J. Chandran et al. / Ultrasonics Sonochemistry 27 (2015) 178–186
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aqueous solutions [19–21]. Fuciarelli et al. reported several base
lesions in DNA solution by the cavitational activity of ultrasound
at a frequency of 2.17 MHz [18]. Studies have also been done on
the sonochemical activity of thymine over a range of US intensities
which also help to understand the kinetics of the reaction [22].
Even though there are many studies on the sonochemically modi-
fied nitrogen bases, sonochemical transformation studies on nucle-
osides would give a better understanding of the mechanistic
aspects of effect of US on cellular DNA. In the present study, we
investigated the effect of US on thymidine (dT), a relevant nucle-
oside used as a target of various ROS generating systems [1,10].
The variation of the sonochemical activity of dT with US frequency
at three different power densities is explained along with the influ-
ence of argon on sonolysis. Moreover, the study is a reinvestigation
of the free radical induced transformation products of dT using
high resolution mass spectrometry. Argon atmosphere simulates
the hypoxic conditions in cells and hence details of the products
in its presence would be highly beneficial. The accurate mass mea-
surement using high resolution mass spectrometer as well as the
MS/MS facility helps to get an insight into the structure [23].
under normal conditions and for 30 min. in the case of argon satu-
rated conditions at a power density of 42 W/mL. The modified nucle-
osides were eluted isocratically with 0.1% formic acid in water and
methanol (95:5) and detected in negative mode of ionization. For
mass spectrometric analyses dT samples were sonolysed for
60 min. at frequency 620 kHz and power density 42 W/mL.
3. Results and discussion
3.1. Effect of frequency and power on the transformation
The dT solutions (10ꢀ4 mol/L) were subjected to four US fre-
quencies (200, 350, 620 and 1000 kHz) at three different power
densities (10.5, 24.5 and 42 W/mL) under aerated conditions. The
degradation profile is given in Fig. 1. At a power density of
10.4 W/mL the degradation of dT was higher at a frequency of
350 kHz, but as power increases to 24.5 W/mL the degradation at
350 and 620 kHz became comparable. At 42 W/mL, the degrada-
tion was found to be almost equal for 350, 620 and 1000 kHz. As
power increases, the rate of degradation increases at all frequen-
cies. The reaction was found to follow pseudo first order kinetics
as inferred from Fig. 1. The experiment was repeated in the pres-
ence of argon atmosphere at 620 kHz and power density of
42 W/mL. The solution was saturated with argon and an argon
atmosphere was maintained throughout the sonolysis. A compara-
tively higher rate of decomposition of dT was observed in argon
atmosphere as shown in Fig. 1(c).
The results clearly indicate that the rate of the sonochemical
reaction depends on the power and frequency of ultrasound. The
increased sonochemical activity observed at frequencies 350 and
620 kHz is expected as it was reported that the overall free radical
production was greater in this range of frequency [24]. When
power density increases, there is an enhancement in the energy
of cavitation facilitating the production of higher number of free
radicals [25] which explains the general increase in the rate of
degradation with power.
2. Materials and methods
2.1. Sonolysis of dT
dT was purchased from Pharma Waldhof and used as such. All
solutions were prepared in water purified by a Cascada™ Lab
Water Systems and of resistivity 18.2 M
X cm. Solutions of dT
(10ꢀ4 mol/L, 250 mL) was subjected to sonolysis at four different fre-
quencies (200, 350, 620 and 1000 kHz) at three different power den-
sities, 10.5, 24.5 and 42 W/mL for 2 h. Two frequencies (620 kHz and
1 MHz), among the four studied, fall under the range used for the
surgical non disruptive applications. The sonolysis was carried out
in a glass reactor with an L3 ELAC Nautik ultrasound generator pow-
ered by an Allied Signal R/F generator (T & C power conversion,
Model AG 1006). For the experiments under argon saturated atmo-
sphere, the solution was bubbled with argon gas for half an hour at a
pressure of 5 psi prior to sonolysis and Ar was bubbled throughout
the experiment. Sonolysis was carried out only at a power density
of 42 W/mL under argon atmosphere, where there was maximum
degradation in the case of aerated conditions. Two transducers were
used with resonances 212 and 620 kHz and 350 and 1000 kHz
respectively. The temperature was maintained at 25 1 °C.
The chemical transformations of molecules on sonolysis are
Å
caused by pyrolysis and reaction with the OH and HÅ produced
by water pyrolysis [14,26,27]. Since dT is non volatile, the contri-
bution from the pyrolytic reactions can be considered negligible
[28] and the major transformation pathway could be the reaction
with free radicals. The intermediate radicals formed from dT on
Å
sonolysis by the addition of OH and HÅ were detected earlier by
spin trapping studies [28] which further support the free radical
mediated mechanism. From the pulse radiolysis studies, it is
reported that ÅOH adds to the base with a reaction rate of the order
of 3–10 ꢁ 109 L/mol s and abstracts hydrogen from the methyl
group of thymine as well as the sugar ring with a rate constant
of 2 ꢁ 109 L/mol s, while the rate constant of HÅ reaction is of the
order ten times lesser than the former (1–5 ꢁ 108 L/mol s) [29].
The pseudo first order nature of the reaction observed in the pre-
sent case proposes that the major reactive species is the highly
2.2. HPLC analysis
The percentage degradation of dT on sonolysis was monitored
by HPLC (Shimadzu prominence UFLC, LC 20 AD) connected with
a diode array detector (SPD-M20 A). An Enable C18 (25 cm ꢁ
4.5 mm ꢁ 5
lm) column was used at a flow rate of 1 mL/min.
The sonolysed mixture was eluted isocratically with water and
methanol (95:5) as mobile phases.
Å
reactive OH radical.
A slightly higher rate of decomposition of dT is observed under
argon atmosphere. Compared to air, argon possesses higher poly-
tropic index and lower thermal conductivity. Also, it has higher
solubility which facilitates more cavitation events to take place
compared to the aerated conditions [30]. This results in an efficient
production of free radicals contributing to the increased rate of
decomposition of dT in the presence of argon.
2.3. Analysis of end products by LC–Q-ToF–MS
The end products were analyzed by Waters Xevo G2 QToF with an
electrospray ionization (ESI) source. It was coupled to Acquity H
class UPLC with BEH C18 (50 mm ꢁ 2.1 mm ꢁ 1.7
lm) column and
Acquity TUV detector. The instrument was tuned with the parent
compound, dT. The ionization conditions are as follows: capillary
voltage: 2.5 V, sampling cone voltage: 30 V and extraction cone volt-
age: 4 V. The source and desolvation temperatures were kept at
135 °C and 350 °C respectively. Nitrogen was used as the desolva-
tion and nebulizing gas at a flow rate of 50 and 900 L/h, respectively.
For the mass spectrometric analyses, the dT was sonolysed for 1 h
3.2. Mass spectrometric analysis of the transformed products
Sonolysis of dT yielded a mixture of products which were ana-
lyzed using LC–Q-ToF–MS. The total ion chromatogram obtained