Rapid Analysis of Aminothiols by UHPLC with
Boron-Doped Diamond Electrochemical Detection
Bruce Bailey, Marc Plante, David Thomas, Qi Zhang and Ian Acworth
Thermo Fisher Scientific, Chelmsford, MA, USA
Thermo Fisher Scientific, 22 Alpha Road, Chelmsford, M
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, simultaneously. A 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 enables the rapid separation of various aminothiols,
disulfides, and thioethers at low levels from whole blood without significant matrix
interferences.
Introduction
A number of biochemically important sulfur-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 detoxifies hydrogen peroxide and lipid hydroperoxides. The high
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., Parkinsonʼs 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 adversely 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
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 Rapid Analysis of Aminothiols by 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 Scientifi
FIGURE 2B. 6041RS ultra Am
FIGURE 2C. Thermo Scientifi
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 de
biocompatible materials in t
contribute to elevated back
introduction of the ECD-300
attached in series after the
ic, 22 Alpha Road, Chelmsford, MA, USA
n of various aminothiols,
blood without significant matrix
ning compounds occur in vivo
[GSH], homocysteine), disulfides
cystine), and thioethers (e.g.,
iols plays numerous physiological
ofactor for glutathione peroxidase,
lipid hydroperoxides. The high
nvironment, essential for its
th cellular toxicity and numerous
nsonʼs disease). Cysteine is also
utathione and is often found in
s an essential amino acid and
S-adenosylmethionine. Although a
for the measurement of thiols,
chnical issues. UV requires
H/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
s using a simple “dilute and shoot”
veral advantages over standard
tween samples; improved
mportant when measuring GSSG
he last peaks eluting in biological
ides better resolution between
ological samples.
(GSH), B) glutathione disulfide
SG (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™ Accucore™ RP-MS column
2.6 μm, 2.1 × 150 mm
0.500 mL/min
0.1% pentafluoropropionic acid, 0.02% ammonium
hydroxide, 2.5% acetonitrile, water
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
supernatant was transferred into an autosampler vial
and placed on the autosampler at 10 °C.
FIGURE 2A. Thermo Scientific™ Dionex™ UltiMate™ 3000 Electrochemical detector
FIGURE 2B. 6041RS ultra Amperometric Analytical Cell with BDD electrode
FIGURE 2C. Thermo Scientific™ Dionex™ nanoViper™ fingertight capillaries
FIGURE 3. Overlay of analytic
3.0
2.8
µA
2.6
2.4
2.2
2.0
GSH
s and thioethers using UHPLC
chemical cell using a BDD
Use of the 6041RS amperome
electrochemical capabilities of
oxidation of organic compound
working electrode materials. Th
voltammetric resolution of com
fittings were employed to cope
particles. These fingertight, virt
operate at pressures up to 14,5
tubing which can slip when usi
tubing and are available in sma
band spreading. Capillaries us
connections made prior to the
after the injector valve.
CysGly
on concerning aminothiols,
scientists require a sensitive
ultaneously. A simple, accurate
analysis of these compounds
perometric electrochemical cell
rode. This allowed the accurate
nsitivity.
Methods
1.8
1.6
1.4
1.2
1.0
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
- WPS-3000TBPL Thermostatted Analytical Autosampler
- 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
biocompatible materials in the flow path to reduce the influence of metal that 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
The direct electrochemical dete
doped diamond electrochemica
potential used for this study (+1
disulfide analytes. Advantages
and method simplicity since no
analysis the electrode surface
+1900 mV. After a 1.5 minute r
again stable and could be used
method, the whole blood samp
and then centrifuged. The clea
autosampler vial and placed on
rapid sample processing thus m
thiols. Rapid UHPLC analysis a
samples within three minutes b
place.
For biological studies, the dete
compounds such as GSH, GSS
illustrates the overlay of calibra
1–20 µg/mL. Peak resolution a
The column used for this meth
material which provides fast, h
pressures. When operated at 0
than 300 bar with the last comp
in Figure 3.
