Quantifying Co in Doping Control Urine Samples – A Pilot Study

Quantifying Co in Doping Control
Urine Samples – A Pilot Study
Daniel Kutscher,1 Oliver Krug,2 Mario Thevis,2 Shona McSheehy Ducos1
1
Thermo Fisher Scientific, Bremen, Germany;
2
German Sports University, Cologne, Germany
Overview
Results
Purpose: To demonstrate the power of ICP-MS for the
analysis of different elements in biological matrices and
demonstrate the use of trace elemental analysis in clinical
applications.
The underlying mechanism in w
improved performance of espec
displayed in figure 2. In 2014, C
World Anti-Doping Agency´s list
so that dishonest sportsmen co
option to increase their oxygen
contrast, testing for elevated lev
e.g. horse racing3.
Methods: Total elemental determinations of Co and other
trace elements in urine were performed using the Thermo
Scientific™ iCAP™ Qc ICP-MS in a single, optimized
operation mode. The excretion of Co in urine was
measured in an elimination study after intake of Co2+ and
vitamin B12 respectively.
Results: Good agreement between the results
determined using a certified reference material was
achieved. The elimination study showed that the
determination of Co in urine can be a potential tool to
screen for its misuse as a performance enhancing
substance.
Introduction
The role of Inductively Coupled Plasma Mass
Spectrometry (ICP-MS) in clinical research is relatively
unknown in the mass spectrometry community. However,
it is a powerful tool for trace elemental analysis thanks to
high sensitivity and robustness when faced with high
matrix samples. ICP-MS provides a fully quantitative
technique for almost all elements in the periodic table,
offers isotopic information and isotope ratio determinations
and can also provide species specific information when
coupled with a separation device such as Ion
Chromatography (IC) or Liquid Chromatography (LC).
The analysis of trace metals in urine is a way to address
potentially harmful exposure to contaminants. One of the
advantages for urine as a sample matrix is that the
specimen is easily derived. Apart from screening for
potential exposure to toxic elements like As, Cd, Hg and
Pb, the determination of Co in urine has gained interest,
as Co2+ could potentially be used as a performing
enhancing substance in endurance sports1.
Methods
Sample Preparation
Urine samples were diluted 10-fold in 2% HNO3, and 50 µl
of an internal standard solution (5 ng.mL-1 Sc, Ge, 2.5
ng.mL-1 Rh and Ir) was added to each sample.
Mass Spectrometry
The iCAP Qc ICP-MS (Figure 1) was used for acquisition
of all data.
An SC-4Q autosampler (Elemental Scientific, Omaha,
NE, USA) was used for all measurements. The iCAP Qc
ICP-MS was operated in single He KED mode for all
analyte ions.
All instrumental parameters can be found in table 1.
2 Quantifying Co in Doping Control Urine Samples – A Pilot Study
Performance Enhancemen
FIGURE 2. Proposed mechan
performance enhancement in
• The effect of 5 µmol Co is eq
Erythropoiesis as 1 IU rhEPO
• Typically, 6-25 IU·L-1 of EPO
• 150 mg CoCl2 leads to an inc
1.19 Mio cells·mm-2 in 7-22 d
Analysis of Trace Elements in
In a pilot study, approx. 200 sam
and various other trace elemen
levels of Co in urine.
Verify
- Co concentrations in urine were found
range [0.1 to 2 ng· mL-1]
All calibration standards were p
and a six point calibration curve
0.025 to 25 ng·L-1 (verification o
to 100 ng·mL-1 (elimination stud
instrumental limit of detection fo
ng L-1. UTAK® Urine Control Sa
Range, UTAK Laboratories, Val
regularly interspersed in the sam
to verify the accuracy of the me
for the repeated analysis of both
NE, USA) was used for all measurements. The iCAP Qc
ICP-MS was operated in single He KED mode for all
analyte ions.
All instrumental parameters can be found in table 1.
and a six point calibration curve w
0.025 to 25 ng·L-1 (verification of re
to 100 ng·mL-1 (elimination study).
instrumental limit of detection for 5
ng L-1. UTAK® Urine Control Samp
Range, UTAK Laboratories, Valenc
regularly interspersed in the samp
to verify the accuracy of the metho
for the repeated analysis of both c
batch (containing 70 samples) are
TABLE 2. Results for the repeate
controls as a quality control with
all values given in µg·L-1.
As
Cd
Ca
Cr
Co
Cu
16.5
0.4
96
1.3
1.9
32
Maximum
14
19
0.34
0.46
82
110
1.1
1.5
1.6
2.2
27
37
Recovery
Reference
Minimum
FIGURE 1. Thermo Scientific iCAP Qc ICP-MS.
TABLE 1. Instrumental Conditions.
