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Mildronate (Meldonium) in professional sports –
monitoring doping control urine samples
using hydrophilic interaction liquid
chromatography –high resolution/high
accuracy mass spectrometry
Christian Görgens,
a
Sven Guddat,
a
*JosefDib,
a
Hans Geyer,
a
Wilhelm Schänzer
a
and Mario Thevis
a,b
To date, substances such as Mildronate (Meldonium) are not on the radar of anti-doping laboratories as the compound is not explicitly
classified as prohibited. However, the anti-ischemic drug Mildronate demonstrates an increase in endurance performance of athletes,
improved rehabilitation after exercise, protection against stress, and enhanced activations of central nervous system (CNS) functions.
In the present study, the existingevidence ofMildronate’s usage in sport, which is arguably not (exclusively) based on medicinal
reasons, is corroborated by unequivocal analytical data allowing the estimation of the prevalence and extent of misuse in profes-
sional sports. Such data are vital to support decision-making processes, particularly regarding the ban on drugs in sport. Due to
the growing body of evidence (black market products and athlete statements) concerning its misuse in sport, adequate test
methods for the reliable identification of Mildronate are required, especially since the substance has been added to the 2015
World Anti-Doping Agency (WADA) monitoring program.
In the present study, two approaches were established using an in-house synthesized labelled internal standard (Mildronate-D
3
).
One aimed at the implementation of the analyte into routine doping control screening methods to enable its monitoring at the low-
est possible additional workload for the laboratory, and another that is appropriate for the peculiar specifics of the analyte, allowing
the unequivocal confirmation of findings using hydrophilic interaction liquid chromatography-high resolution/high accuracy mass
spectrometry (HILIC-HRMS). Here, according to applicable regulations in sports drug testing, a full qualitative validation was
conducted. The assay demonstrated good specificity, robustness (rRT=0.3%), precision (intra-day: 7.0–8.4%; inter-day: 9.9–12.9%),
excellent linearity (R>0.99) and an adequate lower limit of detection (<10 ng/mL). © 2015 The Authors. Drug Testing and Analysis
published by John Wiley & Sons, Ltd.
Keywords: doping; sport; mass spectrometry; monitoring program; HILIC; Mildronate
Introduction
New drug entities in preclinical and clinical trials as well as tradi-
tional drugs, genuinely studied and developed to treat serious dis-
eases, occasionally possess great potential for misuse in elite sports.
Some of these compounds are subject of active discussions on
various Internet platforms and, moreover, are commercially avail-
able but not (yet) prohibited in sports. In the last few years, a pro-
minent example for that scenario was xenon, a hypoxia-inducible
factor (HIF) activator, which was included in the World Anti-Doping
Agency (WADA) Prohibited List in September 2014.
[1–3]
In context of the extended method validation of a novel multi-
target screening assay based on high resolution/high accuracy
mass spectrometry, an interfering signal at m/z 147.1128 repeatedly
appeared in full-scan acquisition mode of selected authentic sports
drug testing specimens. As it was not observed inall urine samples,
an exogenous origin was suspected. The calculated molecular
formula of the unknown compound matched that of Mildronate
(Meldonium), an approved drug in several countries of Eastern
Europe with anti-ischemic properties, which was further in
accordance with several athlete statements on the sample control
form. The use of Mildronate was eventually confirmed by compa-
rison of high resolution/high accuracy mass spectrometric data of
the suspicious urine samples with a Mildronate reference standard.
Mildronate [3-(2,2,2-trimethylhydrazinium)propionate] was origi-
nally developed in the late 1970s as a growth-promoting agent for
* Correspondence to: Sven Guddat, Institute of Biochemistry - Center for Preventive
Doping Research, GermanSport University Cologne, Am Sportpark Müngersdorf 6,
50933 Cologne, Germany. E-mail: s.guddat@biochem.dshs-koeln.de
This is an open access article under the terms of the Creative Commons Attribution-
NonCommercial-NoDerivs License, which permits use and distribution in any
medium, provided the original work is properly cited, the use is non-commercial
and no modifications or adaptations are made.
aInstitute of Biochemistry - Center for Preventive Doping Research, German Sport
University Cologne, Am Sportpark Müngersdorf 6, 50933 Cologne, Germany
bEuropean Monitoring Center for Emerging Doping Agents (EuMoCEDA), Cologne/
Bonn, Germany
Drug Test. Analysis 2015,7, 973–979 © 2015 The Authors. Drug Testing and Analysis published by John Wiley & Sons, Ltd.
