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Thevis M.,German Sport University Cologne | Thevis M.,European Monitoring Center for Emerging Doping Agents | Schanzer W.,German Sport University Cologne
Molecular and Cellular Endocrinology | Year: 2017

The class of selective androgen receptor modulators (SARMs) has been the subject of intense and dedicated clinical research over the past two decades. Potential therapeutic applications of SARMs are manifold and focus particularly on the treatment of conditions manifesting in muscle loss such as general sarcopenia, cancer-associated cachexia, muscular dystrophy, etc. Consequently, based on the substantial muscle- and bone-anabolic properties of SARMs, these agents constitute substances with significant potential for misuse in sport and have therefore been added to the Word Anti-Doping Agency's (WADA's) Prohibited List in 2008. Since then, numerous adverse analytical findings have been reported for various different SARMs, which has underlined the importance of proactive and preventive anti-doping measures concerning emerging drugs such as these anabolic agents, which have evidently been misused in sport despite the fact that none of these SARMs has yet received full clinical approval. In this review, analytical data on SARMs generated in the context of research conducted for sports drug testing purposes are summarized and state-of-the-art test methods aiming at intact drugs as well as diagnostic urinary metabolites are discussed. Doping control analytical approaches predominantly rely on chromatography hyphenated to mass spectrometry, which have allowed for appropriately covering the considerable variety of pharmacophores present in SARMs such as the non-steroidal representatives ACP-105, BMS-564929, GLPG0492 (DT-200), LG-121071, LGD-2226, LGD-4033/VK 5211, ostarine/enobosarm, RAD-140, S-40503, etc. as well as steroidal compounds such as MK-0773 and YK-11. © 2017 Elsevier B.V.


Hoppner S.,German Sport University Cologne | Delahaut P.,CER Groupe | Schanzer W.,German Sport University Cologne | Thevis M.,German Sport University Cologne | Thevis M.,European Monitoring Center for Emerging Doping Agents
Journal of Pharmaceutical and Biomedical Analysis | Year: 2014

The NAD+ depending enzyme SIRT1 regulates the mitochondrial biogenesis, fat and glucose metabolism through catalyzing the deacetylation of several metabolism-related protein-substrates. Recently, synthetic activators of SIRT1 referred to as STACs (Sirtuin activating compounds, e.g. SRT2104) were identified and tested in clinical studies for the treatment of aging-related diseases such as type 2 diabetes, Alzheimer's and obesity. Although the mechanism of SIRT1 activation by small molecules has caused considerable controversy, STACs demonstrated a significant performance enhancement in mice experiments including an improvement of endurance, muscle strength, and locomotor behavior. Due to their potential to increase exercise tolerance in healthy individuals, SIRT1 activators are currently being monitored by anti-doping authorities. In the present study, the in vivo metabolic clearance of three SIRT1 activators was investigated in rats by the collection of urine, DBS (dried blood spots) and plasma samples following a single oral administration. The resulting metabolic products were studied by positive electrospray ionization - (tandem) mass spectrometry and confirmed by the comparison with in vitro generated metabolites using human and rat liver microsomal preparations. Subsequently, a screening procedure for five SIRT1 activators and the metabolite M1-SRT1720 in DBS specimens was developed. Liquid-liquid-extraction and liquid chromatography/tandem mass spectrometry was employed based on diagnostic ion transitions recorded in multiple reaction monitoring mode and two deuterated internal standards namely d8-SRT1720 and d8-M1-SRT1720 were utilized. The doping control assay was characterized with regard to specificity, limit of detection (10-50ng/ml), recovery (65-83%) and imprecision (7-20%) and ion suppression/enhancement effects (<10%), demonstrating its fitness-for-purpose for sports drug testing applications. © 2013 Elsevier B.V.


Thevis M.,German Sport University Cologne | Thevis M.,European Monitoring Center for Emerging Doping Agents | Thomas A.,German Sport University Cologne | Geyer H.,German Sport University Cologne | Schanzer W.,German Sport University Cologne
Growth Hormone and IGF Research | Year: 2014

