Nutritional supplements cross-contaminated and faked with doping substances

Article (PDF Available)inJournal of Mass Spectrometry 43(7):892-902 · July 2008with619 Reads
DOI: 10.1002/jms.1452 · Source: PubMed
Abstract
Since 1999 several groups have analyzed nutritional supplements with mass spectrometric methods (GC/MS, LC/MS/MS) for contaminations and adulterations with doping substances. These investigations showed that nutritional supplements contained prohibited stimulants as ephedrines, caffeine, methylenedioxymetamphetamie and sibutramine, which were not declared on the labels. An international study performed in 2001 and 2002 on 634 nutritional supplements that were purchased in 13 different countries showed that about 15% of the nonhormonal nutritional supplements were contaminated with anabolic-androgenic steroids (mainly prohormones). Since 2002, also products intentionally faked with high amounts of ‘classic’ anabolic steroids such as metandienone, stanozolol, boldenone, dehydrochloromethyl-testosterone, oxandrolone etc. have been detected on the nutritional supplement market. These anabolic steroids were not declared on the labels either. The sources of these anabolic steroids are probably Chinese pharmaceutical companies, which sell bulk material of anabolic steroids. In 2005 vitamin C, multivitamin and magnesium tablets were confiscated, which contained cross-contaminations of stanozolol and metandienone. Since 2002 new ‘designer’ steroids such as prostanozol, methasterone, androstatrienedione etc. have been offered on the nutritional supplement market. In the near future also cross-contaminations with these steroids are expected. Recently a nutritional supplement for weight loss was found to contain the β2-agonist clenbuterol. The application of such nutritional supplements is connected with a high risk of inadvertent doping cases and a health risk. For the detection of new ‘designer’ steroids in nutritional supplements, mass spectrometric strategies (GC/MS, LC/MS/MS) are presented. Copyright
JOURNAL OF MASS SPECTROMETRY
J. Mass Spectrom. 2008; 43: 892902
Published online 19 June 2008 in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/jms.1452
Review
Nutritional supplements cross-contaminated and faked
with doping substances
Hans Geyer,
1
Maria Kristina Parr,
1
Karsten Koehler,
2
Ute Mareck,
1
Wilhelm Sch
¨
anzer
1
and Mario Thevis
1
1
Center for Preventive Doping Research, German Sport University Cologne, Am Sportpark M
¨
ungersdorf 6, 50933 Cologne, Germany
2
German Research Centre of Elite Sports, German Sport University Cologne, Am Sportpark M
¨
ungersdorf 6, 50933 Cologne, Germany
Received 11 April 2008; Accepted 14 May 2008
Since 1999 several groups have analyzed nutritional supplements with mass spectrometric methods
(GC/MS, LC/MS/MS) for contaminations and adulterations with doping substances.
These investigations showed that nutritional supplements contained prohibited stimulants as
ephedrines, caffeine, methylenedioxymetamphetamie and sibutramine, which were not declared on
the labels. An international study performed in 2001 and 2002 on 634 nutritional supplements that were
purchased in 13 different countries showed that about 15% of the nonhormonal nutritional supplements
were contaminated with anabolic-androgenic steroids (mainly prohormones). Since 2002, also products
intentionally faked with high amounts of ‘classic’ anabolic steroids such as metandienone, stanozolol,
boldenone, dehydrochloromethyl-testosterone, oxandrolone etc. have been detected on the nutritional
supplement market. These anabolic steroids were not declared on the labels either. The sources of these
anabolic steroids are probably Chinese pharmaceutical companies, which sell bulk material of anabolic
steroids. In 2005 vitamin C, multivitamin and magnesium tablets were confiscated, which contained cross-
contaminations of stanozolol and metandienone. Since 2002 new ‘designer’ steroids such as prostanozol,
methasterone, androstatrienedione etc. have been offered on the nutritional supplement market. In the
near future also cross-contaminations with these steroids are expected. Recently a nutritional supplement
for weight loss was found to contain the b2-agonist clenbuterol. The application of such nutritional
supplements is connected with a high risk of inadvertent doping cases and a health risk. For the detection
of new ‘designer’ steroids in nutritional supplements, mass spectrometric strategies (GC/MS, LC/MS/MS)
are presented. Copyright 2008 John Wiley & Sons, Ltd.
KEYWORDS: sport; doping; nutritional supplements; GC/MS; LC/MS/MS; designer steroids
INTRODUCTION
The nutritional supplement market has been growing
continuously in the last decades. For 2006, the world wide
market has been estimated at more than US$ 60 billion.
1
In
athletes, the use of supplements is more widespread than
in the general population.
2,3
For elite athletes, figures of
supplement are in the range of 44 and 100%, even though
the prevalence greatly depends on type of sport, age, gender,
and level of competition.
3–6
Some nutritional supplements contain doping sub-
stances, which are declared on the labels. Especially products
marketed as fat burners or activating agents may contain
caffeine (doping substance till 2004) and/or ephedrines.
Whereas the use of caffeine is no longer banned since 2004
Ł
Correspondence to: Hans Geyer, Center for Preventive Doping
Research, German Sport University Cologne, Am Sportpark
M
¨
ungersdorf 6, 50933 Cologne, Germany.