The calibration curves for amin
linearity of response to differen
coefficients ranging from R2 = 0
(Table 1) over the range of 1–2
(%RSD) for the calibration curv
in Table 1. The RSD values ran
electrode provided good stabili
Thermo Scientific Poster Note • PN70532_E 03/13S 3
A
de and Thioether Analysis
ific™ Accucore™ RP-MS column
150 mm
oropropionic acid, 0.02% ammonium
% acetonitrile, water
µA
2.0
1
0.8
0.6
0.4
0.2
1.8
1.6
0
5
CysGly
HCYS
2.2
GSH
CysGly
2.6
2.4
1.2
0
Table 1. Regression data
GSSG
2.8
tiMate™ 3000 Electrochemical detector
ytical Cell with BDD electrode
noViper™ fingertight capillaries
1.4
FIGURE 3. Overlay of analytical standards ranging from 1–20 µg/mL
3.0
blood + 200 µL 0.4 N PCA, mix and
utes at 13,000 RPM. The clear
as transferred into an autosampler vial
the autosampler at 10 °C.
1.6
Methionine
s; 4 µL samples
ific™ 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™
tubing which can slip when using elevated pressures. They are made of PeekSil
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.
1.4
Peak
1.2
20 ug/mL
1.0
CysGly
10 ug/mL
0.8
0.6
5 ug/mL
0.4
2 ug/mL
0.2
15
GSH
15
Meth
15
HCYS
1 ug/mL
-0.0
#
Poin
15
GSSG
15
min
0.0
C
™ 3000 UHPLC system consists of:
Autosampler
umn compartment
Data System software
sis is that the HPLC system must be
in order to achieve optimal sensitivity
em shown above in Figure 2A uses
educe the influence of metal that 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, 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
illustrates the overlay of calibration standards for these compounds ranging from
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
linearity of response to different concentrations was obtained with correlation
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.
4 Rapid Analysis of Aminothiols by UHPLC with Boron-Doped Diamond Electrochemical Detection
Enhanced peak shape (na
of the 6041RS cell with a B
volume of only 50 nL contr
the noise of the electroche
illustrates that amounts les
determined by this method
shown in Table 2 and rang
ratio of 5 are also shown in
5 is also shown in this tabl
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 r
Compound
S/N Ratio
LOD (pg, S/N of 5)
CysG
37
54
FIGURE 4. Calibration curves for standards (1–20 µg/mL, n=3)
1.6
1.2
R² = 0.9997
R² = 0.9892
1
0.8
0
5
10
CysGly
15
0.80
20
25
GSH
Methionine
HCys
GSSG
Slope
CysGly
15
8.9059
0.989
0.0481
Meth
15
3.4526
0.999
1 ug/mL
1.2458
1.00
15
GSSG
1.00
1.9282
15
0.0509
0.0736
1.00
0.0268
0.0170
4.2
4.4
4.6
4.8
5.0
ompounds only using a boronously described.1 The applied
ent to oxidize both thiol and
ude a stable electrode surface
n is required. After each
a 10 second clean cell pulse at
00 mV the electrode was once
he next sample. In the current
perchloric acid media, mixed
en transferred into an
10 C. This approach enabled
ated to the instability of the
enables processing of these
mical transformations take
ol content should include
homocysteine. Figure 3
ese compounds ranging from
ormity were both excellent.
RP-MS 2.6 micron solid-core
ions but with lower system
the backpressure was less
g under 2.8 minutes as shown
shown in Figure 4. Good
obtained with correlation
e compounds evaluated
t relative standard deviation
ns in triplicate) is also shown
9%, indicating that the BDD
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. The 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
µA
0.12
0.50
0.75
1.00
1.25
1.50
1.75
2.0
Figure 6 shows an overlay of chrom
and a sample of deproteinized who
easily measured in small samples
method described. The levels dete
reported levels using other techniq
below the assay LOD using these
detect this compound by simply inc
processed.
