Electrochemical oxidation of zopiclone

Article abstract of DOI:10.1007/s00706-015-1602-9

Electrochemical behaviour of zopiclone was investigated on glassy carbon electrode in static and rotation disc arrangement. Strong influence of kinetics and adsorption phenomena on the electrode processes was proved by voltammetric techniques. Controlled potential electrolysis in off-line and on-line combination with tandem mass spectrometry was employed for investigation of the products of electrochemical oxidation. N-Desmethyl zopiclone was identified and three other oxidation products formed by an introduction of one or two oxygen atom(s) to the molecule of zopiclone (including zopiclone N-oxide) were characterized. Based on mass spectrometric investigation of those products, piperazine moiety was proved as a target of electrochemical oxidation of zopiclone. Since N-desmethyl zopiclone and zopiclone N-oxide have been reported as products of enzymatic metabolism of the drug, the combination of electrochemistry with mass spectrometry may be considered as a reliable tool for simulation of some metabolic transformations. Graphical abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00706-015-1602-9

Monatsh Chem
DOI 10.1007/s00706-015-1602-9
ORIGINAL PAPER
Electrochemical oxidation of zopiclone
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Jakub Taborsky Martin Svidrnoch Ondrej Kurka Lucie Borovcova Petr Bednar Petr Bartak
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Jana Skopalova
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Received: 9 September 2015 / Accepted: 9 November 2015
Ó Springer-Verlag Wien 2015
Abstract Electrochemical behaviour of zopiclone was
investigated on glassy carbon electrode in static and rota-
tion disc arrangement. Strong influence of kinetics and
adsorption phenomena on the electrode processes was
proved by voltammetric techniques. Controlled potential
electrolysis in off-line and on-line combination with tan-
dem mass spectrometry was employed for investigation of
the products of electrochemical oxidation. N-Desmethyl
zopiclone was identified and three other oxidation products
formed by an introduction of one or two oxygen atom(s) to
the molecule of zopiclone (including zopiclone N-oxide)
were characterized. Based on mass spectrometric investi-
gation of those products, piperazine moiety was proved as a
target of electrochemical oxidation of zopiclone. Since N-
desmethyl zopiclone and zopiclone N-oxide have been
reported as products of enzymatic metabolism of the drug,
the combination of electrochemistry with mass spectrom-
etry may be considered as a reliable tool for simulation of
some metabolic transformations.
Graphical abstract
Keywords Electrochemistry Á Mass spectroscopy Á
Voltammetry Á N-Desmethyl zopiclone
Introduction
Zopiclone (ZOP), chemically (RS)-6-(5-chloropyridin-
2-yl)-7-oxo-6,7-dihydro-5H-pyrrolo[3,4-b]pyrazine-5-yl
4-methylpiperazine-1-carboxylate (Fig. 1), is a hyp-
notic drug used for the treatment of insomnia. ZOP
possesses a short duration of action with additional
muscle relaxant and anticonvulsant properties. It was
developed along with other so called Z-drugs, zale-
plon, and zolpidem, as an alternative to the world-wide
used benzodiazepines, which are controversial due to
concerns about adverse psychological and physical
effects, decreasing effectiveness, and physical depen-
dence at their long-term usage.
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-015-1602-9) contains supplementary
material, which is available to authorized users.
´
& Jana Skopalova
jana.skopalova@upol.cz
ZOP is a chiral drug administered as a racemic
mixture although the pharmacological activity is related
to the (?)-(S)-ZOP known as eszopiclone. Both enan-
tiomers are metabolised via cytochrome P450 enzyme
system [1]. The main biotransformation products of
1
Regional Centre of Advanced Technologies and Materials,
Department of Analytical Chemistry, Faculty of Science,
´
Palacky University, 17. listopadu 12, 771 46 Olomouc,
Czech Republic
123

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J. Taborsky et al.