E-mail: h.geyer@biochem.dshs-koeln.de
by the World Anti-Doping Agency (WADA), the use of
ephedrines is prohibited during competition.
7
On the labels
of these products are declared caffeine or ephedrines or their
natural sources. The natural sources are e.g. Guarana and
Ma Huang and ephedra sinica, respectively.
Since 1996 special anabolic androgenic steroids, so-
called prohormones, are legally available on the sports
nutrition market. These substances are advertised to have
enormous properties to increase muscle growth and strength.
According to the doping regulations of the WADA, these
substances belong to the prohibited class of anabolic
agents.
7
The most common prohormones on the market
are the prohormones of testosterone and nandrolone.
8
The
application of a single recommended dose of a nandrolone
prohormone (norandrostendione) may lead to positive
doping findings for the main metabolite norandrosterone
for more than ten days.
8
Several studies have shown that the labelling of
prohormone supplements did not reflect their actual
Copyright 2008 John Wiley & Sons, Ltd.
Nutritional supplements and doping substances 893
content. Many prohormone products contained prohor-
mones as well as concentrations different from those
declared on the labels.
9–13
These mislabelling problems
indicated an insufficient surveillance and quality con-
trol of dietary supplements and a production, which did
not follow Good Manucaturing Practices (GMP) condi-
tions.
On the basis of this knowledge, rumours came up that
nutritional supplements such as vitamins, minerals, and
amino acids may also contain doping substances not declared
on the labels and leading to inadvertent positive doping
cases. Therefore several groups started to analyse nutritional
supplements with mass spectrometric methods (GC/MS,
LC/MS/MS) for nondeclared doping substances.
NUTRITIONAL SUPPLEMENTS
CONTAMINATED AND FAKED WITH
STIMULANTS
First reports on nutritional supplements containing prohib-
ited stimulants, which were not declared on the product
label, were published in 2002. Prior to the Salt Lake City
Olympic Games, 14 of 69 products selected by Dutch
Olympic athletes contained unlabelled stimulants. In twelve
products caffeine was found, three supplements contained
ephedrine. Additionally, one supplement was found to con-
tain methylenedioxymetamphetamie (MDMA).
14,15
These results were confirmed by a similar study by
Parr et al. Out of 110 supplements from the international
market, 12.7% contained caffeine. Furthermore ephedrine
and analogs (pseudoephedrine, methylephedrine) were
detected in two products. In all cases, there was no indication
of the stimulant content on the product label.
16
The use of caffeine is no longer banned by the World
Anti-Doping Agency (WADA) since 2004 but the use
of ephedrines and its analogs and MDMA is prohibited
during competition.
7
Besides the apparent doping risk of
supplements containing banned stimulants, the potential
health risks should not be neglected. Due to structural
similarities with amphetamine, ephedrine exhibits similar
modes of action and side effects (restlessness, tremor,
cardiovascular problems, disturbance of thermoregulation
etc.).
Recently several slimming products adulterated with the
anorectic drug sibutramine appeared on the market. Only
since 2006 sibutramine is on the list of prohibited substances
of the World Anti-Doping Agency. In at least three cases,
sibutramine was detected in ‘pure herbal’ Chinese slimming
capsules.
17–19
Additionally, the same compound was also
found in Chinese slimming tea, which was labelled to be
of solely natural content.
19
Even 50 h following a single
administration of the tea, sibutramine metabolites were
clearly detectable in a volunteer’s urine (Fig. 1) and would
have caused a failed doping test.
It should also be considered that severe side effects such
as increased blood pressure and heart rate may occur and
require regular monitoring of patients using sibutramine.
20
Figure 1. Urinary excretion of sibutramine metabolites
following the consumption of faked Chinese slimming tea
19
sibutramine-M1: desmethyl sibutramine, sibutramine-M2:
bisdesmethyl sibutramine.
NUTRITIONAL SUPPLEMENTS
CONTAMINATED WITH PROHORMONES
In 1999 in connection with follow up studies of posi-
tive doping cases with norandrosterone, the first cases of
nutritional supplements cross-contaminated with anabolic
androgenic steroids (prohormones) were detected.
21
In the
following year further nutritional supplements with non-
declared anabolic androgenic steroids were detected in
connection with positive doping cases.
22
In Fig. 2 are shown
results of the GC/MS analysis of a creatine product contain-
ing seven different prohibited anabolic androgenic steroids
(prohormones) and testosterone not declared on the label.
An international study performed in 2001 and 2002 on 634
nutritional supplements purchased in 13 different countries
showed that about 15 percent of nonhormonal nutritional
supplements, such as minerals, vitamins, proteins, creatine
etc. contained anabolic androgenic steroids (mainly pro-
hormones) not declared on the label.
23,24
In Table 1 the
percentages of nutritional supplements cross-contaminated
with anabolic androgenic steroids (prohormones) in different
countries in 2001/2002 are presented.
