A Fast and Easy Way for Determining
the Level of Oxidative Stress using
UHPLC with Boron-Doped Diamond
Electrochemical Detection
Marc Plante, Bruce Bailey, David Thomas, Qi Zhang, and Ian Acworth
Thermo Fisher Scientific, Chelmsford, MA, USA
Overview
Purpose: In order to obtain satisfactory information concerning aminothiols,
disulfides, and thioethers from biological samples, scientists require a sensitive
approach that can measure key compounds,
compounds simultaneously.
simultaneously A simple
simple, accurate
and rapid UHPLC method was developed for the analysis of these compounds
using isocratic liquid chromatography and an amperometric electrochemical cell
with a boron-doped diamond (BDD) working electrode. This allowed the accurate
quantification of analytes to low picogram (pg) sensitivity.
Methods: Direct analysis of aminothiols, disulfides and thioethers using UHPLC
chromatographic techniques with a robust electrochemical cell using a BDD
electrode is described.
Results: The method provides a fast and easy way for determining the level of
oxidative stress by measuring various aminothiols, disulfides, and thioethers at low
levels from whole blood without significant matrix interferences.
Introduction
A number of biochemically important sulfur
sulfur-containing
containing compounds occur in vivo
including: aminothiols (e.g., cysteine, glutathione [GSH], homocysteine), disulfides
(e.g., cystine, glutathione disulfide [GSSG], homocystine), and thioethers (e.g.,
methionine) as shown in Figure 1. These aminothiols plays numerous physiological
roles. GSH is a major cellular antioxidant and a cofactor for glutathione peroxidase,
an enzyme that
th t detoxifies
d t ifi hydrogen
h d
peroxide
id and
d lilipid
id h
hydroperoxides.
d
id
Th
The hi
high
h
ratio of GSH/GSSG keeps the cell in a reducing environment, essential for its
survival. Decreases in this ratio are associated with cellular toxicity and numerous
diseases including neurodegeneration (e.g., Parkinson’s disease). Cysteine is also
a cellular antioxidant, serves as a precursor to glutathione and is often found in
protein structures as a disulfide link. Methionine is an essential amino acid and
serves as a methyl donor when incorporated into S-adenosylmethionine. Although a
variety of HPLC techniques have been developed for the measurement of thiols,
disulfides, and thioethers most of these exhibit technical issues. UV requires
derivatization which can adverselyy affect the GSH/GSSG ratio,, while some
electrochemical approaches using glassy carbon electrodes suffer from electrode
fouling and loss of response, and require routine maintenance. Although porous
graphite working electrodes are more forgiving, they still need maintenance. Borondoped diamond (BDD) however, enable the direct measurement of these analytes
without electrode issues.1,2 The rapid method presented herein is capable of
accurately determining several aminothiols simultaneously using UHPLC
chromatographic techniques with a BDD working electrode. Examples showing the
analysis of GSH and GSSG from plasma samples using a simple “dilute and shoot”
protocol are provided.
A UHPLC approach was chosen as it provides several advantages over standard
HPLC conditions including: shorter cycle times between samples; improved
resolution, and sharper peaks. A sharper peak is important when measuring GSSG
as it occurs at a low concentration and is one of the last peaks eluting in biological
samples The use of longer UHPLC columns provides better resolution between
samples.
compounds, which is important when analyzing biological samples.
FIGURE 1. Molecular structures of A) glutathione (GSH), B) glutathione disulfide
(GSSG), C) methionine, and D) homocysteine
A) GSH (reduced form)
C) Methionine
B) GSSG (oxidized form)
D) Homocysteine
2 A Fast and Easy Way for Determining the Level of Oxidative Stress using UHPLC with Boron-Doped Diamond Electrochemical Detection
Methods
Analytical Conditions for Am
Column:
Pump Flow Rate:
Mobile Phase:
Column Temperature:
Post Column Temperature
Injection Volume:
Cell Potential:
Filter Constant:
Cell Clean:
Cell Clean Potential:
Cell Clean Duration:
Sample Prep:
FIGURE 2A. Thermo Scientif
FIGURE 2B. 6041RS ultra Am
FIGURE 2C. Thermo Scientif
A
The Thermo Scientific™ Di
- SR-3000 Solvent Rack
- DGP-3600RS Dual Gradi
- WPS-3000TBPL Thermos
- ECD-3000RS Electrochem
with integrated temperatu
- Chromeleon 6.80 SR12 C
Results and Di
An instrumental prerequisit
inert (free from leachable tr
using an electrochemical d
bi
biocompatible
ibl materials
i l iin
contribute to elevated back
introduction of the ECD-300
attached in series after the
ay for determining the level of
, disulfides, and thioethers at low
nterferences.