O
mentioned above. In vivo or in vitro experiments, com-
monly used for elucidation of the drug metabolism, are
usually based on animal experiments which are tedious,
ethically questionable, time consuming, and of limited
reproducibility. Since oxidation reactions play a crucial
role in the CYP-mediated biotransformation, it seems
reasonable to use electrochemical oxidation as a possible
instrumental tool for simulation of the oxidative degrada-
tion processes [19]. Electrochemistry coupled on-line or
off-line to MS (especially to electrospray ionization mass
spectrometry, ESI–MS) has been successfully used in
many research areas including metabolic studies [20] as
well as in peptide, protein, and DNA analysis [21] or
quantification of biomolecules [22].
N
N
N
N
Cl
O
O
N
N
CH3
Fig. 1 Chemical structure of zopiclone
In this paper, electrochemical behaviour of zopiclone
was studied using voltammetric methods on glassy carbon
working electrode. Controlled potential electrolysis of ZOP
solutions containing acetonitrile and aqueous buffer solu-
tions of different pH followed by ESI–MS analysis of the
reaction products as well as on-line coupling of EC flow-
through cell with ESI–MS were used for generation and
characterization of zopiclone oxidation products.
oxidative metabolism are N-desmethyl zopiclone and
zopiclone N-oxide [1–4].
Chemical stability of ZOP was investigated in several
studies. The drug was found to be stable under acidic
conditions in aqueous-acetonitrile solutions [5]. However,
rate of hydrolysis increased with pH and temperature in
aqueous ethanolic media [6]. 2-Amino-5-chloropyridine
was identified as the final degradation product of ZOP in
alkaline solutions [7, 8].
Electrochemical behaviour of ZOP was investigated by
several authors but with no attempt to identify the products
of electrochemical reactions. Electrochemical reduction of
Results and discussion
Electrochemical behaviour of zopiclone
´
ZOP was studied by Vire et al. [9] by different techniques.
ZOP was reducible in two 2-electron steps in the pH range
0 to 12. The electrochemical behaviour was accompanied
by strong adsorption in neutral and acidic solutions.
Adsorption was employed for sensitive determination of
ZOP by adsorptive stripping voltammetry [9]. Yilmaz et al.
[10] have studied oxidation of ZOP at glassy carbon
electrode (GCE) using adsorptive stripping voltammetry.
ZOP oxidation was found to be an irreversible adsorption
controlled process. Square wave voltammetry was used for
determination of submicromolar concentrations of ZOP as
well [10]. Number of other analytical methods has been
used for ZOP determination starting from potentiometric
titrimetry, prescribed by British Pharmacopeia [11] through
the spectrophotometry [12] up to potentiometry with ion
selective electrode [13]. However, chromatographic meth-
ods including gas [14] and liquid chromatography [15–17],
often with mass selective detection, lead the dance in the
field of drug analysis.
Cyclic voltammogram (Fig. 2a) of zopiclone in acetoni-
trile-aqueous buffer solution of pH 4.8 (1:1, v/v) recorded
on glassy carbon electrode (GCE) shows one oxidation
peak at potential Ep = 1.02 V (vs. saturated calomel ref-
erence electrode, SCE). No current response was observed
in the reverse cathodic branch of voltammograms sug-
gesting irreversibility of the electrode process. Similarly,
differential pulse voltammogram (Fig. 2b) revealed one
anodic peak at the potential Ep = 1.04 V.
The effect of the pH of BR buffer solutions on the
oxidation signal of zopiclone was investigated using cyclic
and linear sweep voltammetry (LSV) with GCE in static
and rotating disc (RDE) arrangement, respectively. The
anodic signal of zopiclone occurred in the pH range
2.8–9.5 in both arrangements. The oxidation signal was not
observed in the more acidic buffer solutions (pH 1.9, 2.2,
and 2.5). The limiting current measured on RDE increased
with rising pH values up to pH about 6 and then remained
almost constant up to pH 9.5 (Fig. 3). Oxidation in more
alkaline solutions was not tested due to reported fast
decomposition of ZOP under such conditions [6]. In acidic
solutions with pH below 6, the oxidation of ZOP is a pH-
dependent process. The half-wave potential shifts to lower
values with increasing pH by -59 mV per pH unit (Fig. 3).