Conclusions
• The method for the analysis for th
thioethers proved to be simple an
measurement in deproteinized wh
autoxidation were minimized by r
• Although the analysis of aminothi
the method could be easily adapt
compounds in plasma and tissue
• The levels of GSH and GSSG de
using other techniques.
0.10
0.08
0.06
0.04
0.02
References
GSSG
4.0
Methionine
HCYS
3.8
CysGly
GSH
3.6
0.25
Table 3. Aminothiol levels (µM) o
electrode
CysGly
GSH
Level µM
55.9±13.0 1017
min
3.4
0.20
0.00
Correlation
Coefficient.
HCYS
0.40
-0.20
RSD.
%
1.7446
0.60
-0.00
#
Points
15
GSH
R² = 0.9998
Peak
GSH
2 ug/mL
1.40
1.00
Table 1. Regression data for standard calibration curves
5 ug/mL
1.60
R² = 0.9996
Concentration (µg/mL)
10 ug/mL
µA
CysGly
0.4
20 ug/mL
2.00
1.20
0.6
0
FIGURE 6. Overlay of chromatog
whole blood (blue trace)
1.80
0.2
g from 1–20 µg/mL
3.2
R² = 0.9986
1.4
Response (µA)
rovides the unique
mond which enables the
ode potentials than other
both chromatographic and
er (Figure 2C) fingertight
ures due to smaller column
me (ZDV) capillaries can
safer to use than PEEK™
s. They are made of PeekSil
s to minimize chromatographic
re 150 micron ID for all
d 100 micron ID for those made
0.2 ug/mL
0.00
-0.02
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
min
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, B.A. et al. Direct determin
levels using HPLC-ECD with a no
electrode. Advanced Protocols in
2011, 594, 327-339.
2. Park, H.J. et al. Validation of high
diamond detection for assessing
2010, 407, 151-159.
3. Raffa, M. et al. Decreased glutath
activities in drug-naïve first-episo
11, 124-131.
© 2013 Thermo Fisher Scientific Inc. All rights
trademark of SGE International Pty Ltd. All ot
Inc. and its subsidiaries. This information is n
manners that might infringe the intellectual pr
Thermo Scientific Poster Note • PN70532_E 03/13S 5
/mL, n=3)
R² = 0.9986
R² = 0.9997
R² = 0.9892
µA
1.60
1.40
1.00
R² = 0.9998
0.80
0.40
Methionine
25
CysGly
20
0.60
0.20
GSSG
R² = 0.9996
GSH
1.20
Whole blood
-0.00
GSSG
Slope
.989
0.0509
1.00
0.0268
0.0736
1.00
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
min
4.75
5.00
Table 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
0.0481
1.00
.999
5 ug/mL mix
-0.20
0.00
relation
fficient.
0.0170
improved design features
sensitivity. The small cell
ents which helps minimize
shown in Figure 5
ach compound can be
/N) for this standard are
ection (LOD) with a S/N
on (LOD) with S/N ratio of
ximately 67 pg, while the
d (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
reported levels using other techniques.3 Although the level of homocysteine was
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.
Conclusions
• The method for the analysis for the measurement of thiols, disulfides and
thioethers proved to be simple and reliable with sufficient sensitivity for their
measurement in deproteinized whole blood. Technical issues related to GSH
autoxidation were minimized by rapid sample preparation 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.
References
0.2 ug/mL
4.0
4.5
min
5.0
00 pg on column
e
2.00
1.80
ves
3.5
FIGURE 6. Overlay of chromatograms of standard (black trace) vs.
whole blood (blue trace)
Hcys
7.2
278
GSSG
11.4
175
1. Bailey, B.A. et al. Direct determination of tissue aminothiol, disulfide, and thioether
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.
PO70532_E 03/13S
6 Rapid Analysis of Aminothiols by UHPLC with Boron-Doped Diamond Electrochemical Detection
www.thermofisher.com/dionex
©2016 Thermo Fisher Scientific Inc. All rights reserved. PEEK is a trademark of Victrex PLC. Peeksil is a trademark of SGE
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