ning compounds occur in vivo
GSH], homocysteine), disulfides
cystine), and thioethers (e.g.,
ols plays numerous physiological
ofactor for glutathione peroxidase,
lilipid
id h
hydroperoxides.
d
id
Th
The hi
high
h
nvironment, essential for its
h cellular toxicity and numerous
nson’s disease). Cysteine is also
tathione and is often found in
an essential amino acid and
S-adenosylmethionine. Although a
for the measurement of thiols,
hnical issues. UV requires
/GSSG ratio,, while some
electrodes suffer from electrode
maintenance. Although porous
ey still need maintenance. Boronmeasurement of these analytes
sented herein is capable of
aneously using UHPLC
electrode. Examples showing the
using a simple “dilute and shoot”
veral advantages over standard
tween samples; improved
mportant when measuring GSSG
he last peaks eluting in biological
des better resolution between
ological samples.
(GSH), B) glutathione disulfide
G (oxidized form)
mocysteine
Analytical Conditions for Aminothiol, Disulfide and Thioether Analysis
Column:
Pump Flow Rate:
Mobile Phase:
Column Temperature:
Post Column Temperature
Injection Volume:
Cell Potential:
Filter Constant:
Cell Clean:
Cell Clean Potential:
Cell Clean Duration:
Sample Prep:
Thermo Scientific
Scientific™ Accucore
Accucore™ RP
RP-MS
MS column
2.6 μm, 2.1 × 150 mm
0.500 mL/min
0.1% pentafluoropropionic acid, 0.02% ammonium
y
, 2.5% acetonitrile,, 97.4% water
hydroxide,
50.0 °C
25.0 °C
2 µL standards; 4 µL samples
Thermo Scientific™ Dionex™ model 6041RS ultra
Amperometric Analytical Cell with BDD electrode at
+1600 mV
1.0 s
On
1900 mV
10.0 s
5–20 µL whole blood + 200 µL 0.4 N PCA, mix and
spin for 10 minutes at 13,000 RPM. The clear
s pernatant was
supernatant
as transferred into an a
autosampler
tosampler vial
ial
and placed on the autosampler at 10 °C.
FIGURE 3. Overlay of analytica
3.0
µA
2.8
2.6
24
2.4
2.2
2.0
GSH
H
s and thioethers using UHPLC
chemical cell using a BDD
Use of the 6041RS amperometri
electrochemical capabilities of th
oxidation of organic compounds
working electrode materials. This
voltammetric resolution of compo
fittings were employed to cope w
particles. These fingertight, virtua
operate at pressures up to 14,50
g which can slip
p when using
g
tubing
tubing and are available in small
band spreading. Capillaries used
connections made prior to the au
after the injector valve.
CysGly
y
on concerning aminothiols,
scientists require a sensitive
ltaneously
ltaneously. A simple
simple, accurate
analysis of these compounds
erometric electrochemical cell
rode. This allowed the accurate
nsitivity.
Methods
1.8
1.6
1.4
1.2
1.0
FIGURE 2A. Thermo Scientific™ Dionex™ UltiMate™ 3000 Electrochemical detector
p
Analytical
y
Cell with BDD electrode
FIGURE 2B. 6041RS ultra Amperometric
FIGURE 2C. Thermo Scientific™ Dionex™ nanoViper™ fingertight capillaries
0.8
0.6
0.4
0.2
-0.0
A
B
0.0
C
The Thermo Scientific™ Dionex™ UltiMate™ 3000 UHPLC system consists of:
- SR-3000 Solvent Rack
- DGP-3600RS Dual Gradient Pump
y
Autosampler
p
- WPS-3000TBPL Thermostatted Analytical
- ECD-3000RS Electrochemical Detector
with integrated temperature controlled column compartment
- Chromeleon 6.80 SR12 Chromatography Data System software
Results and Discussion
An instrumental prerequisite for trace analysis is that the HPLC system must be
inert (free from leachable transition metals) in order to achieve optimal sensitivity
using an electrochemical detector. The system shown above in Figure 2A uses
bi
biocompatible
ibl materials
i l iin the
h flflow path
h to reduce
d
the
h iinfluence
fl
off metall that
h can
contribute to elevated background currents at the electrochemical cell. The recent
introduction of the ECD-3000RS detector enables multiple electrodes to be
attached in series after the HPLC column.