This value indicates that the same number of protons and
Electrochemistry coupled to mass spectrometry (EC/
MS) can be used for simulation of some oxidative reactions
involved in the metabolism of drugs and other xenobiotics
[18]. The elucidation of oxidative metabolic reactions is a
crucial point in the drug development. In general, the main
route of drug elimination is oxidative biotransformation via
CYP enzymes family and ZOP is not an exception as
123

Electrochemical oxidation of zopiclone
Fig. 2 Voltammograms of 5 9 10-4 mol dm-3 ZOP (solid line) in
supporting electrolyte (dashed line) containing BR buffer solution pH
4.8 and acetonitrile (1:1, v/v) recorded by cyclic voltammetry (a),
scan rate 50 mV s-1 and differential pulse voltammetry (b) scan rate
20 mV s-1, modulation amplitude 25 mV, pulse width 500 ms
shift in peak potential by 41 mV per log unit was observed
when scan rate exceeded 0.3 V s-1. The fact indicates the
influence of other processes, next to diffusion, in the rate
determining step at higher scan rates. Kinetics of the
electron transfer reaction, kinetics of the chemical reaction
preceding the electron transfer (deprotonation seems to be
the most likely in this case; see pH dependence of limiting
current on Fig. 3) and adsorption on the electrode surface
are the most probable reasons.
The adsorption of zopiclone on electrode surface was
investigated by experiment in which electrode was
immersed into the stock solution of zopiclone for 300 s,
and then the electrode surface was washed with deionized
water. The electrode was placed into electrolyte and CV
voltammogram was recorded (see Supplementary Material,
Fig. S2). A small current signal at 1 V corresponding to
ZOP adsorbed on the electrode can be distinctly recognized
in comparison to the blank measured with clean, mechan-
ically polished, electrode. The signal proved the tendency
of the drug to adsorb on the electrode surface. Also the sign
of the nonlinearity on the calibration dependence of the
peak current on the concentration of ZOP can be ascribed
in large extent to the adsorption on the GCE (Supple-
mentary Material, Fig. S3).
Fig. 3 Dependence of half-wave potential (filled square) and limiting
current (open circle) of zopiclone (c = 5 9 10-4 mol dm-3) on pH
measured on RDE, angular rotation speed: 157 rad s-1, scan rate
0.4 V s-1
electrons are involved in the electrode reaction. At pH
6–9.5, the half-wave potential shift was negligible. The
intersection point of two regression straight lines at pH 5.9
can be related to change in the protolytic forms of ZOP
(pKA = 6.79 [23]).
The effect of scan rate (v) on zopiclone oxidation was
investigated by LSV on GCE in BR buffer pH 4.8 with
acetonitrile (1:1, v/v). The anodic peak of ZOP increased
Dependence of limiting current on square root of rota-
tion velocity of RDE was measured by LSV in solution of
pH 4.8, pH 7.1, and pH 9.4. In neutral and alkaline solu-
tions the limiting current increases with increasing square
root of the rotation speed. The course of the dependence is
typical for the mechanism influenced by both diffusion of
the depolarizer and kinetics of the electron transfer (see
Supplementary Material, Fig. S4). On the contrary, limiting
current measured in acidic solution is almost independent
on square root of rotation velocity. The course is usually
observed in systems controlled by the kinetics of the
electron transfer or the kinetics of the chemical reaction
with increasing scan rates within the range 0.01–0.9 V s-1
.
The dependence of log Ip—log v revealed two linear seg-
ments with the slopes 0.71 (in the scan range
0.01–0.2 V s-1) and 0.89 (in the range of 0.3–0.9 V s-1
)
(see Supplementary Material, Fig. S1). The values of the
slopes are higher than the theoretical value 0.5 for purely
diffusion controlled process indicating the influence of
adsorption on the electrochemical reaction. The depen-
dence of the peak potential Ep on log v resulted in two
straight lines. Peak potential did not change within the slow
scan rates interval up to 0.2 V s-1. However, a significant
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J. Taborsky et al.
preceding the electron transfer (i.e., systems where diffu-
sion is not the rate determining step). The interpretation of
the main course of the dependence fits together with con-
clusion resulting from the dependence of the peak potential
on the scan rate during LSV mentioned above.
is also characteristic for ZOP fragmentation. Fragmentation
pattern corresponds with the already published results [24].