On the basis of the very low and varying concentra-
tions of the prohormones their presence was interpreted as
cross-contaminations and not as intentional admixtures. The
reason for the cross-contaminations was most probably the
fact that manufacturers of prohormones (legally marketed
as nutritional supplements in the USA till 2004) also man-
ufactured other nutritional supplements such as vitamins
on the same production line without a sufficient cleaning of
the production line. Another source of cross-contaminations
could be unclean transport containers from raw material
suppliers of prohormones. The consumption of such con-
taminated nutritional supplements can lead to inadvertent
positive doping cases.
24
These results were confirmed by
several other investigations.
25–27
Copyright 2008 John Wiley & Sons, Ltd. J. Mass Spectrom. 2008; 43: 892902
DOI: 10.1002/jms
894 H. Geyer et al.
Figure 2. SIM-chromatograms of a creatine product with seven prohormones and testosterone not declared on the label (all steroids
are pertrimethylsilylated)
22
5-nordiol: 19-nor-5-androstene-3ˇ,17ˇ-diol, 4-nordiol: 19-nor-4-androstene-3ˇ,17ˇ-diol, DHEA:
dehydroepiandrosterone, 4-nordion: 19-nor-4-androstene-3,17-dione, 4-adiol: 4-androstene-3ˇ,17ˇ-diol, 5-adiol:
5-androstene-3ˇ,17ˇ-diol, 4-adion: 4-androstene-3,17-dione, test: testosterone.
Table 1. Nutritional supplements cross-contaminated with
anabolic-androgenic steroids (prohormones) in different
countries in 2001/2002
23,24
Country
No. of
analyzed
products
No. of cross-
contaminated
products
Percent
age of cross-
contaminated
products
Netherlands 31 8 25.8
Austria 22 5 22.7
UK 37 7 18.9
USA 240 45 18.8
Italy 35 5 14.3
Spain 29 4 13.8
Germany 129 15 11.6
Belgium 30 2 6.7
France 30 2 6.7
Norway 30 1 3.3
Switzerland 13
Sweden 6
Hungary 2
Since that time athletes have been warned by their
federations, information systems have been established
and legislation towards anabolic androgenic steroids as
nutritional supplements has been changed, e.g. by the
Anabolic Steroid Act 2004 in the USA.
NUTRITIONAL SUPPLEMENTS FAKED WITH
HIGH AMOUNTS OF ‘CLASSIC’ ANABOLIC
ANDROGENIC STEROIDS
Since 2002 nutritional supplements have been detected,
which probably were intentionally faked with high amounts
(higher than 1 mg/g) of ‘classic’ anabolic steroids like metan-
dienone, stanozolol, boldenone, oxandrolone, dehydrochlo-
romethyl-testosterone etc. The anabolic steroids are not
declared or declared with nonapproved or fancy names
on the labels. In Table 2 some of these faked nutritional
supplements are listed.
All anabolic steroids found in these faked supplements
are prescription drugs, which are freely available on the
nutritional supplement market. The supplements listed in
Table 2 can be purchased by telephone order and is deliv-
ered by ordinary mail. The consumption of the recommended
dose leads to an administration of supratherapeutic doses
of these steroids. The faked supplements can be purchased
without restrictions by everyone, including women, adoles-
cents, and children. Especially for these groups the use of
these steroids is associated with a large number of adverse
effects. Known symptoms after intake include abnormal
liver function, menstrual disorders, virilization, gynecomas-
tia, severe psychological or psychiatric disorders, and an
increased risk of cardiovascular disease. Long-term applica-
tion of large doses has been shown to result in dependence,
associated with an acute withdrawal syndrome. The use
of these ‘classic’ anabolic steroids in children can lead to
virilization and premature closure of the epiphyses with a
derogation of linear growth. The application can also result
in irreversible adverse effects. In women these effects are
deepening of the voice and growth of the clitoris. During
pregnancy the use of such steroids may result in a viriliza-
tion of the fetus. Due to the fact that most of these steroids
are 17-methylated compounds, their use is associated with a
high-liver toxicity and carcinogenicity.
8,34
With regard to the supratherapeutic doses additional
health risks can be expected.
In addition to enormous health risks the use of the
analyzed supplements can lead to positive doping results.
Copyright 2008 John Wiley & Sons, Ltd. J. Mass Spectrom. 2008; 43: 892–902
DOI: 10.1002/jms
Nutritional supplements and doping substances 895
Table 2. Nutritional supplements faked with ‘classic’ anabolic androgenic steroids since 2002
Product name
(pharmaceutical form) Company Declared ingredients
Not declared ‘classic’ anabolic
androgenic steroids and references
Stanozolon II (powder) Pharmtec AD-4-Complex nutrients, MetX synergistic blend, 1-T
matrix, creatine monohydrate, thermogenic proprietary
blend, ribose- 4 matrix
Metandienone 4 mg/serving (10 g
powder)
28
Parabolon (powder) Pharmtec AD-4-Complex nutrients, MetX synergistic blend, 1-T
matrix, creatine monohydrate, thermogenic proprietary
blend, ribose-4 matrix
Metandienone 10 mg/serving (10 g
powder)
29–31
Met-AD17-diol (capsules) Pharmtec Met-AD17-diol/1-T matrix Metandienone ca.9 mg/capsule
29–31
Stanozolon-S (fizzy tablets) Senesco-Pharma Several prohormones Stanozolol ca. 15 mg/tablet, Boldenone,
DHT
32
Parabolon-S (fizzy tablets) Senesco-Pharma Several prohormones Metandienone ca.17mg/tablet
32
OXA 17-DION (capsules) Muscle Inc. Ltd, Cyprus 5 alpha-androstan-2-oxa-17 alpha-methyl-17,
beta-ol-3-one
Oxandrolone ca.25mg/capsule
33
TESTEXX (capsules) Muscle Inc. Ltd, Cyprus 1,4-Androstadien-4-chloro-17˛-methyl-17ˇ-ol-3-one Dehydrochloro-methyltestosterone ca.