95%
98%
95%
95%
103%
1.8
6.6
1.6
4.6
3.3
2.9
Reference
118
4.6
490
6.2
7.6
81
Maximum
100
136
3.9
5.3
417
564
5.3
7.1
6.5
8.7
69
93
Recovery
94%
96%
91%
105%
98%
95%
3.6
2.9
1.7
1.9
1.6
3.2
Minimum
Parameter
Nebulizer
Nebulizer Gas Flow
Value
PFA-ST
1.06 L·min-1
RF Power
Interface Set-Up
1550 W
Ni Cones, High Matrix
Skimmer Insert
QCell conditions
Cell Gas Flow
KED Voltage
94%
RSD
RSD
IDL
4.8 mL·min-1 100% He
3V
Data Analysis
Thermo Scientific™ Qtegra™ Intelligent Scientific Data
SolutionTM software was used for quantitative assessment
of the data.
0.007 0.0006
0.3
0.001 0.0006 0.0008
Utak 12111 Normal Range
Good agreement between the cert
values was achieved. The low rela
between the different replicates hig
the method.
The obtained results for both group
ng·mL-1 for non elite athletes and 0
endurance athletes) were found to
range, but showing a modest but s
increase for professional enduranc
sum test, p<0.01). However, strong
concentrations were not observed.
Thermo Scientific Poster Note • eWPC • PN43242-EN 0315S 3
Results
he power of ICP-MS for the
ts in biological matrices and
e elemental analysis in clinical
eterminations of Co and other
e performed using the Thermo
MS in a single, optimized
ion of Co in urine was
study after intake of Co2+ and
The underlying mechanism in which Co2+ may lead to
improved performance of especially endurance athletes is
displayed in figure 2. In 2014, Co2+ was not listed on the
World Anti-Doping Agency´s list of prohibited substances2,
so that dishonest sportsmen could potentially use this
option to increase their oxygen transport capacity. In
contrast, testing for elevated levels of Co is already done in
e.g. horse racing3.
Feasibility of Co Urinary Co
Anti Doping Testing
In a second step, an eliminat
verify that intake of Co2+ lead
Co concentration. The results
displayed in figure 3.
Investigate excretio
- 3 participants. s
- 3 participants. mu
Co2+
between the results
reference material was
udy showed that the
can be a potential tool to
erformance enhancing
pled Plasma Mass
linical research is relatively
ometry community. However,
elemental analysis thanks to
ess when faced with high
ovides a fully quantitative
ments in the periodic table,
nd isotope ratio determinations
s specific information when
evice such as Ion
uid Chromatography (LC).
s in urine is a way to address
e to contaminants. One of the
ample matrix is that the
Apart from screening for
elements like As, Cd, Hg and
in urine has gained interest,
used as a performing
durance sports1.
FIGURE 3. Proposed mech
performance enhancement
Performance Enhancement in Endurance Sports
FIGURE 2. Proposed mechanism for Co2+ induced
performance enhancement in enduring sports.
• The effect of 5 µmol Co is equivalent to stimulation of
Erythropoiesis as 1 IU rhEPO.
• Typically, 6-25 IU·L-1 of EPO are circulating in humans.
• 150 mg CoCl2 leads to an increase of RBC from 0.5 to
1.19 Mio cells·mm-2 in 7-22 days.
Analysis of Trace Elements in Urine
10-fold in 2% HNO3, and 50 µl
ion (5 ng.mL-1 Sc, Ge, 2.5
ed to each sample.
re 1) was used for acquisition
In a pilot study, approx. 200 samples were analyzed for Co
and various other trace elements in order to verify baseline
levels of Co in urine.
Verify baseline Co levels in urine
-100 healthy non-elite athletes
- 96 endurance sports athletes
- Co concentrations in urine were found to be in the typically observed
range [0.1 to 2 ng· mL-1]
The results show that the Co
increased in comparison to th
between 0.1-2.0 ng·mL-1. A s
normalization of the determin
to compensate for the specif
are two conclusions to be dra
• A strong increase of the ur
observed within 6 h (betwe
intake of Co2+.
• Multiple doses of Co2+ lea
observed even several da
stopped.
Furthermore, the excretion o
upon regular (daily) intake of
accounting for 22 µg Co). Ho
seem to affect the urinary Co
Conclusion
•
The iCAP Q ICP-MS is a
elements in a challenging
combination of a unique r
single analysis mode for a
method development and
when it comes to routine
samples.