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2
The direct electrochemical detec
doped diamond electrochemical
potential used for this study (+16
disulfide analytes. Advantages o
and method simplicity since no s
analysis the electrode surface wa
+1900 mV. After a 1.5 minute reagain stable and could be used f
method the whole blood sample
method,
and then centrifuged. The clear s
autosampler vial and placed on t
rapid sample processing thus mi
thiols. Rapid UHPLC analysis as
samples within three minutes be
place.
For biological studies, the determ
compounds such as GSH, GSSG
ill
illustrates
the
h overlay
l off calibrati
lib i
1–20 µg/mL. Peak resolution and
The column used for this method
material which provides fast, hig
pressures. When operated at 0.5
than 300 bar with the last compo
in Figure 3.
The calibration curves for amino
y of response
p
to different c
linearity
coefficients ranging from R2 = 0.
(Table 1) over the range of 1–20
(%RSD) for the calibration curve
in Table 1. The RSD values rang
electrode provided good stability
Thermo Scientific Poster Note • EAS2013_PN70904_E 11/13S 3
de and Thioether Analysis
tific
tific™ Accucore
Accucore™ RP
RP-MS
MS column
150 mm
oropropionic acid, 0.02% ammonium
% acetonitrile,, 97.4% water
µA
2.8
2.0
1.2
1
0.8
0.6
0.4
0.2
0
1.8
1.6
0
5
CysGly
G
Table 1. Regression data
GSSG
2.2
HCYS
24
2.4
GSH
H
CysGly
y
2.6
Peak
#
Point
CysGly
15
1.4
1.2
20 ug/mL
1.0
tiMate™ 3000 Electrochemical detector
lytical
y
Cell with BDD electrode
anoViper™ fingertight capillaries
1.4
FIGURE 3. Overlay of analytical standards ranging from 1–20 µg/mL
3.0
e blood + 200 µL 0.4 N PCA, mix and
nutes at 13,000 RPM. The clear
as transferred into an a
autosampler
tosampler vial
ial
the autosampler at 10 °C.
1.6
Me
ethionine
s; 4 µL samples
tific™ Dionex™ model 6041RS ultra
Analytical Cell with BDD electrode at
FIGURE 4. Calibration cu
Response (µA)
Use of the 6041RS amperometric cell (Figure 2B) provides the unique
electrochemical capabilities of the boron-doped diamond which enables the
oxidation of organic compounds using higher electrode potentials than other
working electrode materials. This platform provides both chromatographic and
voltammetric resolution of compounds. The nanoViper (Figure 2C) fingertight
fittings were employed to cope with the higher pressures due to smaller column
particles. These fingertight, virtually zero-dead-volume (ZDV) capillaries can
operate at pressures up to 14,500 psi and are much safer to use than PEEK™
g which can slip
p when using
g elevated p
pressures. They
y are made of PeekSil
tubing
tubing and are available in small internal dimensions to minimize chromatographic
band spreading. Capillaries used on this system were 150 micron ID for all
connections made prior to the autosampler valve and 100 micron ID for those made
after the injector valve.