25 mg/capsule
33
Primo 17-acetoxy (capsules) Muscle Inc. Ltd, Cyprus 17ˇ-Acetoxy-1-methyl-5-alpha-androst-1-en-3-one Metenolone acetate ca.50mg/capsule
33
Parabolon-B (blades) Pharm-Tec P-Blade 17Tren-1.4dien-3-one Beta-Compound Metandienone ca.10mg/blade
33
Stanozolon-B (blades) Pharm-Tec S-Blade 5-Alpha Pyrazol-Compound Stanozolol ca.10mg/blade
33
Copyright 2008 John Wiley & Sons, Ltd. J. Mass Spectrom. 2008; 43: 892902
DOI: 10.1002/jms
896 H. Geyer et al.
Table 3. Chinese companies which offer anabolic androgenic steroids via internet
Company Internet address Online
Tianijn Xinmei Technology Cooperation http://www.worldpharm.com.cn/xinmei/english/hormones.htm September 2006
Zyf Pharm Chemical http://zyongfu.3322.net/ September 2006
Xianju Green Leaf Pharmaceutical Factory http://www.greenleafpharm.com/ September 2006
The sources of these anabolic steroids are probably Chi-
nese pharmaceutical companies, which offer bulk material of
numerous anabolic androgenic steroids via the internet. The
knowledge of China as origin of bulk material of anabolic
steroids is mainly based on our own experience. After pur-
chase of bulk material, shipment tracking led to Chinese
towns as origin of the shipment. In Table 3 some inter-
net addresses of Chinese suppliers of anabolic androgenic
steroids are presented. Probably criminal nutritional supple-
ment companies buy anabolic steroids from such sources
and admix them to their supplements to make them more
effective.
Because the manufacturers ofthesefakedproductsalso
manufacture other nutritional supplements on the same
production line, the risk of cross-contaminations with such
‘classic’ anabolic androgenic steroids is very high. In the
following chapter cases of such cross-contaminations are
presented.
VITAMINS AND MINERALS CONTAMINATED
WITH ‘CLASSIC’ ANABOLIC ANDROGENIC
STEROIDS
At the end of 2005, a German drug surveillance authority con-
fiscated nutritional supplements faked with high amounts of
metandienone and stanozolol from a German manufacturer.
The names of the products were Parabolon-S and Stanozolon-
S from Pharm-Tec. The analyses of vitamin C, multivitamin,
and magnesium tablets, which were produced from the same
manufacturer on the same production line within the same
time interval showed cross-contaminations of these products
with metandienone and stanozolol (Table 4). Metandienone
at a concentration of 50 ng/g corresponding to 0.2
µg/tablet
was detected in vitamin C tablets and stanozolol at concen-
trations of 540 ng/g corresponding to 0.06–0.15
µg/tablet
was detected in multivitamin and magnesium tablets.
35
These vitamin C, multivitamin and magnesium tablets
were sold in ordinary German and Spanish groceries and
drug stores.
35
The consumption of such cross-contaminated
nutritional supplements could lead to inadvertent positive
doping cases, taking into account long-term application, indi-
vidual differences in metabolism and varying concentrations
of the contaminants. On the basis of the same considerations
health risks for young people and females who consume
these vitamins and minerals as nutritional supplements also,
cannot be excluded.
NUTRITIONAL SUPPLEMENTS WITH NEW
‘DESIGNER’ STEROIDS
Since 2002 nutritional supplements with new ‘designer’
steroids such as prostanozol, methasterone, andostatriene-
dione etc. have been offered on the nutritional supplement
market via the internet. In Table 5 are listed such new find-
ings and the corresponding references.
These steroids are neither listed as ingredient in any
currently available medicament nor do their names appear
in the WADA list of prohibited substances. They are only
produced for the nutritional supplement market and are
advertised as anabolic steroids or aromatase inhibitors.
About effects and side effects of these steroids only little
knowledge exists. The legal status of these supplements is not
clear. With these ‘new’ steroids it is possible to circumvent the
Anabolic Steroid Control Act 2004.
53
According to this act
the selling of anabolic steroids as nutritional supplements
is prohibited in the USA, however these new steroids
are not on the list of banned steroids.
