MS/MS and H/D exchange of oxidised melatonin
15 days, displaying no chemical modifications, as checked by UV-
visible spectrophotometry. MLT has a pKa of 12.3[22] that implies
one deprotonation of the nitrogen of the pyrrole ring (NH/N− acid-
base couple), so that at pH 7 MLT is preferentially in the protonated
form (Fig. 1). Irradiations were carried out in test tubes that had
been previously cleaned with hot TFD4 detergent (Franklab S.A.,
France), rinsed thoroughly with ultra-pure water, and then heated
to 400 ◦C for 4 h to avoid any contamination by remaining organic
compounds.
of 12.5 µl min−1. To study deuterium exchange on MLT and its
oxidation end-products, 5 ml of irradiated and non-irradiated MLT
aqueous solutions was lyophilised to dryness and rediluted into
the labelled medium D2O/MeOD (1/1 v/v). Deuterium oxide and
deuterated methanol have minimum isotopic purities of 99.96
and 99.5% D, respectively, and were from Aldrich Chemicals
(Milwaukee, WI, USA).
In addition, high-performance liquid chromatographic/mass
spectrometric (HPLC/MS) experiments were conducted (Surveyor,
Thermoquest, Les Ulis, France) by eluting with either non-
deuterated or deuterated solvents. As much as 5 ml of irradiated
and non-irradiated MLT aqueous solutions was lyophilised to
dryness and rediluted into 500 µl of H2O or D2O. Chromatographic
conditions were as follows: 20 µl of sample was injected onto
the column (Kromasil C18 250 × 2.1 mm, 5 µm, Ait-Chromato,
France), whose temperature was held at 40 ◦C. The elution
was achieved with a mobile phase consisting of a mixture
H2O/acetonitrile or D2O/acetonitrile (80/20, v/v) and delivered
at a flow-rate of 250 µl min−1. The mass spectrometer was
used as a detector, working in the full-scan mode between 100
and 400 Da and in the dependent scan mode, allowing the
fragmentation of selected precursor ions (typical isolation width
of 1 Da and collision energy set at 34%: units as given by the
manufacturer).
Generation of reactive oxygen species by water radiolysis
Radiolysiscorrespondstothechemicaltransformationsofasolvent
because of the absorption of ionising 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 lower than or equal to 10−2 mol l−1
.
[21]
Gamma radiolysis was carried out using an IBL 637 irradiator
(CIS Biointernational, Gif-sur-Yvette, France), a 137Cs source, whose
activity was approximately 222 TBq (6000 Ci). A dose rate
of 9.76 Gy/min was used. The dosimetry was determined by
the Fricke method,[21,23] namely radiooxidation of 10−3 mol l−1
ferrous sulfate solution in 0.4 mol l−1 sulfuric acid (under an
aerated atmosphere) taking λmax (Fe3+) = 304 nm, ε(304 nm)
=
2204 l mol−1 cm−1 at 25 ◦C and a radiolytic yield of G(Fe3+) =
1.62 µmol J−1.Differentradiationdoses,rangingfrom10to440Gy,
were delivered to 5 ml of the aqueous solution of MLT, depending
on the duration of exposure to the γ -ray source: the longer the
time, the higher the radiation dose. For each experimental set,
5 ml of non-irradiated solution was used as a control.
Results and Discussion
Deuterium exchange and fragmentation pathway of
non-oxidised MLT
A collision-induced dissociation (CID) spectrum was obtained for
a solution of MLT (100 µmol l−1) in H2O/MeOH (1/10 v/v) and is
presented in Fig. 1. The precursor ion is detected at m/z 233.1
and main fragments at m/z 216.1, 191.0 and 174.1 corresponding
to losses of 17, 42 and 59 Da, respectively. Initially, these losses
could come from the aliphatic part of MLT (N-acetyl group) and
correspond to NH3, CH2CO and (NH3 + CH2CO or CH3CONH2),
respectively. As shown in Fig. 1 (inset), the ion at m/z 174.1 is
generated by fragmentation of m/z 216.1. However, this fragment
is also known as a direct product ion of protonated MLT,[24,25] by a
loss of CH3CONH2.
Water radiolysis by γ -rays generates the free radical species
e−
,
•OH, •H and the molecular species H2 and H2O2. Under
aq
aerated conditions (oxygen concentration approximately 2 ×
10−4 mol l−1), hydroxyl and superoxide radicals (resulting from
the scavenging of e− and •H species) were simultaneously
aq
produced with the radiolytic yields (G-values expressed in moles
perJoule)of2.8×10−7 and3.4×10−7 mol J−1, respectively. Inthis
study, only hydroxyl radicals were considered as a potent oxidant
of MLT, because superoxide radicals are not able to initiate the
oxidation of MLT.[14,15]
MLT has two potential H/D exchangeable hydrogens (plus one
for protonation), one on the N-pyrrolic group and the other on the
N-acetyl group. An increase of +3 Da is therefore expected and
protonated MLT should be detected exclusively at m/z 236.0 in
D2O/MeOD. However, evenforMLTinpurelabelledmedium, three
peaksatm/z 234, 235and236weredetectedandwerefragmented
(Fig. 2). Although only fully deuterated MLT (m/z 236) is of interest,
the isotopic contribution of m/z 234 and 235 could affect the
abundance of products ions from fragmentation of m/z 236. Since
MLT only contains 13 carbons, this isotopic contribution should
be only due to m/z 235 and should not exceed approximately 13%
(thecontributionatM+2fromm/z234dueeithertoone18Oortwo
13C could be considered as negligible). Thus, the CID spectrum
of m/z 236 is mainly a representative of the fragmentation of
fully deuterated MLT and the isotopic contribution of m/z 235
should be considered as negligible regarding the abundances of
fragmentation products of m/z 236.
Analysis
Analyses were performed on an ion-trap mass spectrometer
(LCQ Advantage, Thermofinnigan, Les Ulis, France) equipped with
an electrospray ionisation (ESI) source. The capillary tempera-
ture was held at 250 ◦C and the relative sheath and auxiliary
gas flow-rates were set at 20 and 5, respectively (sheath gas,
0–100 units corresponding to 0–1.5 l min−1, auxiliary gas, 0–60
units corresponding to 0–18 l min−1, according to the man-
ufacturer’s specifications). 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 positive-ion mode, and each spectrum was
typically an average of 20 scans. MS/MS and MS3 experiments
were performed on mass-selected ions in the ion-trap mass
spectrometer using standard isolation and excitation proce-
dures.
Surprisingly, except for m/z 194.0, the fragmentation of the
unique ion at m/z 236 (Fig. 2(c)) leads to multiple fragments at
m/z 175.2, 176.2, 177.2 and m/z 217.1, 218.1 and 219.1. This
suggests that H/D scrambling could occur during the gas-phase
For direct infusion analysis, irradiated MLT samples were diluted
10 : 1 (v/v) with methanol prior to being infused continuously
into the ESI source with an SGE 250 µl syringe at a flow-rate
c
J. Mass. Spectrom. 2009, 44, 318–329
Copyright ꢀ 2008 John Wiley & Sons, Ltd.