•
The detection of Co in uri
potential tool to investigat
•
Further investigation is re
additionally influencing fa
emental Scientific, Omaha,
measurements. The iCAP Qc
gle He KED mode for all
All calibration standards were prepared in diluted nitric acid
and a six point calibration curve was generated between
0.025 to 25 ng·L-1 (verification of reference levels) and 0.25
to 100 ng·mL-1 (elimination study). The attainable
instrumental limit of detection for 59Co was found to be 0.6
can be found in table 1.
ng L-1. UTAK® Urine Control Samples (Low and High
Range, UTAK Laboratories, Valencia, CA, USA) were
regularly interspersed in the sample list (every 10 samples)
to verify
the
accuracy of the method. The obtained results
– A Pilot
Study
4 Quantifying Co in Doping Control Urine Samples
for the repeated analysis of both control samples within a
ntal Scientific, Omaha,
urements. The iCAP Qc
He KED mode for all
be found in table 1.
All calibration standards were prepared in diluted nitric acid
and a six point calibration curve was generated between
0.025 to 25 ng·L-1 (verification of reference levels) and 0.25
to 100 ng·mL-1 (elimination study). The attainable
instrumental limit of detection for 59Co was found to be 0.6
ng L-1. UTAK® Urine Control Samples (Low and High
Range, UTAK Laboratories, Valencia, CA, USA) were
regularly interspersed in the sample list (every 10 samples)
to verify the accuracy of the method. The obtained results
for the repeated analysis of both control samples within a
batch (containing 70 samples) are displayed in table 2.
As
Reference
telligent Scientific Data
r quantitative assessment
Cr
Co
Cu
Fe
Pb
Mn
Mo
Ni
Se
Zn
0.4
96
1.3
1.9
32
30
0.6
3.5
52
2.6
40
269
Maximum
0.34
0.46
82
110
1.1
1.5
1.6
2.2
27
37
26
35
0.5
0.7
3.0
4.0
44
60
2.2
3.0
34
46
229
309
Recovery
94%
95%
98%
95%
95%
1.8
6.6
1.6
4.6
3.3
2.9
4.9
4.7
2.9
2.2
3.0
2.7
2.5
Minimum
V
Ca
14
19
ions.
8 mL·min-1 100% He
Cd
16.5
Minimum
RSD
FA-ST
06 L·min-1
550 W
Cones, High Matrix
kimmer Insert
•
The iCAP Q ICP-MS is a perfe
elements in a challenging mat
combination of a unique robus
single analysis mode for all an
method development and help
when it comes to routine anal
samples.
•
The detection of Co in urine h
potential tool to investigate its
•
Further investigation is require
additionally influencing factors
complementary sample matric
TABLE 2. Results for the repeated analysis of urine
controls as a quality control within one sample batch,
all values given in µg·L-1.
CAP Qc ICP-MS.
alue
Conclusion
- Co concentrations in urine were found to be in the typically observed
range [0.1 to 2 ng· mL-1]
was used for acquisition
Reference
103% 97% 122%
95%
94% 117% 90% 117%
118
4.6
490
6.2
7.6
81
565
137
4.2
71
27
55
859
Maximum
100
136
3.9
5.3
417
564
5.3
7.1
6.5
8.7
69
93
480
650
116
158
3.6
4.8
60
82
23
31
47
63
730
988
Recovery
94%
96%
91%
105%
98%
95% 87%
89%
3.6
2.9
1.7
1.9
1.6
RSD
IDL
0.007 0.0006
0.3
3.2
3.1
1.0
References
1. O. Krug et al., Quantifying cobalt in
a pilot study, Drug Testing Analysis
2. The Prohibited List, published by th
https://www.wada-ama.org/en/resou
medicine/prohibited-list
3. P. Bartley: in The Sidney Morning H
100% 95% 107% 91% 100%
4.1
3.8
1.7
5.2
2.8
0.001 0.0006 0.0008 0.002 0.0003 0.002 0.0007 0.002 0.02 0.006
Utak 12111 Normal Range
Utak 12110 High Range
Good agreement between the certified and the obtained
values was achieved. The low relative standard deviation
between the different replicates highlights the robustness of
the method.
Utak is the property of Utak Laboratories, Inc. All other trademarks
subsidiaries.
This information is not intended to encourage use of these product
property rights of others.
Presented at the European Winter Plasma Conference on Plasma
The obtained results for both groups (mean values 0.28
ng·mL-1 for non elite athletes and 0.36 ng·mL-1 for listed
endurance athletes) were found to be within the expected
range, but showing a modest but statistically significant
increase for professional endurance athletes (Wilcoxon rank
sum test, p<0.01). However, strongly increased urinary Co
concentrations were not observed.
Thermo Scientific Poster Note • eWPC • PN43242-EN 0315S 5
which Co2+ may lead to
ecially endurance athletes is
Co2+ was not listed on the
st of prohibited substances2,
could potentially use this
en transport capacity. In
evels of Co is already done in
Feasibility of Co Urinary Concentration as a Tool for
Anti Doping Testing
In a second step, an elimination study was performed to
verify that intake of Co2+ leads to an increase of the urinary
Co concentration. The results obtained for both groups are
displayed in figure 3.