The differences in intensities of fragments found in this
work and those published in the above mentioned paper are
due to different setup of collision cells. It is worth noting
that ion with m/z = 263 has been reported as the product of
zopiclone hydrolysis [8].
MS analysis of oxidation products of zopiclone
Figure 5a shows MS spectrum of electrolysed ZOP
solution, containing the mixture of oxidation products and
the rest of ZOP, which was recorded in off-line setup. Four
main signals of proposed oxidation products can be
observed. First oxidation product (designated as P1, m/
z = 375) corresponds to N-desmethyl zopiclone (difference
14 Da from the m/z of ZOP). In MS/MS spectrum the loss of
CO (m/z = 347) and CO2 (m/z = 331), respectively, are
observed (Fig. 5b). Besides, intensive signals at m/z = 263
and m/z = 245 confirm that both pyridine and the pyrrolo-
pyrazine rings remain unchanged and thus, the oxidative
demethylation occurs on piperazine skeleton. The frag-
mentation pattern of the product P1 corresponds to those
reported for N-demethylated metabolite of zopiclone [24].
Second product (P2, m/z = 405) corresponds with the
addition of one oxygen atom to the ZOP molecule. Pres-
ence of fragments at m/z = 263 and m/z = 245 suggests
that the oxidation takes place on the piperazine ring as well
(Fig. 5c). Formation of N-oxide related to oxidative
metabolism of ZOP has been already described in literature
[1]. Electrochemical formation of N-oxide(s) has also been
reported for another drug possessing tertiary amine group
[25]. Oxidation of one of nitrogen atoms in the piperazine
ring is thus a reasonable possibility explaining this type of
ZOP oxidation. However, oxidation of N-methyl group or
the carbons of the piperazine skeleton accompanied by
piperazine ring opening cannot be excluded in this case.
Third product (P3, Fig. 5d, m/z = 403) corresponds
with a gain of one oxygen and loss of two hydrogen atoms.
The formation of fragments at m/z = 263 and m/z = 245
suggests that pyridine and pyrrolo-pyrazine rings remain
For more detailed characterization of the oxidation prod-
ucts two experimental arrangements were used. First
arrangement consisted of an off-line controlled potential
electrolysis of zopiclone on the large surface Pt electrode
combined with a tandem mass spectrometric measurement
of the electrolysed zopiclone solutions. Second one
involved on-line coupling of electrochemical flow-through
cell containing the working porous graphite electrode with
mass spectrometer (EC/MS). In both types of experiment,
electrolysis was performed in acetonitrile/ammonium
acetate buffer solution pH 3.5, 6.8, and 9.5 (1:1, v/v).
For off-line experiment the controlled potential values
suitable for exhaustive electrolysis were chosen according to
courses of cyclic voltammograms of zopiclone recorded in
different media. To eliminate the influence of nonelectrolytic
reaction of zopiclone, the mass spectra of electrolysed solu-
tions were compared to those of control samples. The control
samples were obtained by electrolysis of zopiclone solutions
at potentials where no electrochemical reaction occurs.
Figure 4a shows mass spectrum of ZOP standard in
which the pseudomolecular ion [M ? H]? at m/z = 389 is
evident. MS/MS spectrum (Fig. 4b) revealed the main
fragment ion at m/z = 345 corresponding to a loss of CO2.
This unusual scission of CO2 from the molecule, giving
evidence of rather strong association of the piperazine ring
with another heterocyclic part of the ZOP structure, has
already been reported [24]. Formation of fragments at m/
z = 245 and m/z = 263 is described in the inset of Fig. 5b.