53
According to
Table 4. Nutritional supplements from the German and Spanish market cross-contaminated with ‘classic’ anabolic steroids
35
Product Market Expiry date Tube Cross-contaminations
Vitamin C Germany July 2005 Tube 1 Metandienone ca 50 ng/g
Tube 2 Metandienone ca 50 ng/g
Calcium Germany July 2005 Tube 1 Negative
Tube 2 Negative
Magnesium C Vitamin C Germany January 2006 Tube 1 Negative
Tube 2 Negative
Tube 3 Negative
Multivitamins Spain July 2006 Tube 1 Stanozolol ca 10 ng/g
Tube 2 Stanozolol ca 15 ng/g
Tube 3 Stanozolol ca 10 ng/g
Magnesium Germany January 2005 Tube 1 Stanozolol ca 40 ng/g
Tube 2 Stanozolol ca 40 ng/g
Tube 3 Stanozolol ca 5ng/g
Copyright 2008 John Wiley & Sons, Ltd. J. Mass Spectrom. 2008; 43: 892–902
DOI: 10.1002/jms
Nutritional supplements and doping substances 897
Table 5. New steroids detected in nutritional supplements since 2002
Chemical name (IUPAC) Trivial name References
17ˇ-Hydroxy-2˛,17˛-dimethyl-5˛-androstan-3-one Methasterone 3640
17ˇ-Hydroxy-17˛-methyl-5˛-androst-1-en-3-one Methyl-1-testosterone 36
4-Hydroxyandrost-4-ene-3,17-dione Formestane 4143
4,17ˇ-Dihydroxyandrost-4-ene-3-one 4-Hydroxytestosterone 4143
5˛-Androstane-3ˇ,17˛-diol 44
Androst-4-ene-3ˇ,17˛-diol 44
5ˇ-Androst-1-ene-3ˇ,17ˇ-diol 44
5ˇ-Androst-1-ene-3˛,17ˇ-diol 44
17ˇ-Hydroxy-5˛-androstano-[3,2-c]-pyrazol Prostanozol 38, 40
6˛-Methylandrost-4-ene-3,17-dione 6˛-Methylandrostendione 38, 40, 45
3ˇ-Hydroxy-5ˇ-androstan-17-one Epietiocholanolone 40
17ˇ-Hydroxy-17˛-methyl-5ˇ-androstan-3-one 5ˇ-Mestanolone 40
17˛-Methyl-5˛-androst-2-en-17ˇ-ol Desoxymethyltestosterone 40
4-Chloro-17˛-methylandrost-4-ene-3,17ˇ-diol 39
Androst-4-ene-3,6,17-trione 6-Oxo-androstendione 46, 47
Androsta-1,4,6-trien-3,17-dione Androstatrienedione 33, 48, 49
3ˇ-Hydroxyandrost-4-ene-7,17-dione 7-Keto-dehydroepiandrosterone 50
6-Bromandrost-4-ene-3,17-dione 51
17˛-Methyl-5˛-androstane-3˛,17ˇ-diol 51
17ß-Hydroxy-5˛-androstano-[3,2-c]-isoxazol 52
17ß-Hydroxy-5˛-androstano-[2,3-d]-isoxazol 52
Estra-4,9-diene-3,17-dione 52
8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.
2000
4000
6000
8000
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
Time
Abund.
TI
6-Oxo-androstenedione
metabolites
Boldenone-
metabolite
Androsta-
trienedione
Figure 3. TIC Chromatogram of a doping control sample containing metabolites of the ‘new’ steroids androstatrienedione and
6-oxo-androstendione.
33
European legislation these products have to be classified
as nonlicensed pharmaceuticals even if they are marketed as
nutritional supplements. In sports these new steroids belong
to the prohibited classes of anabolic androgenic steroids or
aromatase inhibitors.
7
In most cases the labeling of these products contains
nonapproved or fancy names of the steroids.
The sources of the new anabolic steroids are not
known. Most probably Chinese pharmaceutical companies
are involved because some of the new steroids detected in
nutritional supplements were offered on their product lists
on the internet.
The use of such nutritional supplements with new
steroids by athletes is shown in the following examples.
In September 2006 in several urine samples of athletes,
unknown signals were observed, raising concerns about
their relevance for doping control. The mass spectrometric
analyses of these signals identified them as metabolites
of androstatrienedione and 6-oxo-androstendione (Fig. 3).
The results were attributed to a consumption of the
nutritional supplement Novedex Xtreme from Gaspari,
which is advertised as testosterone booster and aromatase
inhibitor. This product contained androstatrienedione and
6-oxo-androstendione.
33
Another case is presented in Fig. 4. In a doping control
sample was detected hitherto an unknown signal which
was identified as metabolite of the steroids 6˛-methyl-
androstenedione. Additionally an increased ratio of the
endogenous steroids testosterone and epitestosterone was
detected. This result was attributed to the administration of
Copyright 2008 John Wiley & Sons, Ltd. J. Mass Spectrom. 2008; 43: 892902
DOI: 10.1002/jms
898 H. Geyer et al.
8.
9. 10. 11. 12. 13. 14. 15. 16. 17.
9. 10. 11. 12. 13. 14. 15. 16. 17.