Research article
Drug Testin
g
and Anal
y
sis
Received: 3 February 2015 Revised: 20 February 2015 Accepted: 23 February 2015 Published online in Wiley Online Library: 5 April 2015
(www.drugtestinganalysis.com) DOI 10.1002/dta.1788
973
animals in the Latvian Institute of Organic Synthesis.
[4–6]
In recent
years, several studies and clinical trials identified Mildronate as an
effective anti-ischemic drug with multiple indications besides its
cardioprotective properties, including the treatment of neurode-
generative disorders, bronchopulmonary diseases and application
as an immunomodulator.
[7,8]
Most of Mildronate’s clinical benefits
are mediated through its modulation of the carnitine metabolism,
which is the essentialfactor in regulating thecellular energy metab-
olism through fatty acid β-oxidation and glycolysis in the myocar-
dium, as carnitine is the key molecule in fatty acid metabolism in
mitochondria. As an analogue of carnitine, Mildronate inhibits the
last step of carnitine biosynthesis by inhibition of γ-butyrobetaine
hydroxylase, which catalyzes the formation of L-carnitine from
γ-butyrobetaine (GBB). Furthermore, Mildronate inhibits the trans-
port of carnitine through the cell membranes of liver and kidneys
and reduces carnitine palmitoyl transferase-I (CPT-I) activity in the
outer mitochondrial membrane.
[9]
Under aerobic conditions, carnitine improves myocardial func-
tioning through enhancement of fatty acid β-oxidation that sup-
plies about 80% of myocardial ATP generation.
[8]
However,
under oxygen deficiency, cytotoxic intermediates can accumulate
in the cell due to insufficient oxygen supply. A reduced
intracellular concentration of free carnitine leads to suppression
of fatty acid metabolism and therefore enhances glycolysis
during ischemia, which has a cytoprotective effect and increases
the effectiveness of ATP-generation, as carbohydrate oxidation
requires less oxygen per ATP molecule than β-oxidation of free
fatty acids.
[10–14]
Moreover, glycolysis is stimulated directly via
Mildronate by increasing the expression of hexokinase type 1,
which catalyzes the formation of glucose-6-phosphate from
glucose.
[9]
Under sport-physiological aspects, reports on positive effects on
the physical working capacity of elite athletes were published and
dosages of Mildronate (per os between 0.25 and 1.0 g twice a day
over 2–3 weeks during the training period and 10–14 days before
competition) were discussed. Further studies demonstrated an in-
crease in endurance performance of athletes, improved rehabilita-
tion after exercise, protection against stress, and enhanced
activations of central nervous system (CNS) functions.
[15,16]
More-
over, Mildronate shows mood-improving effects as well as an in-
creased learning and memory performance, which are properties
athletes may also benefit from.
[8,17,18]
In dubious online shops, the
performance-enhancing effects of Mildronate are advertised
overtly.
[19]
Additionally, Mildronate-containing products can also
be obtained from well-known online auction platforms as an
over-the-counter (OTC) drug, which give easy access to the drug
worldwide.
Since January 2015, Mildronate is subject of WADA’s Monitoring
Program to assess its prevalence and misuse in sport, necessitating
methodologies capable of measuring and confirming the presence
and absence of this drug in human urine.
[20]
In recent years, differ-
ent assays were presented for the identification of Mildronate in hu-
man plasma and urine, while most of them were based on
hydrophilic interaction liquid chromatography-tandem mass spec-
trometry (HILIC-MS/MS).
[21–24]
In the present study, two approaches were established. One
aimed at the implementation of the analyte into an existing initial
test method to enable the detection at lowest possible additional
workload for the laboratory and another allowing the unequivocal
confirmation of findings using isotope-dilution hydrophilic interac-
tion liquid chromatography high resolution/high accuracy mass
spectrometry (HILIC-HRMS).
Experimental
Chemicals and reagents
1,1-Dimethylhydrazine, tert-butylhydroquinone, methyl-acrylate,
dimethylsulphate-D
6
, calcium hydroxide and the reference com-
pound of Mildronate dihydrate were obtained from Sigma-Aldrich
(Deisendorf, Germany), while Mildronate-D
3
was synthesized in
house for the use as internal standard (IS). Acetonitrile and acetone
were bought from VWR International (Darmstadt, Germany). Acetic
acid, ammonium acetate, and ethanol were supplied by Merck
(Darmstadt, Germany). Deionized water was obtained from a water
purification system (Sartorius Stedim Biotech S.A., Aubagne, France).