The minor fragment at m/z = 217 corresponding to loss of
CO from cyclopyrrolon moiety of the ion with m/z = 245
Fig. 4 MS (a) and MS/MS spectrum (b) of zopiclone (5 9 10-4 mol dm-3) in water/CH3CN (1:1, v/v)
123

Electrochemical oxidation of zopiclone
Fig. 5 MS spectrum of electrolysed ZOP (a), containing a mixture of
oxidation products and the rest of ZOP, and MS/MS spectra of main
oxidation products: P1 at m/z = 375 (b), P2 at m/z = 405 (c), P3 at
m/z = 403 (d), and P4 at m/z = 419 (e) acquired in solution of
zopiclone (c = 5 9 10-4 mol dm-3) electrolysed in acetonitrile/
ammonium acetate buffer solution pH 6.8 (1:1, v/v) 30 min on Pt
gauze electrode at E = 1.2 V (vs. SCE)
intact as in the case of the previously discussed oxidation
products P1 and P2. The fragment at m/z = 359 corre-
sponds with loss of CO2 typical for ZOP derivatives. Minor
fragment at m/z = 330 corresponds probably with a
cleavage of formyl radical indicating the presence of
aldehyde group preferentially linked to one of the piper-
azine nitrogen atoms.
Fourth product (P4, m/z = 419) can be explained by the
addition of two oxygen and loss of two hydrogen atoms.
MS/MS spectrum of P4 (Fig. 5e) shows intensive signal
corresponding with the loss of CO2 typical for ZOP
derivatives (dominant fragment at m/z = 375). As men-
tioned above, fragments at m/z = 263 and m/z = 245,
respectively, suggest oxidative process occurring at the
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J. Taborsky et al.
piperazine ring. The introduction of two oxygen atoms into
the piperazine skeleton can proceed by several ways
leading to different products. One of the theoretical pos-
sibilities involves a formation of an N-carboxylic group.
Since the loss of CO2 from the fragment at m/z = 375 is
missing in the MS/MS spectrum, this possibility is not
likely. A formation of N-oxide and aldehyde group either
on terminal methyl group or on piperazine carbon skeleton
(accompanied by a ring opening) can be considered as
another possibility. A fragment at m/z = 345 corresponds
with a loss of formaldehyde from fragment at m/z = 375
suggesting the presence of aldehyde group preferentially
linked to one of the nitrogen atoms from piperazine part.
Fragment at m/z = 329 can be explained by consequent
loss of oxygen atom from the fragment at m/z = 345.
Similarly to previously discussed oxidation product P2, N-
oxide formed on one of the piperazine nitrogen atoms is the
most probable option. Thus, the oxidation product P4
presumably contains one aldehyde group and one oxygen
atom linked to piperazine nitrogen atoms.
When compared the signals of particular oxidation prod-
ucts in acidic conditions, the signal of P1 reaches the
highest value. This oxidation product exhibits the highest
decrease of relative intensity with increasing pH as well.
This fact points out different chemical property of P1
compared to the rest of detected oxidation products (e.g.,
pK value and proton-affinity in gas phase).
On-line connection of electrochemical cell directly to
mass spectrometry interface was performed and compared
with the results from the above discussed off-line experi-
ments. Zopiclone solution in 1:1 (v/v) mixture of
acetonitrile and ammonium acetate buffer of pH 3.5, pH
6.8, and pH 9.5 was continuously pumped through flow cell
containing porous graphite electrode. Constant potential
within the range from 0 to 0.8 V (vs. Pd/H2 reference
electrode) in 100 mV (0–0.4 V) and 50 mV (0.4–0.8 V)
increments, respectively, was applied on the electrode and
appropriate mass spectra were collected. The electrode
potential of 0 V vs. Pd/H2 reference electrode corre-
sponded to 0.234 V vs. SCE at pH 7.0. Oxidation products
were detected within 4 min after their formation in the cell
at the flow rate 4 mm3 min-1. Resulting ‘‘mass voltam-
mograms’’ are shown in Fig. 6. For better lucidity, the
mass voltammograms are displayed in the m/z range from
In off-line experiments the highest relative intensities
(intensity of particular oxidation product divided by the
sum of intensities of all signals in given spectrum) are
observed in acidic conditions for all oxidation products.
Fig. 6 Mass voltammograms of zopiclone (c = 5 9 10-4 mol dm-3), 1:1 (v/v) acetonitrile/ammonium acetate buffer solution of pH 3.5 (a), pH
6.8 (b), and pH 9.5 (c) acquired in on-line EC/MS
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Electrochemical oxidation of zopiclone
300 to 450, in which the changes in the intensities of the
signals of ZOP and its oxidation products were observed.