5000
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
Time
Abund.
TIC
20
0
60
100
140
180
220
260
300
340
380
420
460
500
Time
Abund.
m/z 432
6α-Methylandrostendione-
metabolite
Testosterone
Epitestosterone
Figure 4. TIC and single-ion chromatograms of a doping control sample containing metabolites of the new steroid
6-alpha-methyl-androstenedione (upper trace) and showing an increased testosterone/epitestosterone ratio (lower trace).
33
the nutritional supplement methyl-1-pro from Proline, which
contains the ‘new’ steroid 6-alpha-methyl-androstenedione.
Additionally this supplement contains the prohormone
androstenedione. As for the steroids in the Novedex product
the 6-˛-methyl-androstenedione is neither listed on the
prohibited list of the WADA nor as ingredient in any
currently available medicament and is only produced for
the nutritional supplement market.
33
As the manufacturers of these products also manufacture
other nutritional supplements on the same production line,
cross-contaminations of ‘normal’ nutritional supplements
with ‘new’ steroids can be expected possibly causing
inadvertent doping in the near future.
NUTRITIONAL SUPPLEMENTS WITH THE
b2-AGONIST CLENBUTEROL
Recently nutritional supplements for weight loss, labeled to
contain as active ingredient ‘30
µg Clenbuterol Enetat’ per
tablet, appeared on the market. After the GC/MS analysis
the tablets were found to contain clenbuterol in an estimated
amount of 30
µg per tablet.
54
The ˇ2-agonist clenbuterol is
only legally available on prescription and is classified as
prohibited doping substance in sports.
7
After administration
of one tablet, clenbuterol was detected in a urine sample,
collected 3 h after ingestion, in a concentration of 2 ng/ml.
54
Table 6. Characteristic fragment ions of selected trimethylsilylated steroids using electron ionization
Steroid nucleus Representative compound
Common ions
(m/z)
3-Keto 5˛-Dihydrotestosterone-bis-TMS 143 142
3-Keto-4-ene Testosterone-bis-TMS (androsta-3,5-dien-3,17ˇ-diol-bis-O-TMS) 208 129
Testosterone-bis-TMS (androsta-2,4-dien-3,17ˇ-diol-bis-O-TMS) 194 129
Nandrolone-bis-TMS (estra-3,5-dien-3,17ˇ-diol-bis-O-TMS) 194 129
3-Keto-1-ene 1-Testosterone (androsta-1,3-diene-3,17ˇ-diol-bis-O-TMS) 194 129
3-Keto-1,4-diene 1-Dehydrotestosterone-bis-TMS
(androsta-1,3,5-triene-3,17ˇ-diol-bis-O-TMS)
206 191
3-Hydroxy-1-ene Androst-1-ene-3˛,17ˇ-diol-bis-O-TMS 143 142
17-Methyl 17˛-Methyl-5˛-androstane-3˛,17ˇ-diol-bis-O-TMS 143 130
17-Keto Androsterone-bis-O-TMS 169 105
Etiocholanolone-bis-O-TMS 169 105
Copyright 2008 John Wiley & Sons, Ltd. J. Mass Spectrom. 2008; 43: 892–902
DOI: 10.1002/jms
Nutritional supplements and doping substances 899
50 100 150 200 250 300 350 400
73
143
179
194
206
231
246
301
341
356
431
446
m/z
20%
40%
60%
80%
100%
Relative abundance
O
O
H
CH
3
TMS
TMS
Figure 5. Mass spectrum (EI) of methyl-1-testosterone, bis-TMS (M
C
D 446).
36
STRATEGIES FOR THE DETECTION OF NEW
AND UNKNOWN STEROIDS IN NUTRITIONAL
SUPPLEMENTS
The issue of ‘designer steroids’ has become a serious prob-
lem in sports-drug testing, and compounds being available
and advertised as nutritional supplements have frequently
been found to contain one or more modified steroids with
unknown structure, physicochemical and biological prop-
erties. Hence comprehensive screening tools to determine
thepresenceofsuchhormoneswererequired,andmass
spectrometry has demonstrated to provide the necessary
sensitivity and selectivity to enable the determination of
conserved core structures of steroid hormones.
55,56
Common screening methods for the detection of steroidal
agents are commonly based on either GC/MS or LC/MS/MS
methods. GC/MS necessitates the derivatization of most ana-
lytes, which is commonly accomplished using trimethylsily-
lation. Consequently the dissociation behavior of the analytes
upon electron ionization (EI) is directed and influenced by
the introduced trimethylsilyl (TMS) residues, but character-
istic fragment ions or neutral losses are found indicating
the principle structure and functional group(s) of steroids.
A list of common fragment ions derived from EI-MS anal-
yses of typical steroidal structures is presented in Table 6.
