Rapid Analysis of Aminothiols by UHPLC with Boron ... - Dionex

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
Analytical Conditions for Am
Column:
Pump Flow Rate:
Mobile Phase:
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.
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
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)
B) GSSG (oxidized form)
C) Methionine D) Homocysteine
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
2 Rapid Analysis of Aminothiols by UHPLC with Boron-Doped Diamond Electrochemical Detection

c, 22 Alpha Road, Chelmsford, MA, USA
n concerning aminothiols,
scientists require a sensitive
ltaneously. A simple, accurate
nalysis of these compounds
erometric electrochemical cell
ode. This allowed the accurate
sitivity.
and thioethers using UHPLC
hemical cell using a BDD
of various aminothiols,
blood without significant matrix
ing compounds occur in vivo
GSH], homocysteine), disulfides
ystine), and thioethers (e.g.,
ols plays numerous physiological
factor for glutathione peroxidase,
lipid hydroperoxides. The high
vironment, essential for its
h cellular toxicity and numerous
sonʼs disease). Cysteine is also
tathione and is often found in
an essential amino acid and
-adenosylmethionine. Although a
for the measurement of thiols,
hnical issues. UV requires
/GSSG ratio, while some
lectrodes suffer from electrode
aintenance. Although porous
y still need maintenance. Boronmeasurement
of these analytes
ented herein is capable of
neously using UHPLC
lectrode. Examples showing the
using a simple “dilute and shoot”
eral advantages over standard
ween samples; improved
portant when measuring GSSG
e last peaks eluting in biological
des better resolution between
logical samples.
GSH), B) glutathione disulfide
G (oxidized form)
ocysteine
Methods
Analytical Conditions for Aminothiol, Disulfide and Thioether Analysis
Column:
Thermo Scientific Accucore RP-MS column
2.6 μm, 2.1 × 150 mm
Pump Flow Rate:
0.500 mL/min
Mobile Phase:
0.1% pentafluoropropionic acid, 0.02% ammonium
hydroxide, 2.5% acetonitrile, water
Column Temperature: 50.0 °C
Post Column Temperature 25.0 °C
Injection Volume:
2 µL standards; 4 µL samples
Cell Potential:
Thermo Scientific Dionex model 6041RS ultra
Amperometric Analytical Cell with BDD electrode at
+1600 mV
Filter Constant:
1.0 s
Cell Clean:
On
Cell Clean Potential: 1900 mV
Cell Clean Duration: 10.0 s
Sample Prep:
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
A
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.
B
C
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.
FIGURE 3. Overlay of analytic
3.0 µA
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
-0.0
CysGly
GSH
0.0 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 R 2 = 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

e and Thioether Analysis
fic Accucore RP-MS column
50 mm
ropropionic acid, 0.02% ammonium
acetonitrile, water
; 4 µL samples
fic Dionex model 6041RS ultra
nalytical Cell with BDD electrode at
blood + 200 µL 0.4 N PCA, mix and
tes at 13,000 RPM. The clear
s transferred into an autosampler vial
he autosampler at 10 °C.
iMate 3000 Electrochemical detector
tical Cell with BDD electrode
oViper fingertight capillaries
3000 UHPLC system consists of:
utosampler
mn compartment
ata System software
is is that the HPLC system must be
n order to achieve optimal sensitivity
m shown above in Figure 2A uses
duce the influence of metal that can
t the electrochemical cell. The recent
ables multiple electrodes to be
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.
FIGURE 3. Overlay of analytical standards ranging from 1–20 µg/mL
3.0
µA
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
CysGly
GSH
Methionine
HCYS
-0.0
min
0.0 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 R 2 = 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.
GSSG
20 ug/mL
10 ug/mL
5 ug/mL
2 ug/mL
1 ug/mL
Response (µA)
0.12
0.10
0.08
0.06
0.04
0.02
0.00
-0.02
FIGURE 4. Calibration cu
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0 5
CysGly
Table 1. Regression data
Poin
Peak #
CysGly 15
GSH 15
Meth 15
HCYS 15
GSSG 15
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
CysGly
GSH
0.0 0.5 1.0 1.5
Table 2. Signal-to-noise r
Compound CysG
S/N Ratio 37
LOD (pg, S/N of 5) 54
4 Rapid Analysis of Aminothiols by UHPLC with Boron-Doped Diamond Electrochemical Detection

ovides the unique
ond which enables the
de potentials than other
oth chromatographic and
r (Figure 2C) fingertight
res due to smaller column
e (ZDV) capillaries can
safer to use than PEEK
. They are made of PeekSil
to minimize chromatographic
e 150 micron ID for all
100 micron ID for those made
from 1–20 µg/mL
Response (µA)
FIGURE 4. Calibration curves for standards (1–20 µg/mL, n=3)
1.6
R² = 0.9986
1.4
1.2
R² = 0.9997
1
R² = 0.9892
0.8
0.6
R² = 0.9996
0.4
R² = 0.9998
0.2
0
0 5 10 15 20 25
Concentration (µg/mL)
CysGly GSH Methionine HCys GSSG
FIGURE 6. Overlay of chromatog
whole blood (blue trace)
2.00
µA
1.80
1.60
1.40
1.20
1.00
0.80
0.60
0.40
0.20
-0.00
-0.20
CysGly
GSH
20 ug/mL
10 ug/mL
5 ug/mL
2 ug/mL
1 ug/mL
min
.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0
mpounds only using a boronusly
described. 1 The applied
nt to oxidize both thiol and
de a stable electrode surface
is required. After each
10 second clean cell pulse at
0 mV the electrode was once
e next sample. In the current
erchloric acid media, mixed
n transferred into an
0 C. This approach enabled
ted to the instability of the
nables processing of these
ical transformations take
l content should include
omocysteine. Figure 3
se compounds ranging from
rmity were both excellent.
P-MS 2.6 micron solid-core
ons but with lower system
the backpressure was less
under 2.8 minutes as shown
hown in Figure 4. Good
btained with correlation
compounds evaluated
relative standard deviation
s in triplicate) is also shown
%, indicating that the BDD
0.12
0.10
0.08
0.06
0.04
0.02
0.00
-0.02
Table 1. Regression data for standard calibration curves
Peak #
Points
RSD.
%
Correlation
Coefficient.
Slope
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
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
CysGly
GSH
Methionine
HCYS
0.2 ug/mL
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 CysGly GSH Methionine Hcys GSSG
S/N Ratio 37.0 29.4 20.4 7.2 11.4
LOD (pg, S/N of 5) 54 67 98 278 175
GSSG
0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.0
Table 3. Aminothiol levels (µM) o
electrode
CysGly GSH
Level µM 55.9±13.0 1017
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.
References
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
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
es
GSSG
R² = 0.9996
R² = 0.9998
20 25
elation Slope
ficient.
989 0.0481
.00 0.0509
999 0.0736
.00 0.0268
.00 0.0170
improved design features
ensitivity. The small cell
nts which helps minimize
shown in Figure 5
ch compound can be
N) for this standard are
ction (LOD) with a S/N
n (LOD) with S/N ratio of
imately 67 pg, while the
(400 pg on column)
1.00
0.80
0.60
0.40
0.20
-0.00
-0.20
CysGly
GSH
Methionine
Whole blood
5 ug/mL mix
0.00 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 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
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 (

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