Oxidation end-products of trans-resveratrol 635
ꢀaq
.
2
ꢀ7
of e
and H by O
), with respective yields of 2.8 ꢂ 10 and
ꢀ
7
ꢀ1 11
3
.4 ꢂ 10 mol.J
.
It is well known that hydroxyl radicals
1
5
are highly reactive species, whereas superoxide radicals are
2
1
poorly oxidizing or reducing species. Jia et al. have shown
that RVT is only able to scavenge superoxide free radicals
ꢀ
1 22
.
in vitro for concentrations much higher than 100 mmol.L
As a consequence, under our conditions, hydroxyl radicals
are the only radical species initiating the oxidation process.
Volumes of 1.5 mL of a non-irradiated solution were
systematically taken as a control for each experiment. Prior
to each set of experiments, glassware was carefully washed
with TFD4 soap (Franklab, France), rinsed with ultra-pure
water (resistivity 18.2 MV, Maxima Ultra Pure Water, ELGA)
and finally heated at 4008C for 4 h to avoid any pollution by
organic compounds.
Figure 1. Chemical structure of trans-resveratrol.
assisted by the hydroxyl groups of the aromatic rings of RVT.
In our study, the identification of these oxidation products was
n
performed by MS experiments and deuterium labelling.
EXPERIMENTAL
Chemicals and reagents
trans-Resveratrol (trans-RVT) (ref. 70675) was purchased
from Cayman Chemical Company (Spi-Bio, Montigny-
le-Bretonneux, France). All aqueous solutions of trans-RVT
Mass spectrometric analyses
Mass spectrometry was performed on an ion-trap mass
spectrometer (LCQ Advantage, ThermoFinnigan, Les Ulis,
France) equipped with an ESI source. The capillary
temperature was held at 2508C and the relative sheath and
auxiliary gas flow rates were set at 20 and 5, respectively
ꢀ
1
(
M ¼ 228 g.mol ) were prepared at a concentration of
ꢀ
1
1
00 mmol.L
(pH 7.0) with ultra-pure water (resistivity
1
8.2 MV, Maxima Ultra Pure Water, ELGA), using sonication
for 2 h. Aqueous solutions of trans-RVT are stable for at least
week when protected from light to avoid chemical
modifications and isomerization into cis-resveratrol, as
ꢀ
1
1
(sheath gas, 0–100 units corresponds to 0–1.5 L.min ;
ꢀ
1
auxiliary gas, 0–60 units corresponds to 0–18 L.min ). Other
parameters, such as lens or capillary voltages, were tuned
systematically to obtain the best signal intensities for each ion
of interest. All experiments were performed in the negative-
ion mode, and each spectrum was typically an average of 20
acquired scans. For tandem mass spectrometry (MS/MS)
experiments, a typical isolation width of 1 Da was used.
For direct infusion analysis, irradiated aqueous solutions
of trans-RVT were diluted in acetonitrile (1:1, v/v) prior to
being infused continuously into the ESI source with an SGE
1
9
checked by UV-visible spectrophotometry. Piceatannol
ref. AGP-1015-50) was from ABCys (Paris, France). 3,5-
(
Dihydroxybenzoic acid (ref. D110000), 4-hydroxybenzoic
acid (ref. 240141), 3,5-dihydroxybenzaldehyde (ref. 144088)
and 4-hydroxybenzaldehyde (ref. 368113) were purchased
from Sigma-Aldrich (St. Louis, Mo., USA).
.
Generation of HO free radicals by gamma
radiolysis of water
ꢀ
1
Radiolysis corresponds to the chemical transformations of a
solvent because of the absorption of ionizing radiation,
which produces a homogeneous solution of free radicals
within a few nanoseconds. Radiolytically generated free
radicals are independent of the nature and concentration of
the dissolved compound as long as its concentration remains
250 mL syringe at a flow rate of 12.5 mL.min . To study
deuterium exchange on trans-RVT and its oxidation end-
products, 3 mL of irradiated and non-irradiated trans-RVT
aqueous solutions were lyophilized to dryness and rediluted
2
into the labelled medium D O/acetonitrile (1:1, v/v).
Deuterium oxide has minimum isotopic purities of 99.96%
and was from Aldrich Chemicals (Milwaukee, WI, USA).
In addition, some experiments were conducted by coupling
a high-performance liquid chromatograph (Surveyor, Thermo-
quest, Les Ulis, France) to the mass spectrometer. As much of
3 mL of irradiated and non-irradiated RVT aqueous solutions
ꢀ2
ꢀ1 12
lower than or equal to 10 mol.L .
Gamma irradiations were performed with an IBL 637
irradiator (CIS Biointernational, Gif-sur-Yvette, France)
1
37
using a
Cs g-ray source whose activity was ꢁ222 TBq
(
6000 Ci). The main advantage of gamma radiolysis is that it
allows the modulation of the cumulated amount of ROS
produced by increasing or decreasing the radiation dose
were lyophilized to dryness and rediluted into 200 mL of H O/
2
acetonitrile (20:80, v/v). Chromatographic conditions were as
follows: 20mL of sample were injected onto the column
(Kromasil C18 250 ꢂ 2.1 mm, 5 mm, A.I.T. Chromato, France),
whose temperature was held at 308C. Gradient elution was
ꢀ
1
(
expressed in Gy; 1 Gy ¼ 1 J.kg ), i.e. by selecting the time
during which the sample is exposed to the g-ray source:
the longer the exposure, the higher the radiation dose. It can
be noted that for diluted solutions, no direct interaction of g-
radiation with the molecule under focus occurs and the
radiolytic effect is only due to the radical species produced
by water radiolysis. The dosimetry was determined by the
2
carried out with H O (mobile phase A) and acetonitrile (mobile
ꢀ1
phase B) at a flow rate of 150 mL.min . The mobile phase
gradient was programmed with the following time course:
20% mobile phase B at 0 min, linear increase to 80% mobile
phase B at 20 min, 20% mobile phase B at 21 min and held
5 min. The mass spectrometer was used as a detector, working
in the full-scan mode between 50 and 500 Da and in a
dependent scan mode, allowing the fragmentation of selected
precursor ions (typical isolation width of 1 Da and collision
energy set at 35%, units as given by the manufacturer).
2
0
method of Fricke and Morse and the dose rate was found to
ꢀ
1
be 10 Gy.min in our experiments. A radiation dose of
4
00 Gy was delivered to the aqueous solutions of trans-RVT.
In the presence of oxygen, i.e. for aerated solutions, water
radiolysis leads to the generation of both hydroxyl and
:
ꢀ
superoxide free radicals (O coming from the scavenging
2
Copyright # 2010 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2010; 24: 634–642
DOI: 10.1002/rcm