Such ions that commonly distinguish a particular steroid
structure are for instance m/z 129, 130, 143, 169, 194, and
206 as well as the elimination of 103. The ion at m/z
129 represents a valuable indicator for the presence of a
3- or 17-hydroxyl function
57,58
while fragments found at
m/z 130 and 143 have frequently been observed with 17-
methyl-steroids
59–61
such as methyl-1-testosterone depicted
in Fig. 5. A fragment at m/z 169 is a characteristic item of
steroids comprising a 17-oxo function, which is converted to
its enol-TMS ether,
62,63
and the ions at m/z 194 and 206 are
indicators for ˛,ˇ-unsaturated 3-keto steroids such as testos-
terone and metandienone, respectively.
64–66
A loss of 103 u
was observed in cases of hydroxylated methyl residues such
as 19-hydroxytestosterone
67
or 17ˇ-hydroxymethyl,17˛-
methyl-androst-1,4,13-trien-3-one, a long-term metabolite of
metandienone.
68
Table 7. Characteristic product ions of selected steroids using
electrospray ionization and CID
Steroid nucleus
Representative
compound
Product ions
(m/z)
3-Keto 5˛-Dihydrotestosterone 255 215
5ˇ-Dihydrotestosterone 255 215
17˛-Methyl-5˛-
androstan-17ˇ-ol-3-one
269 229
1˛-Methyl-5˛-androstan-
17ˇ-ol-3-one
269 229
3-Keto-4-ene Testosterone 109 97
Nandrolone 109 91
3-Keto-1-ene 1-Testosterone 187 145
Metenolone 187 145
Androst-1-en-3,17-dione 185 143
3-Keto-1,4-diene 1-Dehydrotestosterone 135 121
3-Keto-4,6-diene 6-Dehydrotestosterone 133 97
3-Keto-4,9-diene Androsta-4,9(11)-dien-
17ˇ-ol-3-one
147 145
3-Keto-4,9,11-triene Trenbolone 227 199
Gestrinone 241 199
Tetrahydrogestrinone 241 199
Propyltrenbolone 227 199
The utility of LC/MS/MS in particular for steroids with
marginal gas-chromatographic properties was demonstrated
several times.
69–71
Especially employing the precursor ion
scanning option of triple-quadrupole mass analyzers pro-
vided a useful tool for the detection of unknown steroids
when focusing on product ions derived from common steroid
structures and nuclei.
70
For instance, anabolic steroids with
3-keto-4-ene core (e.g. testosterone) commonly yield ions at
m/z 97 and 109, 3-keto-1,4-diene nuclei (e.g. metandienone)
give rise to abundant ions at m/z 121 and 135, and ions at
m/z 227 and 241 are observed with 3-keto-4,9,11-triene struc-
tures such as trenbolone and gestrinone, respectively.
72,73
A
summary of characteristic product ions of anabolic steroids
is shown in Table 7. The applicability is demonstrated with
the product-ion spectrum of androsta-1,4,6-triene-3,17-dione
Copyright 2008 John Wiley & Sons, Ltd. J. Mass Spectrom. 2008; 43: 892902
DOI: 10.1002/jms
900 H. Geyer et al.
80 100 120 140 160 180 200 220 240 260 280
m/z
20%
40%
60%
80%
100%
147.0
173.1
171.0
97.0
161.0
121.0
145.0
109.0
265.1
187.1
133.0
209.1
223.1
79.0
199.1
239.1
283.2
[M+H]
+
O
O
Relative abundance
Figure 6. ESI-product-ion spectrum of androsta-1,4,6-triene-3,17-dione, [M C H]
C
D 283.
48
(Fig. 6). Here the ions at m/z 121 and 133 indicate the com-
bined presence of 1,4-and 4,6-ene structures, which add up
to a possible steroid nucleus with three double bonds.
Using the discriminating and identification power of
mass spectrometry enables the detection of an enormous
variety of unknown steroids based on common structural
features. This way more analytes are covered in screen-
ing methods than with conventional targeted assays that
necessitate the knowledge of the molecular weight.
SOURCES OF ‘LOW RISK’ SUPPLEMENTS
To prevent inadvertent doping, athletes should take nutri-
tional supplements only from sources of ‘low-risk sup-
plements’. Such sources exist, for example, for athletes
in the Netherlands and Germany. In these countries ath-
letes have access to databases, which contain nutritional
supplements from companies, which perform quality con-
trol for anabolic steroids and stimulants and/or guarantee
that they have no contact to these substances in the pro-
duction and transportation processes. In the Netherlands,
this data base (NZVT database) was installed by the anti-
doping authority of the Netherlands and is available under
<http://antidoping.nl/nzvt>. In Germany this database
(K
¨
olner Liste D Cologne List) is under the umbrella of the
Olympic Center Rhineland (www.koelnerliste.de).
Other sources of ‘low-risk supplements’ are the lists of
therapeutics from pharmaceutical companies. An investi-
gation in Germany has shown that analogs of nutritional
supplements listed on the index of therapeutics from the
German pharmaceutical industry, the so called ‘Rote Liste’,
did not contain anabolic androgenic steroids.
74
CONCLUSION
The situation on the nutritional supplement market has
got worse because not only cross-contaminations with
prohormones and stimulants but also with ‘classic’ anabolic
androgenic steroids and ˇ2 agonists can be observed.
The reason for this situation is the increasing trade and
availability of bulk material of anabolic steroids, especially
from Chinese companies. Within the next years cross-
contaminations with new ‘designer’ steroids are expected.