Synthesis of labelled IS
For in-house synthesis of Mildronate-D
3
(3-(2,2,2-trimethy-
lhydrazinium)propionate-D
3
) as IS, 37.9 g (0.63 mol) of 1,1-
dimethylhydrazine was added to 0.19 g (1.1 mmol) of stirred and
heated (50±5°C) tert-butylhydroquinone. To form the intermediate
product of 3-(2,2-dimethylhydrazino)methylpropionate, 51.6 g (0.6
mol) of methyl acrylate was added and the mixture was heated
up to 80±5°C for 2.5 hs. To stop the reaction process, the mixture
was cooled in ice bath, and subsequently, 100 mL of acetone and
hexadeuterated dimethylsulphate was added. Thereafter, the reac-
tion mixture was maintained at 50-60°C for 5 h. After removing the
solvent by distillation, the emulsive product of hexadeuterated
3-(2,2,2-trimethylhydrazinium)methylpropionate methylsulphate
was obtained. To remove the methylsulphate-D
3
residue,
500 mL of distilled water, 200 mL of ethanol (96%), and calcium
hydroxide (54.2 g, 0.73 mol) was added to the reaction
mixture. After heating at 50–60°C for 2 h the formed calcium
sulphate dehydrate precipitated and was removed by filtration.
The filtrate was concentrated under reduced pressure and
the 3-(2,2,2-trimethylhydrazinium)propionate-D
3
dihydrate was
obtained (Figure 1).
[25]
The synthesized internal standard,
Mildronate-D
3
was characterized using high resolution/high
accuracy mass spectrometry (Figure 2).
Sample preparation
For the initial testing assay, two aliquots of urine samples each with
45 μL were pooled and fortified with 10 μL of Mildronate-D
3
(IS,
conc. 1 μg/mL). After shaking, the mixture was injected into the in-
strument (reversed phase liquid chromatography-tandem mass
spectrometry; RPLC-MS/MS).
For confirmatory analysis, suspicious urine samples were diluted
appropriately using deionized water. Subsequently, an aliquot of
270 μL of the diluted urine sample was fortified with 30 μLinternal
standard (Mildronate-D
3
,conc.1μg/mL). The mixture was further
diluted with 700 μL of acetonitrile and 100 μL of a 100 mM ammo-
nium acetate solution. The samples were mixed and an aliquot of
20 μL was injected into the instrument. Mildronate concentrations
were estimated semi-quantitatively using a single-point calibrator
(Mildronate reference standard).
LC-MS/MS
Initial testing
For initial testing, an ion transition diagnostic for Mildronate was
implemented in a frequently used multi-target LC-MS/MS method
for the determination of diuretics, stimulants, masking agents,
C. Görgens et al.
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Analysis published by John Wiley & Sons, Ltd.
Drug Test. Analysis 2015,7, 973–979
974
SARMs, and others, which is based on direct injection of urine
samples.
[26]
In brief, a linear gradient with 5 mM ammonium acetate
buffer containing 0.1% glacial acetic acid (pH = 3.5, solvent A) and
acetonitrile (solvent B) on a Nucleodur C18 Pyramid analytical col-
umn (2 x 50 mm, 3 mm particle size; Macherey-Nagel, Düren,
Germany) was used. For mass spectrometric detection, a hybrid tri-
ple quadrupole/linear ion trap mass spectrometer (AB Sciex 5500
QTrap; Darmstadt, Germany) interfaced by an electrospray ioniza-
tion ion-source operating in both positive and negative ionization
mode was applied. The analyte and the internal standard
Mildronate-D
3
were detected utilizing multiple reaction monitoring
(MRM) of the diagnostic ion transitions at m/z 147→58 (Mildronate)
and at m/z 150→61 (Mildronate-D
3
)(CE=30eV,dwelltime=10ms).
Confirmatory analysis
To confirm suspicious initial testing results, a HILIC-HRMS approach
was developed. The method is based on direct injection of diluted
urine specimens followed by an effective online sample-clean up,
accomplished by a dual pump setup in combination with a HILIC
trapping column (Nucleodur HILIC, 20 x 2 mm, particle size 3 μm,
Macherey-Nagel, Düren, Germany) and a HILIC analytical column
(Nucleodur HILIC, 100 x 2 mm, particle size 1.8 μm, Macherey-Nagel,
Düren, Germany). The LC dual pump system consisted of an Agilent
1100 Series binary pump operating in isocratic mode and an Accela
1250 quaternary pump (Thermo Fisher Scientific, Bremen,
Germany) for gradient elution. For sample injection a Thermo PAL
autosampler (Thermo Fisher Scientific, Bremen, Germany) was uti-
lized. Both pumps operated at a flow rate of 250 μL/min. Mobile
phase was composed of deionized water (solvent A), acetonitrile
(solvent B) and a 200 mM ammonium acetate buffer containing
0.15% glacial acetic acid (pH = 5.5, solvent C). The Initial conditions
of 0% A, 95% B and 5% C were isocratically held for 1 min before
switching valve positions to backflush from trapping to analytical
column. The content of 5% C was maintained stable throughout
the gradient, while the content of solvent B was decreased linear
from 95% to 40% within 10 min. After 2 min of isocratic elution,
re-equilibration started for 5 min at initial mobile phase conditions.