0.8
10 ug/mL
0.6
5 ug/mL
0.4
0.2
-0.0
GSH
15
2 ug/mL
Meth
15
HCYS
15
1 ug/mL
GSSG
15
min
0.0
C
™ 3000 UHPLC system consists of:
Autosampler
p
umn compartment
Data System software
n
sis is that the HPLC system must be
in order to achieve optimal sensitivity
em shown above in Figure 2A uses
educe
d
the
h iinfluence
fl
off metall that
h can
at the electrochemical cell. The recent
nables multiple electrodes to be
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
2.2
2.4
2.6
2.8
3.0
3.2
3.4
3.6
3.8
4.0
4.2
4.4
4.6
4.8
5.0
The direct electrochemical detection of aminothiol compounds only using a borondoped diamond electrochemical cell has been previously described.1 The applied
potential used for this study (+1600 mV) was sufficient to oxidize both thiol and
disulfide analytes. Advantages of this approach include a stable electrode surface
and method simplicity since no sample derivatization is required. After each
analysis the electrode surface was regenerated by a 10 second clean cell pulse at
+1900 mV. After a 1.5 minute re-equilibration at +1600 mV the electrode was once
again stable and could be used for the analysis of the next sample. In the current
method the whole blood sample was added to the perchloric acid media
method,
media, mixed
and then centrifuged. The clear supernatant was then transferred into an
autosampler vial and placed on the autosampler at 10 °C. This approach enabled
rapid sample processing thus minimizing issues related to the instability of the
thiols. Rapid UHPLC analysis as shown in Figure 3 enables processing of these
samples within three minutes before any major chemical transformations take
place.
For biological studies, the determination of aminothiol content should include
compounds such as GSH, GSSG, methionine, and homocysteine. Figure 3
ill
illustrates
the
h overlay
l off calibration
lib i standards
d d ffor these
h
compounds
d ranging
i ffrom
1–20 µg/mL. Peak resolution and retention time uniformity were both excellent.
The column used for this method was the Accucore RP-MS 2.6 micron solid-core
material which provides fast, high resolution separations but with lower system
pressures. When operated at 0.5 mL/min at 50.0 °C the backpressure was less
than 300 bar with the last compound (GSSG) eluting under 2.8 minutes as shown
in Figure 3.
The calibration curves for aminothiol standards are shown in Figure 4. Good
y of response
p
to different concentrations was obtained with correlation
linearity
coefficients ranging from R2 = 0.989–1.00 for the five compounds evaluated
(Table 1) over the range of 1–20 µg/mL. The percent relative standard deviation
(%RSD) for the calibration curves (five concentrations in triplicate) is also shown
in Table 1. The RSD values ranged from 1.2% to 8.9%, indicating that the BDD
electrode provided good stability during this study
study.
4 A Fast and Easy Way for Determining the Level of Oxidative Stress using UHPLC with Boron-Doped Diamond Electrochemical Detection
Enhanced peak shape (nar
of the 6041RS cell with a B
volume of only 50 nL contri
the noise of the electrochem
illustrates that amounts les
determined by this method.
method
shown in Table 2 and range
ratio of 5 are also shown in
5 is also shown in this table
LOD for GSSG is 175 pg o
FIGURE 5. Sensitivity of
0.15
µA
0.12
0.10
0.08
0.06
0.04
CysGly
GSH
B
0.02
0.00
-0.02
0.0
0.5
1.0
1.5
Table 2. Signal-to-noise ra
Compound
S/N Ratio
LOD (pg, S/N of 5)
CysG
37.0
54
FIGURE 4. Calibration curves for standards (1–20 µg/mL, n=3)
1.6
1.2
R² = 0.9997
1
R² = 0.9892
0.8
R² = 0.9998
2 ug/mL
1 ug/mL
10
15
Concentration (µg/mL)
GSH
Methionine
20
25
GSSG
RSD.
%
Correlation
Coefficient.
Slope
4.0
4.2
4.4
4.6
4.8
5.0
ol compounds only using a boroneviously described.1 The applied
ficient to oxidize both thiol and
nclude a stable electrode surface
ation is required. After each
by a 10 second clean cell pulse at
+1600 mV the electrode was once
of the next sample. In the current
he perchloric acid media
media, mixed
then transferred into an
at 10 °C. This approach enabled
related to the instability of the
e 3 enables processing of these
hemical transformations take
CysGly
15
8.9059
0.989
0.0481
GSH
15
1.7446
1.00
0.0509
Meth
15
3.4526
0.999
0.0736
HCYS
15
1.9282
1.00
0.0268
GSSG
15
1.2458
1.00
0.0170
othiol content should include
nd homocysteine. Figure 3
these
h
compounds
d ranging
i ffrom
uniformity were both excellent.
ore RP-MS 2.6 micron solid-core
arations but with lower system
°C the backpressure was less
ting under 2.8 minutes as shown
re shown in Figure 4. Good
was obtained with correlation
five compounds evaluated
cent relative standard deviation
ations in triplicate) is also shown
8.9%, indicating that the BDD
yy.
