies Co., Tokyo,
Japan) and an on-line photodiode array detector
(Waters Assoc., Milford, USA). The mass spectra
were obtained using a Micromass triple quadrupole
ion-tunnel mass spectrometer equipped
with a Zspray ESI source (Micromass UK Ltd.,
Manchester, UK). Approximately 100 µl of the
eluate from HPLC (conditions were similar as with
HPLC analysis above) was delivered to the ESI
source by a micro splitter valve for ESI/MS and
MS/MS analysis. Standard cyanidin 3-rutinoside
was used to tune the instrument. For the precursor
ion scan, the instrument was tuned to maximum
abundance of the daughter ion (m/z 287) and molecular
cation (m/z 595) signals (approximately in
equal abundance). For the product ion scan, the
precursor ion (m/z 595) was attenuated to 50%. The
quadruple instrument was operated at the following
settings: capillary voltage 3.0 kV; cone voltage
35 V; RF lense 1 50 V; desolvation gas temperature
500°C at a flow of 17 l/min; source temperature
105°C; collision gas (argon) pressure, 7 psi; collision
energy was set at 18 eV. The relative amount
of each anthocyanin was reported as the mean of
three replicates. The detection limits (S/N > 3) of
approximately 1 femtomol were obtained during
LC/MS/MS analysis.
1
HNMR spectral data were recorded on a Bruker
Advance 400 instrument operating at 400 MHz in
CD3
OD/CF3
OD (9:1) with TMS (tetramethylsilane)
as internal standard. The sample temperature was