The overall runtime was 18 min (injection-to-injection).
For the detection of Mildronate a Q Exactive hybrid quadrupol-
orbitrap® mass spectrometer (Thermo Fisher Scientific, Bremen,
Germany) interfaced by an electrospray ionization ion-source (ESI)
operating in positive mode (+4 kV, source temperature 300 °C, cap-
illary temperature 350 °C) was utilized. The positively charged pre-
cursor ion at m/z 147.1128 was detected using full-scan mode,
while the diagnostic product ions (m/z 58.0651, m/z 59.0730 and
m/z 132.0893) were detected using targeted higher energy collision
dissociation (t-HCD) acqusition mode with a resolution of 17,500
FWHM and an isolation window for the quadrupole of 1 Da. Auto-
mated gain control value was set to 2 ×10
5
with a maximum
allowed fill time of 100 ms. The applied normalized collision energy
was set to 50%. The collision gas utilized was nitrogen provided by
a CMC nitrogen generator (CMC Instruments, Eschborn, Germany).
Method validation
Method validation of the analytical approaches were carried out ac-
cording to WADA guidelines considering the parameters specificity,
intra- and inter-day precision at low, medium and high concentra-
tion levels (initial testing:1μg/mL, 10 μg/mL, 100 μg/mL; confirma-
tion:1μg/mL, 5 μg/mL, 10 μg/mL) linearity (initial testing: 0.5, 1.0,
5.0, 10.0, 50.0, 100.0 μg/mL; confirmation:1.0,2.5,5.0,7.5,10.0,
12.5 μg/mL), lower limit of detection (LLOD), ion suppression /
enhancement effects and robustness.
[27]
Routine doping control samples
Over a period of six months, analysis of a total of 8320 routine dop-
ing control samples (female/male/unknown: 2455/5846/19) from
elite athletes covering different classes of sport as well as in- and
out-of-competition samples (IC/OOC: 4459/3861) were analyzed
for the presence of Mildronate.
Results and discussion
Mass spectrometry
The MS behaviour of Mildronate andthe IS Mildronate-D
3
was stud-
ied using high resolution/high accuracy mass spectrometry. In
Figure 2, the t-HCD product ion spectra of the positively charged
precursor ions at m/z 147.1129 (C
6
H
15
N
2
O
2
,[M
+
]) and m/z
Figure 1. Synthesis of Mildronate-D
3
(3-(2,2,2-trimethylhydrazinium)propionate-D
3
)asinternalstandard.
Mildronate (Meldonium) in professional sports
Drug Testin
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and Anal
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Drug Test. Analysis 2015,7, 973–979 © 2015 The Authors. Drug Testing and
Analysis published by John Wiley & Sons, Ltd.
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975
150.1317 (C
6
H
12
N
2
O
2
D
3
,[M
+
]) are depicted. When subjected to a
normalized collision energy (NCE) of 50% the Mildronate precursor
ion dissociates leading to at least 3 product ions: m/z 58.0654
(C
3
H
8
N), m/z 59.0732 (C
3
H
9
N) and m/z 132.0894 (C
5
H
12
N
2
O
2
). The
most abundant product ions at m/z 58.0654 and m/z 59.0732 re-
sulted from the loss of the trimethylamine residue, while the only
additional fragment at m/z 132.0894 is formed by elimination of a
methyl group (-15 Da) from the precursor ion. The proposed
collision-induced fragmentation pathway of Mildronate could be
further confirmed by HRMS data of the IS Mildronate-D
3
. Here, for
the precursor ion as well as for the described product ions a 3 Da
shift of the mass-to-charge ratio was observed due to the substitu-
tion with a deuterated methyl group at the amino residue (m/z
150.1317 (C
6
H
12
N
2
O
2
D
3
,[M
+
]), m/z 61.0842 (C
3
H
5
ND
3
), m/z
62.0920 (C
3
H
6
ND
3
), m/z 135.1083 (C
5
H
12
N
2
O
2
D
3
). Moreover, for
the IS an additional fragment at m/z 60.0779 (C
3
H
6
ND
2
)wasde-
tected, according to proton/deuteron substitution. Our results,
based on accurate mass measurements, are in accordance with
proposed collision-induced fragmentation pathways of Mildronate
in recent publications.