Investigate excretion after intake of Co2+
- 3 participants. single dose; 500 µg
- 3 participants. multiple doses of 500 µg
Co2+
FIGURE 3. Proposed mechanism for Co2+ induced
performance enhancement in enduring sports.
ent in Endurance Sports
nism for Co2+ induced
in enduring sports.
equivalent to stimulation of
PO.
O are circulating in humans.
ncrease of RBC from 0.5 to
days.
in Urine
amples were analyzed for Co
nts in order to verify baseline
y baseline Co levels in urine
-100 healthy non-elite athletes
- 96 endurance sports athletes
d to be in the typically observed
The results show that the Co concentration in urine is
increased in comparison to the reference values found
between 0.1-2.0 ng·mL-1. A similar picture was found after
normalization of the determined Co concentration in order
to compensate for the specific gravity of the samples. There
are two conclusions to be drawn from the results:
• A strong increase of the urinary Co concentration is
observed within 6 h (between 40 and 318 ng·mL-1 ) after
intake of Co2+.
• Multiple doses of Co2+ lead to elevated levels of Co2+
observed even several days after the intake was
stopped.
Furthermore, the excretion of Co in urine was monitored
upon regular (daily) intake of Vitamin B12 (500 µg·day-1,
accounting for 22 µg Co). However, Vitamin B12 does not
seem to affect the urinary Co level.
Conclusion
•
The iCAP Q ICP-MS is a perfect tool to determine trace
elements in a challenging matrix such as urine. The
combination of a unique robust interface design and a
single analysis mode for all analytes greatly simplifies
method development and helps to improve throughput
when it comes to routine analysis of a large number of
samples.
prepared in diluted nitric acid
ve was generated between
of reference levels) and 0.25
udy). The attainable
for 59Co was found to be 0.6
Samples
(Low and
Co inHigh
Doping Control Urine Samples
A Pilotdetection
Study
6 Quantifying
• –The
of Co in urine has been shown to be a
alencia, CA, USA) were
potential tool to investigate its abuse in sports doping.
seem to affect the urinary Co level.
eline Co levels in urine
100 healthy non-elite athletes
96 endurance sports athletes
Conclusion
n the typically observed
red in diluted nitric acid
generated between
erence levels) and 0.25
he attainable
Co was found to be 0.6
s (Low and High
a, CA, USA) were
list (every 10 samples)
. The obtained results
ntrol samples within a
isplayed in table 2.
•
The iCAP Q ICP-MS is a perfect tool to determine trace
elements in a challenging matrix such as urine. The
combination of a unique robust interface design and a
single analysis mode for all analytes greatly simplifies
method development and helps to improve throughput
when it comes to routine analysis of a large number of
samples.
•
The detection of Co in urine has been shown to be a
potential tool to investigate its abuse in sports doping.
•
Further investigation is required to elucidate the role of
additionally influencing factors and to establish tests for
complementary sample matrices like erythrocytes.
d analysis of urine
n one sample batch,
e
Pb
Mn
Mo
Ni
Se
Zn
0
0.6
3.5
52
2.6
40
269
6
5
0.5
0.7
3.0
4.0
44
60
2.2
3.0
34
46
229
309
7% 122%
95%
94% 117% 90% 117%
.9
4.7
2.9
2.2
3.0
2.7
2.5
65
137
4.2
71
27
55
859
80
50
116
158
3.6
4.8
60
82
23
31
47
63
730
988
7%
89%
.1
1.0
References
1. O. Krug et al., Quantifying cobalt in doping control urine samples –
a pilot study, Drug Testing Analysis 6 (2014), 1186-1190
2. The Prohibited List, published by the World Anti Doping Agency,
https://www.wada-ama.org/en/resources/sciencemedicine/prohibited-list
3. P. Bartley: in The Sidney Morning Herald, February 4th 2014.
100% 95% 107% 91% 100%
4.1
3.8
1.7
5.2
2.8
002 0.0003 0.002 0.0007 0.002 0.02 0.006
Utak 12110 High Range
ed and the obtained
ve standard deviation
lights the robustness of
Utak is the property of Utak Laboratories, Inc. All other trademarks are the property of Thermo Fisher Scientific 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.
Presented at the European Winter Plasma Conference on Plasma Spectrochemistry, Münster, Germany 02/2015.
(mean values 0.28
36 ng·mL-1 for listed
e within the expected
atistically significant
athletes (Wilcoxon rank
y increased urinary Co
www.thermofisher.com
©2016 Thermo Fisher Scientific Inc. All rights reserved. Utak is the property of Utak Laboratories, Inc. All other trademarks are the property of
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