Objective: Since Goldspink and colleagues identified the expression of the mRNA of an insulin-like growth factor 1 (IGF-1) isoform in response to mechanical stress in 1996, substantial research into the so-called mechano growth factor and its modus operandi followed until today. Promising preclinical results were obtained by using the synthetic, 24-amino acid residues spanning peptide translated from the exons 4-6 of IGF-1Ec (which was later referred to as the mechano growth factor (MGF) peptide), particularly with regard to increased muscle myoblast proliferation. Consequently, the MGF peptide represented a promising drug candidate for the treatment of neuromuscular disorders; however, its misuse potential in sport was also identified shortly thereafter, and the substance (or class of substances) has been considered prohibited according to the regulations of the World Anti-Doping Agency (WADA) since 2005. While various MGF peptide versions have been known to sports drug testing authorities, the occurrence of a 'full-length MGF' as offered via illicit channels to athletes or athletes' managers was reported in 2014, arguably being undetectable in doping controls. Methods: An aliquot of the product was obtained and the content characterized by state-of-the-art analytical approaches including gel electrophoretic and mass spectrometric (top-down and bottom-up) sequencing approaches. Upon full characterization, its implementation into modified routine doping controls using ultrafiltration, immunoaffinity-based isolation, and nanoliquid chromatography-high resolution/high accuracy mass spectrometry was established. Results: A protein with a monoisotopic molecular mass of 12264.9. Da and a sequence closely related to IGF-1Ec (lacking the signal- and propeptide moiety) was identified. The C-terminus was found to be modified by the elimination of the terminal lysine and a R109H substitution. With the knowledge of the compound's composition, existing doping control assays targeting peptide hormones such as IGF-1 and related substances were assessed as to their capability to detect the full-length MGF. The analyte was detectable at concentrations of 0.25. ng/mL using adapted routine test methods employing immunoaffinity purification followed by nanoscale liquid chromatography-high resolution/high accuracy (tandem) mass spectrometry. Conclusions: A potentially performance enhancing 'full-length' MGF derivative was identified and successfully implemented into sports drug testing protocols. Future tests are indicated probing for optimized/dedicated detection methods and assessment of efficacy and elimination kinetics of the substance. © 2014 Elsevier Ltd.


Thevis M.,German Sport University Cologne | Thevis M.,European Monitoring Center for Emerging Doping Agents | Thomas A.,German Sport University Cologne | Thomas A.,European Monitoring Center for Emerging Doping Agents | And 2 more authors.
Expert Review of Proteomics | Year: 2014

With the growing availability of mature systems and strategies in biotechnology and the continuously expanding knowledge of cellular processes and involved biomolecules, human sports drug testing has become a considerably complex field in the arena of analytical chemistry. Proving the exogenous origin of peptidic drugs and respective analogs at lowest concentration levels in biological specimens (commonly blood, serum and urine) of rather limited volume is required to pursue an action against cheating athletes. Therefore, approaches employing chromatographic-mass spectrometric, electrophoretic, immunological and combined test methods have been required and developed. These allow detecting the misuse of peptidic compounds of lower (such as growth hormone-releasing peptides, ARA-290, TB-500, AOD-9604, CJC-1295, desmopressin, luteinizing hormone-releasing hormones, synacthen, etc.), intermediate (e.g., insulins, IGF-1 and analogs, 'full-length' mechano growth factor, growth hormone, chorionic gonadotropin, erythropoietin, etc.) and higher (e.g., stamulumab) molecular mass with desired specificity and sensitivity. A gap between the technically possible detection and the day-to-day analytical practice, however, still needs to be closed. © 2014 Informa UK, Ltd.


Thevis M.,German Sport University Cologne | Schanzer W.,European Monitoring Center for Emerging Doping Agents
Journal of Pharmaceutical and Biomedical Analysis | Year: 2014

The number and diversity of potentially performance-enhancing substances is continuously growing, fueled by new pharmaceutical developments but also by the inventiveness and, at the same time, unscrupulousness of black-market (designer) drug producers and providers. In terms of sports drug testing, this situation necessitates reactive as well as proactive research and expansion of the analytical armamentarium to ensure timely, adequate, and comprehensive doping controls. This review summarizes literature published over the past 5 years on new drug entities, discontinued therapeutics, and 'tailored' compounds classified as doping agents according to the regulations of the World Anti-Doping Agency, with particular attention to analytical strategies enabling their detection in human blood or urine. Among these compounds, low- and high-molecular mass substances of peptidic (e.g. modified insulin-like growth factor-1, TB-500, hematide/peginesatide, growth hormone releasing peptides, AOD-9604, etc.) and non-peptidic (selective androgen receptor modulators, hypoxia-inducible factor stabilizers, siRNA, S-107 and ARM036/aladorian, etc.) as well as inorganic (cobalt) nature are considered and discussed in terms of specific requirements originating from physicochemical properties, concentration levels, metabolism, and their amenability for chromatographic-mass spectrometric or alternative detection methods. © 2014 Elsevier B.V. All rights reserved.