[28,29]
Liquid chromatography
Since Mildronate is of comparably low molecular mass and
comprises a permanent charge, its retention on conventional
reversed-phase materials is modest. However, the sensitivity and
specificity of the applied initial analytical approach was sufficient
to allow screening for Mildronate-containing urine samples, espe-
cially in consideration of the recommended daily dose of a few hun-
dred to thousand mg per day. Due to Mildronate’shighlypolar
chemical structure (Figure 2), the analyte elutes at the very begin-
ning of the chromatographic run (RT: 0.52 min). Here, a deuterated
internal standard is necessarily recommended to compensate for
matrix effects. For confirmatory analysis, a more sophisticated chro-
matography using a HILIC stationary phase was established. In
Figure 3, theextracted ion chromatograms generated with the con-
firmatory HILIC-HRMS approach are shown, representing a blank
urine sample, a spiked urine sample containing Mildronate at
1μg/mL, and an authentic doping control urine sample found to
contain Mildronate. The data illustrate that the chosen methodology
provides the required chromatographic retention of the target
m/z
0
10
20
30
40
50
60
70
80
90
100
Relative Abundance
58.0654
147.1129
132.0894
C6H15O2N2
C5H12O2N2
C3H8N
59.0732
C3H9N
0
10
20
30
40
50
60
70
80
90
100
61.0842
150.1317
135.1083
C6H12O2N2D3
C5H9O2N2D3
62.0920
60.0779
C3H5ND3
C3H6ND3
C3H6ND2
0.8467 ppm
0.7696 ppm
3.7891 ppm
4.1838 ppm
3.5549 ppm
3.343 ppm
2.9878 ppm
-0.6836 ppm
0.6027 ppm
50 60 70 80 90 100 110 120 130 140 150 160 170
m/z
50 60 70 80 90 100 110 120 130 140 150 160 170
Relative Abundance
Figure 2. Product ion t-HCD mass spectra of the permanent positively charged Mildronate and Mildronate-D
3
(IS) precursor ions at m/z 147.1129 and m/z
150.1317 measured in a spiked urine sample by means of high resolution/high accuracy mass spectrometry using a Q Exactive hybrid quadrupole-orbitrap®
mass spectrometer (17,500 FWHM, NCE: 50%).
C. Görgens et al.
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y
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Analysis published by John Wiley & Sons, Ltd.
Drug Test. Analysis 2015,7, 973–979
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analyte and unambiguous mass spectrometric data for the identifi-
cation of the xenobiotic.
Method validation
The fitness-for-purpose of the employed analytical approaches for
the initial testing and confirmation of Mildronate was determined
according to WADA guidelines.
[27]
The results of the method valida-
tion are summarized in Table 1. Analysis of 10 different blank urine
specimens (5 female and 5 male) demonstrated good specificity
without any interfering signals for both assays and almost zero bio-
logical noise using high resolution/high accuracy mass spectromet-
ric detection. Furthermore, both assays allowed the reliable
detection of Mildronate in 10 different spiked urine specimens at
a concentration of 1 μg/mL with reproducible retention times.
Moreover, the confirmation assay provides stable product ion ratios
according to relevant WADA criteria.
[30]
Both approaches are char-
acterized by good intra- and inter-day precisions at low, medium
and high concentration levels (intra-day: initial testing: 5.9 –
12.3%; confirmation: 7.0 –8.4%; inter-day: initial testing: n.d. ; confir-
mation: 9.9 –12.9%).
The lower limit of detection (LLOD) was estimated either by mea-
suring the respective signal to noise ratio (S/N >3; initial testing) or
by estimation of the lowest concentration that could reliably be
detected in urine samples with reproducible RT and mass accuracy
of less than 5 ppm (confirmation assay). Regarding the adminis-
tered amount of up to 2000 mg per day and the half-life of around
6.5 h, the estimated LLOD at 200 ng/mL for the initial testing assay
9.0 10.0 11.0 12.0
Time (min)
RT: 10.61
RT: 10.61
RT: 10.61
RT: 10.61
RT: 10.61
9.0 10.0 11.0 12.0
Time (min)
RT: 10.65
RT: 10.65
RT: 10.65
RT: 10.65
9.0 10.0 11.0 12.0
Time (min)
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
RT: 10.63 RT: 10.66
blank urine spiked urine sample Mildronate containing urine
sample
m/z 147.1128
m/z 61.0848
IS
m/z 58.0651
m/z 59.0730
m/z 132.0893
m/z 147.1128
m/z 61.0848
IS
m/z 58.0651
m/z 59.0730
m/z 132.0893
m/z 147.1128
m/z 61.0848
IS
m/z 58.0651
m/z 59.0730
m/z 132.0893
Figure 3. Extracted ion chromatograms of Mildronate (m/z 147.1128; resolution: 17,500 FWHM, NCE: 50%) of a blank urine sample,a spiked urine sample at
1μg/mL and a diluted urine sample (142 μg/mL).