Fragment ions with m/z = 245 and m/z = 263, common to
ZOP and its oxidation products, were observed on the
voltammograms at all pH and potentials (data not shown).
Oxidation of zopiclone evaluated as a decrease of ZOP
signal in acetate buffer solution of pH 3.5 and 6.8 starts at
potential 0.5 V vs. Pd/H2 reference electrode, in acetate
buffer solution of pH 9.5 the oxidation starts at lower
potential. Unlike acidic and neutral media, in alkaline
solution of pH 9.5 on-line experiments reveal one more
oxidation product at m/z = 420 (designated as P5) beside
the above mentioned products P1–P4 (Fig. 6c). Even m/
z value suggests the presence of odd number of nitrogen
atoms. In the case of the discussed product P5 of ZOP
oxidation it corresponds to incorporation of one nitrogen
atom into the structure. Collision spectrum (MS2, Supple-
mentary Material, Fig. S5) of P5 provided three dominant
fragments at m/z = 402, 390, and 358. The fragment at m/
z = 402 corresponds to a loss of water suggesting the
presence of one extra –OH group. Subsequent loss of CO2,
typical for ZOP derivatives, leads to the most abundant
fragment at m/z = 358. The fragment at m/z = 390 can be
ascribed to a loss of CH2O, NO or CH2-NH2. Besides, a
minor fragment at m/z = 403 was also observed during
fragmentation of P5. Tentatively, this fragment can be
explained as a loss of ammonia from parent ion. The
structure of the P5 product was not revealed from the
acquired MS data. Note that P5 was observed in on-line
EC/MS experiments conducted in alkaline ammonium
buffer. We suggest that P5 arises from a side reaction of
ZOP with ammonia under oxidative conditions. When
sodium ions were used as buffer constituent instead of
ammonium ones, P5 product was not observed. This
observation supports the proposed introduction of one
nitrogen atom from ammonia to ZOP structure.
formation of all remaining products (P2-P4, as well as a
specific product P5 formed solely in alkaline solution in the
presence of ammonia). Some oxidation products identified
in electrochemical oxidation are similar to oxidation
products from in vitro metabolic experiments (i.e., N-des-
methyl zopiclone and zopiclone N-oxide) [1].
Conclusion
Voltammetric measurements reveal that electrochemical
oxidation of zopiclone is quite complicated process influ-
enced by kinetics and adsorption phenomena. Controlled
potential electrolysis combined with mass spectrometry
identification of the reaction products is a powerful tool for
elucidation of electrochemical oxidation of zopiclone. Four
products were found in electrolysed samples by means of
off- and on-line tandem mass spectrometry. N-Desmethyl
zopiclone was identified and three other oxidation products
formed by an introduction of oxygen atom(s) to the
molecule of zopiclone (including zopiclone N-oxide) were
characterized. N-Desmethyl zopiclone and zopiclone N-
oxide were formerly confirmed as in vitro metabolic
products of zopiclone. The fact gives great credibility to
the combination of electrochemistry with mass spectrom-
etry for metabolic studies.
Experimental
Reagents
Zopiclone (ZOP) was obtained from Farmak (98 %, Olo-
mouc, Czech Republic), zopiclone N-oxide (European
Pharmacopoeia Reference Standard) was purchased from
Fluka. A stock solution of 1 9 10-3 mol dm-3 ZOP was
prepared in acetonitrile (HPLC grade, Sigma-Aldrich
Czech Republic) and kept in a refrigerator. Britton-
Robinson (BR) buffer solutions were prepared from phos-
phoric acid, acetic acid, and boric acid (0.04 mol dm-3
each, analytical grade, Lachema, Czech Republic). Desired
pH values were adjusted with sodium hydroxide
In on-line experiments the relatively sharp decrease of
ZOP intensity and increase of oxidation products is
observed (Supplementary Material, Fig. S6–S8). The pro-
cesses occurring in acidic, neutral, and alkaline conditions
reveal similar main features. The increase of P1 response
starts at lower potentials compared to other oxidation
products (P2–P4), intensity of signal reaches its maximum
faster and at higher potentials ([0.6 V) decreases fast. The
formation of the other products occurs at higher potentials
and changes in intensities are less dependent on potentials
compared to P1. Therefore, it can be concluded (in
agreement with off-line experiments, see above) that
chemical nature of P1 and process of its formation differ
from those of the other oxidation products. The fact is
obvious since P1 is formed by demethylation of ZOP (no
extraneous substituent incomes to its molecule) while
introduction of one or two oxygen atom(s) leads to
(0.2 mol dm-3
,
analytical grade, Lach-Ner, Czech
Republic). Ammonium acetate (p.a., [98.0 %, Lach-Ner,
Czech Republic) was used as supporting electrolyte for MS
experiments. Desired pH values were adjusted with acetic
acid or ammonia (p.a., 25 % in water, Lach-Ner, Czech
Republic).