Enhanced peak shape (narrow peak width) in addition to improved design features
of the 6041RS cell with a BDD electrode provides good sensitivity. The small cell
volume of only 50 nL contributes to low background currents which helps minimize
the noise of the electrochemical cell. The chromatogram shown in Figure 5
illustrates that amounts less than 400 pg on column of each compound can be
determined by this method.
method The signal-to-noise
signal to noise ratios (S/N) for this standard are
shown in Table 2 and range from 7.2–37. The limit of detection (LOD) with a S/N
ratio of 5 are also shown in this table. The limit of detection (LOD) with S/N ratio of
5 is also shown in this table. For GSH the LOD is approximately 67 pg, while the
LOD for GSSG is 175 pg on column.
FIGURE 5. Sensitivity of low level analytical standard (400 pg on column)
0.15
0.20
0.25
0.50
0.75
1.00
1.25
1.50
1.75
Table 3
3. Aminothiol levels (µM)
electrode
CysGly
GS
Level µM
55.9±13.0 101
µA
0.12
Figure 6 shows an overlay of chr
and a sample of deproteinized w
easily measured in small sample
method described. The levels de
p
levels using
g other techn
reported
below the assay LOD using thes
detect this compound by simply
processed.
C
Conclusions
l i
• A simple and sensitive method
stress was developed via the an
deproteinized whole blood. Tec
minimized by rapid sample prep
• Although the analysis of aminot
the method could be easily ada
compounds in plasma and tissu
• The levels of GSH and GSSG d
using other techniques
techniques.
0.10
0.08
0.06
0.04
0.02
References
GSSG
3.8
0.60
0.00
#
Points
Methionine
HCYS
3.6
0.80
-0.20
Peak
CysGly
GSH
3.4
1.00
-0.00
HCys
min
3.2
1.40
CysGly
5
Table 1. Regression data for standard calibration curves
5 ug/mL
1.60
0.40
0
CysGly
10 ug/mL
µA
GSH
R² = 0.9996
0.4
20 ug/mL
2.00
1 20
1.20
0.6
0
FIGURE 6. Overlay of chromat
whole blood (blue trace)
1.80
0.2
ging from 1–20 µg/mL
3.0
R² = 0.9986
1.4
Response (µA)
) provides the unique
diamond which enables the
ctrode potentials than other
es both chromatographic and
oViper (Figure 2C) fingertight
essures due to smaller column
olume (ZDV) capillaries can
uch safer to use than PEEK™
ures. They
y are made of PeekSil
ons to minimize chromatographic
were 150 micron ID for all
and 100 micron ID for those made
0.2 ug/mL
0.00
-0.02
min
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
Table 2. Signal-to-noise ratio values calculated for 400 pg on column
Compound
S/N Ratio
LOD (pg, S/N of 5)
CysGly
37.0
54
GSH
29.4
67
Methionine
20.4
98
Hcys
7.2
278
GSSG
11.4
175
1 Bailey,
1.
B il
B
B.A.
A ett al.
l Di
Directt d
determ
t
levels using HPLC-ECD with a
electrode. Advanced Protocols
2011, 594, 327-339.
2. Park, H.J. et al. Validation of hig
diamond detection for assessin
2010, 407, 151-159.
3. Raffa, M. et al. Decreased gluta
activities in drug-naïve first-epis
11,, 124-131.
© 2013 Thermo Fisher Scientific Inc. All rig
trademark of SGE International Pty Ltd. All
Inc. and its subsidiaries. This information is
manners that might infringe the intellectual
Thermo Scientific Poster Note • EAS2013_PN70904_E 11/13S 5
ards (1–20 µg/mL, n=3)
R² = 0.9986
FIGURE 6. Overlay of chromatograms of standard (black trace) vs.
whole blood (blue trace)
2.00
µA
1.80
R² = 0.9997
R² = 0.9892
1.60
1.40
1 20
1.20
0.80
nine
25
CysGly
20
0.20
GSSG
0.60
0.40
15
tion (µg/mL)
Methionine
R² = 0.9998
1.00
GSH
R² = 0.9996
Whole blood
-0.00
HCys
GSSG
5 ug/mL mix
-0.20
min
alibration curves
0.00
D.
Correlation
Coefficient.