Table 1. Validation results
intra-day precision
(n = 6/6/6)
inter-day precision
(n = 18/18/18)
LLOD [ng/mL]
(n = 6)
ion suppression
(n = 6)
calibration curve robustness
(rRT, n = 6)
levels
[μg/mL]
CV [%] levels
[μg/mL]
CV [%]
initial testing <200 71 –93% slope: 1.0034 intercept:
3.5106 (R
2
= 0.9901)
1.7% 1.0 12.3 ——
10.0 5.9
100.0 10.9
confirmation <10 73 –97% slope: 0.4554 intercept:
0.0440 (R
2
= 0.9971)
0.3% 1.0 8.4 1.0 9.9
5.0 7.0 5.0 12.9
10.0 7.0 10.0 12.1
Mildronate (Meldonium) in professional sports
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and 10 ng/mL for the confirmation assay is suggested to be suffi-
cient for a reliable Mildronate detection over a couple of days after
administration.
[29]
Linearity was tested in the range between
0.5 μg/mL and 100 μg/mL (initial testing) as well as 1.0 μg/mL
and 12.5 μg/mL (confirmation), providing correlation coefficients
>0.99 between Mildronate concentration and signal response.
Ion suppression / enhancement effects were determined by
comparing six different urine samples spiked with 1 μg/mL of
Mildronate with matrix-free standard solutions at corresponding
concentrations. The matrix effect ranged from 71 to 93% (initial
testing) and from 73 to 97% (confirmation). Although the method
demonstrated sufficient robustness under these conditions, the
usage of the in-house synthesized Mildronate-D
3
as internal stan-
dard is recommended.
Routine doping control samples
A total of 8320 random doping control urine samples covering dif-
ferent classes of sport either from in- or out-of-competition were
analyzed for the presence of Mildronate. In total 182 positive
Mildronate findings (2.2%) in a concentration range between 0.1
and 1428 μg/mL were detected and confirmed using the employed
HILIC-HRMS assay. Mildronate was found in both in- and out-of-
competition samples (IC: 135 (74%); OOC: 47 (26%)), and noconsid-
erable gender specific differences regarding the number of findings
(female: 85 (47%); male: 97 (53%))(Figure 4) orthe mean Mildronate
concentrations (female: 120.9 μg/mL; male: 136.0 μg/mL) were ob-
served. Within the investigated classes of sport Mildronate findings
were found to a greater extend in samples from strength sports
(67%) and endurance sports (25%). However, the misuse of
Mildronate is not limited to a particular sport or to a group of sports
but is rather used in a wide range of sports. In the absence of further
information as to why the drug was (mis)used, the findings of high
Mildronate concentrations in samples originating not from so-
called ‘high-risk’sport disciplines are alarming.
Conclusion
Mildronate, an approved drug with multiple indications besides its
anti-ischemic properties, is known to have a positive effect on the
endurance performance of athletes, improves rehabilitation after
exercise, protect against stress and activates CNS functions. So
far, Mildronate is not part of WADA’s Prohibited List. Effects on
the human organism similar to those exhibited by the prohibited
substance Trimetazidine are given, as both substances cause a sig-
nificant inhibition of the β-oxidation of free fatty acids.
[1,31]
Origi-
nally included in Section S6.b of the WADA Prohibited List,
Trimetazidine was categorized under sub-section S4.5.3 in January
2015 due to its function of a metabolic modulator of the cardiac
metabolism.
Based on a growing body of evidence concerning Mildronate
misuse in sport and the inclusion of the substance into the 2015
WADA monitoring program, adequate test methods for both initial
testing and confirmation of the analyte were established. The mol-
ecule’s specific physico-chemical properties and the fact that the
drug is mostly excreted unchanged via renal route makes
Mildronate an ideal analyte for the presented ‘dilute-and-inject’
HILIC-HRMS approach. The reduced sample preparation, the flexi-
bility of the present approach and the compatibility of the initial
testing assay with existing analytical protocols enables the simple
implementation of Mildronate into screening assays of fellow
anti-doping laboratories for utmost comprehensive monitoring of
the substance’smisuse.
Furthermore, the present study indicates the wide prevalence of
Mildronate in international elite sports and demonstrates the
alarming extent of administered dosages, finding more than 180
cases of Mildronate use within numerous different sport disciplines
and urinary concentration levels of more than 1 mg/mL. Addition-
ally, the easy access to Mildronate from numerous online shops cer-
tainly plays an important role for the widespread use in
international elite sports. Under medical and pharmacological as-
pects as well as to preserve the integrity of sport the ban of
Mildronate from sport is deemed indicated.