Voltammetric experiments
An AutoLab PGSTAT128N electrochemical analyser
(Metrohm Autolab, Utrecht, The Netherlands) with
123

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J. Taborsky et al.
software NOVA 1.10 was used for voltammetric experi-
ments with three-electrode system consisting of a glassy
carbon working electrode (GCE, disc diameter 3.0 mm,
Bioanalytical Systems, USA) or rotating GCE (disk
diameter 2.0 mm, Metrohm, The Netherlands), platinum
wire auxiliary electrode, and saturated calomel reference
electrode. GCE was polished using aqueous suspension of
alumina powder (0.05 lm particles, Sigma-Aldrich) on a
wet microcloth (Buehler, USA) and sonicated in distilled
water for 30 s prior to each measurement. Cyclic voltam-
metry (CV) and linear sweep voltammetry (LSV) were
performed at different scan rates over the range 0.02–0.8 V
s-1 and at different pH values of the supporting buffer
solution. Differential pulse voltammograms were recorded
at pulse amplitude of 25 mV, pulse width 500 ms and scan
rate 0.02 V s-1. Hydrodynamic measurements were car-
ried out with angular velocity ranged from 52 to
314 rad s-1 and scan rates 0.02 and 0.4 V s-1. All exper-
iments were performed in supporting electrolytes
containing BR buffer solutions of desired pH and ace-
tonitrile (1:1, v/v). Hydrodynamic voltammograms were
processed using el-Chem Viewer software [26].
(ESA, Chelmsford, MA, USA) connected to the poten-
´
ˇ´
tiostat Detector ADLC 1 (Laboratornı prıstroje Praha,
Czech Republic). Working ZOP solution (5 9 10-4
mol dm-3) in 1:1 (v/v) acetonitrile/ammonium acetate
buffer solution (pH 3.5, pH 6.8, and pH 9.5) was used for
on-line oxidation in the potential range from 0 to 0.8 V (vs.
Pd/H2).
Acknowledgments The authors gratefully acknowledge the finan-
cial support by the Ministry of Education, Youth and Sports of the
Czech Republic (project LO1305).
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Potentiostat 100 mA (L-Chem, Horka nad Moravou, Czech
Republic) with three-electrode system consisted of plat-
inum gauze working electrode, platinum auxiliary
electrode placed in a separate cathode compartment and
reference SCE electrode separated from the bulk solution
with a porous ceramic frit. The electrolysis was performed
in mixture of acetonitrile/ammonium acetate buffer solu-
tion (1:1, v/v) of different pH values and at different
potentials: pH 3.5 (0.2 V, 1.4 V), pH 6.8 (0.2 V, 1.2 V),
pH 9.5 (0.2 V, 1.2 V). All samples were electrolysed in
stirred solutions containing 5 9 10-4 mol dm-3 ZOP for
30 min.
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¨
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Mass spectrometer Agilent 1100 Series LC/MSD Trap
(Agilent Technologies, Palo Alto, CA, USA) with elec-
trospray ionization (ESI) was used for analysis of
electrolysed solutions of ZOP. Parameters of ESI source
working in the positive mode were as follows: capillary
voltage 2.4 kV, source temperature 150 °C, pressure of
desolvation gas 10 psi, desolvation gas flow rate 180 dm3
h-1. Nitrogen was used as desolvation gas and helium as
collision gas.
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On-line electrochemistry/mass spectrometry measure-
ments were performed using ESA Conditioning cell 5021A
123