Slope
59
0.989
0.0481
46
1.00
0.0509
26
0.999
0.0736
82
1.00
0.0268
58
1.00
0.0170
0.25
0.50
0.75
1.00
1.25
1.50
1.75
2.00
2.25
2.50
2.75
3.00
3.25
3.50
3.75
4.00
4.25
4.50
4.75
5.00
Table 3
3. Aminothiol levels (µM) observed in whole blood (n=3) using the BDD
electrode
CysGly
GSH
Methionine
GSSG
Level µM
55.9±13.0 1017.2±26.2
107.4±52.7
71.1±25.9
) in addition to improved design features
rovides good sensitivity. The small cell
ckground currents which helps minimize
hromatogram shown in Figure 5
n column of each compound can be
noise ratios (S/N) for this standard are
The limit of detection (LOD) with a S/N
limit of detection (LOD) with S/N ratio of
LOD is approximately 67 pg, while the
ytical standard (400 pg on column)
Figure 6 shows an overlay of chromatograms for a standard mixture of aminothiols
and a sample of deproteinized whole blood. The levels of GSH and GSSG was
easily measured in small samples of whole blood (<20 µL) using the UHPLC
method described. The levels detected as shown in Table 3 are within the range of
3 Although
p
levels using
g other techniques.
q
g the level of homocysteine
y
was
reported
below the assay LOD using these small sample volumes, it would be possible to
detect this compound by simply increasing the volume of sample collected and
processed.
C
Conclusions
l i
• A simple and sensitive method for the determination of the levels of oxidative
stress was developed via the analysis of thiols, disulfides and thioethers from
deproteinized whole blood. Technical issues related to GSH autoxidation were
minimized by rapid sample preparation techniques.
techniques
• Although the analysis of aminothiols in this work was related to whole blood,
the method could be easily adapted to determine the levels of these
compounds in plasma and tissue samples as well.
• The levels of GSH and GSSG detected are within the range of reported levels
using other techniques
techniques.
GSSG
References
0.2 ug/mL
min
3.0
3.5
4.0
4.5
5.0
culated for 400 pg on column
Methionine
20.4
98
Hcys
7.2
278
GSSG
11.4
175
1 Bailey,
1.
B il
B
B.A.
A ett al.
l Di
Directt d
determination
t
i ti off ti
tissue aminothiol,
i thi l disulfide,
di lfid and
d thi
thioether
th
levels using HPLC-ECD with a novel stable boron-doped diamond working
electrode. Advanced Protocols in Oxidative Stress II, Methods in Molecular Biology,
2011, 594, 327-339.
2. Park, H.J. et al. Validation of high-performance liquid chromatography–boron-doped
diamond detection for assessing hepatic glutathione redox status. Anal. Biochem.
2010, 407, 151-159.
3. Raffa, M. et al. Decreased glutathione levels and impaired antioxidant enzyme
activities in drug-naïve first-episode schizophrenic patients BMC Psychiatry, 2011,
11,, 124-131.
© 2013 Thermo Fisher Scientific Inc. All rights reserved. PEEK is a trademark of Victrex PLC. Peeksil is a
trademark of SGE International Pty Ltd. All other trademarks are the property of Thermo Fisher Scientific
Inc. and its subsidiaries. This information is not intended to encourage use of these products in any
manners that might infringe the intellectual property rights of others.
PO70904_E11/13S
6 A Fast and Easy Way for Determining the Level of Oxidative Stress using UHPLC with Boron-Doped Diamond Electrochemical Detection
www.thermofisher.com
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Europe-Other +43 1 333 50 34 0
Finland +358 9 3291 0200
France +33 1 60 92 48 00
Germany +49 6103 408 1014
India +91 22 6742 9494
Italy +39 02 950 591
Japan +81 6 6885 1213
Korea +82 2 3420 8600
Latin America +1 561 688 8700
Middle East +43 1 333 50 34 0
Netherlands +31 76 579 55 55
New Zealand +64 9 980 6700
Norway +46 8 556 468 00
Russia/CIS +43 1 333 50 34 0
Singapore +65 6289 1190
Sweden +46 8 556 468 00
Switzerland +41 61 716 77 00
Taiwan +886 2 8751 6655
UK/Ireland +44 1442 233555
USA +1 800 532 4752
EAS2013_PN70904_E 07/16S