Acknowledgements
This project was supported in part by funding from the Partnership
for Clean Competition Research Collaborative. The content of
this publication does not necessarily reflect the views or policies
of the Research Collaborative. Further support was received
from the Federal Ministry of the Interior of the Federal Republic
of Germany.
References
[1] World Anti-Doping Agency. The 2014 Prohibited List. Available at:
http://www.antidoping.cz/documents/WADA-Prohibited-List-2014-EN.
pdf [16 January 2015].
Figure 4. Mildronate findings in official doping control samples (n = 8320) and distribution between in- and out-of-competition samples (IC/OOC),
gender (f = female; m = male) and type of sports (team sports, endurance sports, strength sports, others)
C. Görgens et al.
Drug Testin
g
and Anal
y
sis
wileyonlinelibrary.com/journal/dta © 2015 The Authors. Drug Testing and
Analysis published by John Wiley & Sons, Ltd.
Drug Test. Analysis 2015,7, 973–979
978
[2] W. Jelkmann. Xenon misuse in sports –Increase of hypoxia-inducible
factors and erythropoietin, or nothing but “hot air“?Dtsch. Z.
Sportmed. 2014,65, 267.
[3] M. Thevis, T. Kuuranne, H. Geyer, W. Schänzer. Annual banned-
substance review: Analytical approaches in human sports drug
testing. Drug Test. Anal. 2015,7,1.
[4] S. Aronovich, A. Vasilevich, I. Yanovich, V. Gavrilovna, E. Amilovich,
Y. Yanovich. 3-(2,2,2-Trimethylhydrazine) propionate, method of
obtaining it and use //. World Patent No. 80/01068 [29 May 1980].
[5] S.A. Hiller, A.V. Jeremeev, I. Kalvinsh, V.G. Semenihina, E.E. Liepinsh, Y.
Y. Latvietis, A.A. Anderson, E.B. Astapenok, Y.Y. Spruzh, P.
T. Trapentsier, P.I. Podoprigora. Le 3-(triméthyl-2,2,2-hydrazinium)-3
propionate, procédé de préparation et application. Belgian Patent
No. 880831 (1980).
[6] A. Eremeev, I. Kalvinsh, V. Semenikhina, E.E. Liepinsh, Y.Y. Latvieti,
P.P. Anderson, E.B. Astapenok, Y.Y. Spruzh, P.T. Trapentsiers,
G.I. Podoprigora, S.A. Giller. 3-(2,2,2-Trimethylhydrazinium)
propionate and method for the preparation and use
thereof //. US Patent No. 4481218 [06 November 1984].
[7]B.Z.Simkhovich,Z.V.Shutenko,D.V.Meirena,K.B.Khagi,
R. J. Mezapuķe,T.N.Molodchina,I.J.Kalviņs, E. Lukevics. 3-(2,2,2-
trimethylhydrazinium) propionate (THP): A novel gammabutyrobetaine
hydroxylase inhibitor with cardioprotective properties. Biochem.
Pharmacol. 1988,37, 195.
[8] N. Sjakste, I. Kalvinsh. Mildronate: An Antiischemic drug with multiple
indications. Pharmacologyonline.2006,1,1.
[9] M. Dambrova, E. Liepinsh, I. Kalvinsh. Mildronate: Cardioprotective
action through carnitine-lowering effect. Trends Cardiovasc. Med.
2002,12, 275.
[10] E. Liepinsh, I. Kalvinsh, M. Dambrova. The regulation of energy
metabolism pathways through L carnitine homeostasis. in Role of the
Adipocyte in Development of Type 2 Diabetes, (Ed: D.C. Croniger),
Intech, Latvia, 2011, pp. 107.
[11] Y. C. Hwang, S. Bakr, R. Ramasamy, S. R. Bergmann. Relative importance
of enhanced glucose uptake versus attenuation of long-chain acyl
carnitines in protecting ischemic myocardium. Coron. Artery Dis.
2002,13, 313.
[12] R. Lerch, C. Tamm, I. Papageorgiou, R. H. Benzi. Myocardial fatty acid
oxidation during ischemia and reperfusion. Mol. Cell. Biochem. 1992,
116, 103.
[13] H. Rupp, A. Zarain-Herzberg, B. Maisch. The use of partial fatty acid
oxidation inhibitors for metabolic therapy of angina pectoris and
heart failure. Herz.2002,27, 621.