Reichel C.,Doping Control Laboratory | Reichel C.,European Monitoring Center for Emerging Doping Agents | Thevis M.,German Sport University Cologne | Thevis M.,European Monitoring Center for Emerging Doping Agents
Drug Testing and Analysis | Year: 2012

The neonatal Fc receptor (FcRn) has been under investigation for several years as a pharmaceutical drug target. Clinical studies have shown that fusion proteins consisting of human recombinant erythropoietin (rhEPO) and the Fc-part of IgG can be transported after pulmonary administration via FcRn across the airway epithelium to the blood stream. So far, no clinically approved pharmaceutical formulation of EPO-Fc is available. Since various forms of recombinant erythropoietins have been frequently misused by athletes as performance-enhancing agents, EPO-Fc might play a similar role in sports in the future. In order to investigate the detectability of EPO-Fc in human blood, different strategies were tested and developed. Only two of them fulfilled the necessary requirements regarding sensitivity and specificity. A rapid protocol useful for screening purposes first enriches EPO-Fc from human serum via high capacity protein A beads and subsequently detects EPO-Fc in the eluate with a commercial EPO ELISA kit. The limit of detection (LOD) of the method is about 5 pg (45 amol) EPO-Fc and is independent of the serum volume used. For screening and/or confirmation purposes a second protocol was evaluated, which consists of a fast EPO immunopurification step followed by sodium dodecyl sulfate or sarcosyl polyacrylamide gel electrophoresis (SDS-PAGE, SAR-PAGE) and Western double-blotting with chemiluminescence detection - a method already established in routine EPO anti-doping control. The latter strategy allows the detection of EPO-Fc in serum together with all other recombinant erythropoietins and with an identical LOD (5 pg/45 amol) as for the rapid screening protocol. © 2012 John Wiley & Sons, Ltd.


Beuck S.,German Sport University Cologne | Schanzer W.,German Sport University Cologne | Thevis M.,German Sport University Cologne | Thevis M.,European Monitoring Center for Emerging Doping Agents
Drug Testing and Analysis | Year: 2012

Increasing the blood's capacity for oxygen transport by erythropoiesis-stimulating agents (ESAs) constitutes a prohibited procedure of performance enhancement according to the World Anti-Doping Agency (WADA). The advent of orally bio-available small-molecule ESAs such as hypoxia-inducible factor (HIF) stabilizers in the development of novel anti-anaemia therapies expands the list of potential ESA doping techniques. Here, the erythropoiesis-stimulating properties and doping relevance of experimental HIF-stabilizers, such as cobaltous chloride, 3,4-dihydroxybenzoic acid or GSK360A, amongst others, are discussed. The stage of clinical trials is reviewed for the anti-anaemia drug candidates FG-2216, FG-4592, GSK1278863, AKB-6548, and BAY85-3934. Currently available methods and strategies for the determination of selected HIF stabilizers in sports drug testing are based on liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). For the support of further analytical assay development, patents claiming distinct compounds for the use in HIF-mediated therapies are evaluated and exemplary molecular structures of HIF stabilizers presented. Moreover, data concerning the erythropoiesis-enhancing effects of the GATA inhibitors K7174 and K11706 as well as the lipidic small-molecule ESA PBI-1402 are elucidated the context of doping analysis. © 2012 John Wiley & Sons, Ltd.


Thevis M.,German Sport University Cologne | Thevis M.,European Monitoring Center for Emerging Doping Agents | Thomas A.,German Sport University Cologne | Schanzer W.,German Sport University Cologne
Analytical and Bioanalytical Chemistry | Year: 2013

Urine samples have been the predominant matrix for doping controls for several decades. However, owing to the complementary information provided by blood (as well as serum or plasma and dried blood spots (DBS)), the benefits of its analysis have resulted in continuously increasing appreciation by anti-doping authorities. On the one hand, blood samples allow for the detection of various different methods of blood doping and the abuse of erythropoiesis-stimulating agents (ESAs) via the Athlete Biological Passport; on the other hand, targeted and non-targeted drug detection by means of chromatographic-mass spectrometric methods represents an important tool to increase doping control frequencies out-of-competition and to determine drug concentrations particularly in in-competition scenarios. Moreover, blood analysis seldom requires in-depth knowledge of drug metabolism, and the intact substance rather than potentially unknown or assumed metabolic products can be targeted. In this review, the recent developments in human sports drug testing concerning mass spectrometry-based techniques for qualitative and quantitative analyses of therapeutics and emerging drug candidates are summarized and reviewed. The analytical methods include both low and high molecular mass compounds (e.g., anabolic agents, stimulants, metabolic modulators, peptide hormones, and small interfering RNA (siRNA)) determined from serum, plasma, and DBS using state-of-the-art instrumentation such as liquid chromatography (LC)-high resolution/high accuracy (tandem) mass spectrometry (LC-HRMS), LC-low resolution tandem mass spectrometry (LC-MS/MS), and gas chromatography-mass spectrometry (GC-MS). © 2002 Springer-Verlag Berlin Heidelberg.


