Supplemental Material
Identification of black market products and potential doping agents in Germany 2010-2013
Oliver Krug1,2, Andreas Thomas1, Katja Walpurgis1, Thomas Piper1, Gerd Sigmund1, Wilhelm
Schänzer1, Tim Laussmann3, Mario Thevis1,2*
1Center
for preventive Doping Research/Institute for Biochemistry, German Sport University
Cologne, Germany; 2European Monitoring Center for Emerging Doping Agents EuMoCEDA,
Cologne/Bonn, Germany; 3Centre of Education and Science of the Federal Revenue
Administration, Cologne, Germany
Materials and methods
Chemicals
Glacial acetic acid and ammonium acetate were purchased from Merck (Darmstadt,
Germany). 12% Bis-Tris gels (8 cm x 8 cm x 0.1 cm, 10-well), MOPS running buffer (10x) as
well as lithium dodecyl sulfate (LDS) buffer (4x) came from Life Technologies (Darmstadt,
Germany). GelCode Blue Stain Reagent (Coomassie G-250) was supplied by Pierce (Rockford,
IL, USA) and N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) by Chem. Fabrik Karl
Bucher (Waldstetten, Germany). Trypsin was obtained from Promega (Madison, WI) and
dithiothreitol (DTT), N-methyl-bis-trifluoroacetamide (MBTFA) as well as all buffer
ingredients (electrophoresis grade/analytical grade) came from Sigma-Aldrich (Steinheim,
Germany). Acetonitrile (ACN) and methanol (MeOH) were bought from VWR International
GmbH (Darmstadt, Germany) and were of analytical or high-performance liquid
chromatography (HPLC) grade. Aqueous buffers were prepared by using deionized water
(Sartorius Stedim Biotech S. A., Aubagne, France).
LC-MS/MS screening
As more than 80 percent of the confiscated and analyzed black market products contain AAS
or stimulants, the initial LC-MS/MS screening is performed in selected reaction monitoring
(SRM) mode including ion transitions for the most common drugs. These analyses are
conducted on a TSQ Vantage (Thermo Fisher Scientific, Dreieich, Germany) coupled to an
Agilent (Waldbronn, Germany) 1100 HPLC system equipped with an electrospray ionization
(ESI) source and a C-18 column (2.1 x 50 mm, particle size 3.0 µm, Macherey Nagel, Düren,
Germany). For LC, 5 mM ammonium acetate with 0.1% acetic acid is used as eluent A and
ACN or MeOH as eluent B. A gradient is set as follows: Starting conditions 0% B; increasing to
100% B within 5 min, remaining at 100 % for 5 min, 6 min re-equilibration with 0% B. The
flow is set to 300 µL/min and the injection volume to 10 µL. Depending on the samples, the
ESI source is operated both in positive and negative mode at a source temperature of 350 °C.
Identified analytes are confirmed and quantified by using the corresponding reference
standard solutions and product ion scan experiments.
LC-HRMS (Full-scan mode)
Samples returning no or negative results in initial LC-MS/MS screening analyses are
scrutinized by complementary measurements using LC-HRMS. In order to detect unusual or
unknown ingredients, measurements are conducted in full-scan mode from m/z 80 to 1500.
Amongst others, the following systems were employed: a) Exactive MS interfaced to an
Accela LC system (both Thermo Fisher Scientific, Bremen, Germany); b) Q Exactive coupled
to a Thermo Dionex UltiMate 3000 RS Column Compartment UHPLC+ (both Thermo Fisher
Scientific, Bremen, Germany); c) AB Sciex TripleTOF 5600 (Applied Biosystems, Darmstadt,
Germany) in combination with an Agilent (Waldbronn, Germany) 1260 Infinity HPLC; and d)
Agilent 6550 iFunnel Q-TOF connected to an Agilent 1290 Infinity HPLC system.
Full-scan data are manually assessed with regard to unfamiliar or only sporadically occurring
drugs and drug candidates including anabolic agents, stimulants, growth factors, natural and
synthetic insulins, IGF-1 and synthetic analogs as well as growth hormone releasing factors.
GC-MS
In cases where the different LC-MS approaches yield no identification, the black market
products are further analyzed by two different GC-MS experiments. The analytes comprising
stimulants, beta-2-agonists, cannabinoids, narcotics and AAS are derivatized with MSTFA
and/or MBTFA [12] (30 min, 60 °C) and are analyzed either on a 7200 Accurate-Mass Q-TOF
interfaced via EI to a 7890A GC System (Agilent, Waldbronn, Germany) or an Agilent 5973
Mass Selective Detector coupled to a HP 6890 series GC-system equipped with an nitrogen
phosphorus detector (NPD). For chromatography an Agilent J&W DB-17MS column (30m,
inner diameter 0.25 mm, film thickness 0.25 µm) is used. Instrument temperatures are
280°C for the inlet, 300°C for the transfer line, and 150°C for the injector. Helium (purity
grade 4.6) is used as carrier gas, injections are conducted in splitless mode at a volume of
0.5 µL. The GC temperature is set to 60°C for 1.5 min followed by a ramp of 30°C/min to
220°C and a ramp of 10°C/min to 310°C. Temperature gradients are adjusted if needed.
The GC-MS data are manually evaluated and for confirmation and quantification, the
corresponding reference standard solutions and reference databases are used.
SDS-PAGE analysis
Black market products with assumed peptidic content are subjected to SDS-PAGE analysis on
12% Bis-Tris gels. A total of 20 µL of the dissolved lyophilisates are mixed with 7 µL of LDS
sample buffer as well as 3 µL of DTT (1 M) and subsequently heated for 10 min at 70°C to
achieve denaturation of proteins and reduction of disulfide bonds according to standard
protocols. Gels are run in MOPS electrophoresis buffer for 90 min at a constant voltage of
125 V (XCell Surelock Mini-Cell, Life Technologies, Darmstadt, Germany). Following
Coomassie staining, gels are digitalized with a light transmission scanner (GE Healthcare,
Munich, Germany) operated in transparent mode using a red filter and a resolution of 300
dpi.
For protein identification, gel bands are excised from the gel and subjected to in-gel tryptic
digestion and nano-LC-HRMS [13]. Following destaining with 200 µL of 100 mM
NH4HCO3/ACN (1:1) (2 x 10 min, 750 rpm, RT), gel slices are dehydrated in 100 µL of ACN and
dried under reduced pressure for 15 min (Jouan 10.22, Thermo Fisher Scientific). Afterwards,
bands are rehydrated in 10 µL of a trypsin solution (20 µg/mL in 50 mM NH4HCO3, pH 8) for
45 min at 4°C and incubated for 3-16 h at 37 °C. Tryptic peptides are extracted with 100 µL of
50% ACN/1% TFA (3 x 10 min, 1000 rpm) and combined extracts dried in a vacuum
concentrator. Dried extracts are then reconstituted in 50 µL of 2% acetic acid and subjected
to LC-MS/MS analysis.
An LTQ Orbitrap and Q Exactive (Thermo Fisher Scientific) mass spectrometer coupled to a
Waters nanoAcquity LC-system (Waters, Eschborn, Germany) were used for LC-MS/MS
measurements. In the data-dependent MS/MS experiments, only the three most abundant
ions with a charge state of more than 1 were selected for fragmentation and MS/MS spectra
were recorded with a resolution of 35,000 (at m/z 200; Q Exactive) and 30,000 (at m/z 400,
LTQ Orbitrap), respectively. MS data were evaluated by using Proteome Discoverer Software
(Thermo Fisher Scientific) in combination with SEQUEST (HT) search algorithm and UniProt
database.