[14] R. S. Schofield, J. A. Hill. Role of metabolically active drugs in the
management of ischemic heart disease. Am. J. Cardiovasc. Drugs.
2001,1, 23.
[15] Z. Kakhabrishvili, N. Chabashvili, V. Akhalkatsi, T. Skhirtladze,
T. Chutkerashvili. Mildronate effect on physical working capacity
among highly qualified judokas. Ann. Biomed. Res. Edu. 2002,2, 551.
[16] M. Dzintare, I. Kalvins. Mildronate increases aerobic capabilities of
athletes through carnitine-lowering effect. Curr. Issues New Ideas Sport
Sci. 2012,5, 59.
[17] L. Stoenica, O. Senkov, R. Gerardy-Schahn, B. Weinhold, M. Schachner,
A. Dityatev. In vivo synaptic plasticity in the dentate gyrus of mice
deficient in the neural cell adhesion molecule NCAM or its polysialic
acid. Eur. J. Neurosci. 2006,23, 2255.
[18] V. Klusa, U. Beitnere, J. Pupure, S. Isajevs, J. Rumaks, S. Svirskis,
Z. Dzirkale, I. Kalvinsh. Mildronate and its neuroregulatory
mechanisms: Targeting the mitochondria, neuroinflammation, and
protein expression. Medicina (Kaunas).2013,49, 301.
[19] Melodian Olainfarm. [Website]. Available at: http://meldoniumolain
farm.lv/?valoda=en [19 February 2015].
[20] World Anti-Doping Agency. The 2015 Monitoring Program. Available
at: https://wada-main-prod.s3.amazonaws.com/resources/files/wada-
2015-monitoring-program-en.pdf [16 January 2015].
[21] Y. V. Pidpruzhnykov, V. E. Sabko, V. V. Iurchenko, I. A. Zupanets. UPLC-
MS/MS method for bioequivalence study of oral drugs of meldonium.
Biomed. Chromatogr. 2012,6, 599.
[22] J. Hmelnickis, O. Pugovics, H. Kazoka, A. Viksna, I. Susinskis, K. Kokums.
Application of hydrophilic interaction chromatography for simultaneous
separation of six impurities of mildronate substance. J. Pharmaceut.
Biomed. 2008,48,649.
[23] Y. F. Lv, X. Hu, K. S. Bi. Determination of mildronate in human plasma
and urine by liquid chromatography-tandem mass spectrometry.
J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2007,852, 35.
[24] O. Sahartova, V. Shatz, I. Kalvins. HPLC analysis of mildronate and its
analogs in plasma. J. Pharmaceut. Biomed. 1993,11, 1045.
[25] D. Zicane, M. Turks. New process for the preparation of 3-(2,2,2-
Trimethylhydrazinium) propionate dihydrate. EU Patent No. 2 128
126 A1 [2 December 2009].
[26] S.Guddat,E.Solymos,A.Orlovius,A.Thomas,G.Sigmund,H.Geyer,
M. Thevis, W. Schänzer. High-throuput screening for various classes of
doping agents using a new ‘dilute-and-shoot’liquid chromatography-
tandem mass spectrometry multi-target approach. Drug Test. Anal.
2011,3,836.
[27] World Anti Doping Agency. International Standard for Laboratories.
Available at: https://wada-main-prod.s3.amazonaws.com/resources/
files/WADA-ISL-2015-Final-v8.0-EN.pdf [16 January 2015].
[28] L. J. Cai, J. Zhang, W. X. Peng, R. H. Zhu, J. Yang, G. Cheng, X. M. Wang.
Determination of mildronate in human plasma and urine
by UPLC-positive ion electrospray tandem mass spectrometry.
Chromatographia.2011,73, 659.
[29] Y. Peng, J. Yang, Z. Wang, J. Wang, Y. Liu, Z. Luo, A. Wen.
Determination of mildronate by LC–MS/MS and its application to a
pharmacokinetic study in healthy Chinese volunteers. J. Chromatogr.
B.2010,878, 551.
[30] World Anti-Doping Agency. Technical Document –TD2010IDCR. Avail-
able at: https://wada-main-prod.s3.amazonaws.com/resources/files/
WADA_TD2010IDCRv1.0_Identification%20Criteria%20for%20Qualita-
tive%20Assays_May%2008%202010_EN.doc.pdf [29 January 2015].
[31] World Anti-Doping Agency. The 2015 Prohibited List. Available at:
http://www.fia.com/sites/default/files/basicpage/file/20141224/wada-
2015-prohibited-list-en.pdf [16 January 2015].
Mildronate (Meldonium) in professional sports
Drug Testin
g
and Anal
y
sis
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Analysis published by John Wiley & Sons, Ltd.
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