Thevis M.,German Sport University Cologne | Thevis M.,European Monitoring Center for Emerging Doping Agents | Piper T.,German Sport University Cologne | Beuck S.,German Sport University Cologne | And 2 more authors.
Rapid Communications in Mass Spectrometry | Year: 2013

RATIONALE Anabolic agents have been top-ranked for many years among statistics of adverse analytical findings compiled by the World Anti-Doping Agency (WADA). Besides archetypical anabolic-androgenic steroids (AAS), alternative substances with similar effects concerning bone and muscle anabolism have been therapeutically pursued. A prominent emerging class of drugs is the chemically heterogeneous group of selective androgen receptor modulators (SARMs), some of which have been detected in doping control samples between 2009 and 2012 despite missing clinical approval. METHODS In order to support the momentum of expanding the preventive and proactive measures among anti-doping laboratories, the analytical characterization of substances with misuse potential is of great importance. In the present study, the SARM drug candidates RAD140 (comprising a 5-phenyloxadiazole nucleus) and ACP-105 (bearing an N-substituted tropanol pharmacophore) were studied regarding their mass spectrometric behavior under ESI-MS(/MS) and EI-MS(/MS) conditions. Reference material was synthesized according to established protocols and dissociation pathways of RAD140 and ACP-105 were elucidated with liquid chromatography/ electrospray ionization quadrupole/time-of-flight or iontrap/orbitrap and gas chromatography/electron ionization quadrupole/time-of-flight high resolution/high accuracy mass spectrometry. RESULTS Fragmentation pathways to diagnostic product ions of RAD140 (e.g. m/z 223 and 205 using ESI-MS/MS and m/z 421 and 349 using EI-MS/MS) and ACP-105 (such as m/z 233 and 193 or 231 and 217 for ESI-MS/MS and EI-MS/MS measurements, respectively) were proposed as substantiated by determined elemental compositions and MSn experiments as well as comparison to spectra of a structural analog. Notably, for the formation of the characteristic fragment ion at m/z 421 of RAD140, the comparably seldom intramolecular migration of a trimethylsilyl residue triggered by electron ionization was suggested as corroborated by all of the above-mentioned analytical means. CONCLUSIONS The obtained data will support future sports drug testing methods and facilitate and accelerate the implementation of this analyte and related compounds or metabolites in both GC/MS(/MS)- and LC/MS(/MS)-based routine doping control procedures. Copyright © 2013 John Wiley & Sons, Ltd. Copyright © 2013 John Wiley & Sons, Ltd.


Geyer H.,German Sport University Cologne | Geyer H.,European Monitoring Center for Emerging Doping Agents | Schanzer W.,German Sport University Cologne | Thevis M.,German Sport University Cologne | Thevis M.,European Monitoring Center for Emerging Doping Agents
British Journal of Sports Medicine | Year: 2014

According to the World Anti-Doping Agency (WADA) Prohibited List, anabolic agents consist of exogenous anabolic androgenic steroids (AAS), endogenous AAS and other anabolic agents such as clenbuterol and selective androgen receptor modulators (SARMs). Currently employed strategies for their improved detection include the prolongation of the detection windows for exogenous AAS, non-targeted and indirect analytical approaches for the detection of modified steroids (designer steroids), the athlete's biological passport and isotope ratio mass spectrometry for the detection of the misuse of endogenous AAS, as well as preventive doping research for the detection of SARMs. The recent use of these strategies led to 4-80-fold increases of adverse analytical findings for exogenous AAS, to the detection of the misuse of new designer steroids, to adverse analytical findings of different endogenous AAS and to the first adverse analytical findings of SARMs. The strategies of the antidoping research are not only focused on the development of methods to catch the cheating athlete but also to protect the clean athlete from inadvertent doping. Within the past few years several sources of inadvertent doping with anabolic agents have been identified. Among these are nutritional supplements adulterated with AAS, meat products contaminated with clenbuterol, mycotoxin (zearalenone) contamination leading to zeranol findings, and natural products containing endogenous AAS. The protection strategy consists of further investigations in case of reasonable suspicion of inadvertent doping, publication of the results, education of athletes and development of methods to differentiate between intentional and unintentional doping.

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