With mass spectrometric methods focusing on fragment and
product ions from common steroid structures and nuclei it
is possible to detect the new ‘designer’ steroids.
To protect athletes from inadvertent doping, databases of
low-risk supplements as in the Netherlands and Germany,
should be installed also in other countries.
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DOI: 10.1002/jms
    • "In some cases, users expect the supplements to fulfill nutritional needs created by an incomplete diet, while in other cases they expect the supplements to enhance their sports performance. However, there is not a consensus among physicians and trainers regarding the benefits of nutritional supplements, because of an enormous quantity of contaminated , faked, or ineffective supplements that might pose serious risks to an athlete's health or lead to evidence of doping in adverse analytical findings [1][2][3][4][5][6][7][8][9][10]. Whey protein (WP), which is the soluble protein fraction in milk serum that is obtained during cheese and casein production, has been studied since the 1970s as a source of high biological-value proteins, as well as bioactive peptides, which might act as antimicrobial, antihypertensive, and immune-response modulator agents [11][12][13]. "
    [Show abstract] [Hide abstract] ABSTRACT: Significance: The present work shows how shotgun proteomics can be used to provide reliable answers in quality control matters, especially focusing on Whey Protein nutritional supplements which are a very popular subject in food and nutrition. In order to achieve an appropriate methodology, careful evaluation was performed applying extremely rigorous quality criteria, established for the proteomic analysis. These criteria and the methodological approach used in this work might serve as a guide for other authors seeking to use proteomics in quality control.
    Full-text · Article · Apr 2016
    • "Public concern later of the safety of prohormone precursors of testosterone prompted their classification also as class III substances through enactment of the Anabolic Steroid Control Act of 2004, thus essentially banning the use of these products in the US [167]. In order to circumvent controlled substance laws, new forms of anabolic steroids began to be developed, synthesized and modified from a parent steroid, thus referred to as " designer steroids "168169170171. These synthetic anabolic steroids then were added to and sold as dietary supplements, but their presence was not shown on the label. "
    [Show abstract] [Hide abstract] ABSTRACT: In the United States (US), the risk of hepatotoxicity linked to the widespread use of certain herbal products has gained increased attention among regulatory scientists. Based on current US law, all dietary supplements sold domestically, including botanical supplements, are regulated by the Food and Drug Administration (FDA) as a special category of foods. Under this designation, regulatory scientists do not routinely evaluate the efficacy of these products prior to their marketing, despite the content variability and phytochemical complexity that often characterizes them. Nonetheless, there has been notable progress in the development of advanced scientific methods to qualitatively and quantitatively measure ingredients and screen for contaminants and adulterants in botanical products when hepatotoxicity is recognized.
    Full-text · Article · Mar 2016
    • "It is well documented worldwide that discrepancies can exist between ingredients listed on supplement labels and the actual ingredients the products contain (Abbate et al., 2014; Andrews et al., 2007; Baume et al., 2006; Cohen et al., 2013a; Cohen et al., 2013b; de Hon et al., 2007; Geyer et al., 2008; Green et al., 2001; Judkins et al., 2010; Kamber et al., 2001; Khazan et al., 2014; Kwiatkowska et al., 2015; Lanzarotta et al., 2012; Maughan et al., 2004; Odoardi et al., 2015; Outram et al., 2015; Vaclavik et al., 2014; van der Bijl et al., 2013; van der Merwe et al., 2005; Van Thuyne et al., 2006; Venhuis et al., 2014). Many dietary supplements have been found to contain unlabeled additives, contaminants, and adulterants (Austin et al., 2013; Cellini et al., 2013; Cohen, 2009; Cohen et al., 2013c; Petroczi et al., 2010; Wu et al., 2013). "
    [Show abstract] [Hide abstract] ABSTRACT: Many studies have found that some dietary supplement product labels do not accurately reflect the actual ingredients. However, studies have not been performed to determine if ingredients in the same dietary supplement product vary over time. The objective of this study was to assess the consistency of stimulant ingredients in popular sports supplements sold in the United States over a nine-month period. Three samples of nine popular sports supplements were purchased over the nine-month period. The 27 samples were analyzed for caffeine and several other stimulants (including adulterants). The identity and quantity of stimulants were compared to stimulants listed on the label and stimulants found at earlier time points to determine the variability in individual products over the nine-month period. The primary outcome measure was the variability of stimulant amounts in the products examined. Many supplements did not contain the same number and quantity of stimulants at all time points over the nine-month period. Caffeine content varied widely in five of the six caffeinated supplements compared to the initial measurement (-7% to +266%). In addition, the stimulants - synephrine, octopamine, cathine, ephedrine, pseudoephedrine, strychnine, and methylephedrine - occurred in variable amounts in eight of the nine products. The significance of these findings is uncertain: the sample size was insufficient to support statistical analysis. In our sample of nine popular sports supplements, the presence and quantity of stimulants varied over a nine-month period. However, future studies are warranted to determine if the variability found is significant and generalizable to other supplements.
    Article · Feb 2016
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