Wilhelm Schänzer’s research while affiliated with Deutsche Sporthochschule Köln and other places

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Publications (332)


Fig. 4 Product ion mass spectra and possible structure of molecules (A) S-M5 as 16-oxo-Stan ([M + H] + = 343) and (B) S-M6 ([M + H] + = 359)
Investigations into the concentration and metabolite profiles of stanozolol and LGD-4033 in blood plasma and seminal fluid using liquid chromatography high-resolution mass spectrometry
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November 2022

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66 Reads

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6 Citations

Analytical and Bioanalytical Chemistry

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Andreas Thomas

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Mario Thevis

Potential scenarios as to the origin of minute amounts of banned substances detected in doping control samples have been a much-discussed problem in anti-doping analysis in recent years. One such debated scenario has been the contamination of female athletes’ urine with ejaculate containing doping agents and/or their metabolites. The aim of this work was to obtain complementary information on whether relevant concentration ranges of doping substances are excreted into the ejaculate and which metabolites can be detected in the seminal fluid (sf) and corresponding blood plasma (bp) samples. A method was established to study the concentration and metabolite profiles of stanozolol and LGD-4033—substances listed under anabolic substances (S1) on the World Anti-Doping Agency’s Prohibited List—in bp and sf using liquid chromatography high-resolution mass spectrometry (LC-HRMS). For sf and bp, methods for detecting minute amounts of these substances were developed and tested for specificity, recovery, linearity, precision, and reliability. Subsequently, sf and bp samples from an animal administration study, where a boar orally received stanozolol at 0.33 mg/kg and LGD-4033 at 0.11 mg/kg, were measured. The developed assays proved appropriate for the detection of the target substances in both matrices with detection limits between 10 and 40 pg/mL for the unmetabolized drugs in sf and bp, allowing to estimate the concentration of stanozolol in bp (0.02–0.40 ng/mL) and in sf (0.01–0.25 ng/mL) as well as of LGD-4033 in bp (0.21–2.00 ng/mL) and in sf (0.03–0.68 ng/mL) post-administration. In addition, metabolites resulting from different metabolic pathways were identified in sf and bp, with sf resembling a composite of the metabolic profile of bp and urine. Graphical Abstract

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Investigations on the in vivo metabolism of 5α‐androst‐2‐en‐17‐one

June 2022

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29 Reads

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2 Citations

Rapid Communications in Mass Spectrometry

Rationale: The anabolic steroid 5α-androst-2-en-17-one (2EN) is sold as a prohormone and has been investigated regarding its potential as steroidal aromatase inhibitor. The administration of 2EN was detected in a doping control sample in 2015, and investigations into its metabolism allowed for the identification and characterization of three urinary metabolites. Unfortunately, the utility of the main metabolite 2β,3α-dihydroxy-5α-androstan-17-one for doping control purposes was hampered under routine doping control conditions due to chromatographic issues, thus warranting further studies on the metabolism of the prohibited substance. Methods: The metabolism of 2EN was re-investigated after oral administration of two-fold deuterated 2EN employing hydrogen isotope ratio mass spectrometry (IRMS) in combination with high accuracy/high resolution mass spectrometry. After a single dose of 50 mg of doubly labeled 2EN, urine samples were collected for 9 days. All samples were processed using routine doping control methods for IRMS analysis, and all detected metabolites were further characterized by mass spectrometry-based investigations. Results: More than 15 different metabolites still comprising the deuterium label were detected after administration. The presence of steroids exhibiting a 5β-configuration was unexpected as the administered 2EN features a 5α-configured pharmacophore. Further investigations corroborated a significant impact of the administered 2EN on etiocholanolone and 5β-androstanediol. Seven metabolites of 2EN not present as endogenous compounds were identified as potential candidates for routine doping controls and could be detected for up to 9 days after administration. Conclusion: The new metabolites identified in this study enable to detect the misuse of 2EN for up to 9 days. The conversion of a 5α-steroid to urinary metabolites with 5β-configuration has not been reported so far and should be further investigated.


Stanozolol‐N‐glucuronide metabolites in human urine samples as suitable targets in terms of routine anti‐doping analysis

June 2021

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104 Reads

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12 Citations

Drug Testing and Analysis

The exogenous anabolic‐androgenic steroid (AAS) stanozolol stays one of the most detected substances in professional sports. Its detection is a fundamental part of doping analysis and the analysis of this steroid has been intensively investigated for a long time. This contribution to the detection of stanozolol doping describes for the first time the unambiguous proof for the existence of 17‐epistanozolol‐1'N‐glucuronide and 17‐epistanozolol‐2'N‐glucuronide in stanozolol‐positive human urine samples due to the access to high quality reference standards. Examination of excretion study samples shows large detection windows for the phase‐II metabolites stanozolol‐1'N‐glucuronide and 17‐epistanozolol‐1'N‐ glucuronide up to 12 days and respectively up to almost 28 days. In addition, we present appropriate validation parameters for the analysis of these metabolites using a fully automatic method online solid‐phase extraction (SPE) method already published before. Limits of identification (LOI) as low as 100 pg/ml as well as other validation parameters like accuracy, precision, sensitivity, robustness and linearity are given.


Current Insights into the Steroidal Module of the Athlete Biological Passport

May 2021

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181 Reads

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28 Citations

International Journal of Sports Medicine

For decades, the class of anabolic androgenic steroids has represented the most frequently detected doping agents in athletes’ urine samples. Roughly 50% of all adverse analytical findings per year can be attributed to anabolic androgenic steroids, of which about 2/3 are synthetic exogenous steroids, where a qualitative analytical approach is sufficient for routine doping controls. For the remaining 1/3 of findings, caused by endogenous steroid-derived analytical test results, a more sophisticated quantitative approach is required, as their sheer presence in urine cannot be directly linked to an illicit administration. Here, the determination of urinary concentrations and concentration ratios proved to be a suitable tool to identify abnormal steroid profiles. Due to the large inter-individual variability of both concentrations and ratios, population-based thresholds demonstrated to be of limited practicability, leading to the introduction of the steroidal module of the Athlete Biological Passport. The passport enabled the generation of athlete-specific individual reference ranges for steroid profile parameters. Besides an increase in sensitivity, several other aspects like sample substitution or numerous confounding factors affecting the steroid profile are addressed by the Athlete Biological Passport-based approach. This narrative review provides a comprehensive overview on current prospects, supporting professionals in sports drug testing and steroid physiology.



Studies on the in vivo metabolism of Methylstenbolone and detection of novel long term metabolites for doping control analysis

November 2019

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307 Reads

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19 Citations

Drug Testing and Analysis

The anabolic‐androgenic steroid methylstenbolone (MSTEN; 2α,17α‐dimethyl‐17β‐hydroxy‐5α‐androst‐1‐en‐3‐one) is available as a so‐called designer steroid or nutritional supplement. It is occasionally detected in doping control samples, predominantly tested and confirmed as the glucuronic acid conjugate of MSTEN. The absence of other meaningful metabolites reported as target analytes for sports drug testing purposes can be explained with the advertised metabolic stability of MSTEN. In 2013, a first investigation into the human metabolism of MSTEN was published, and 2 hydroxylated metabolites were identified as potential targets for initial testing procedures in doping controls. These metabolites were not observed in recent doping control samples that yielded adverse analytical findings for MSTEN, and in the light of additional data originating from a recent publication on the in vivo metabolism of MSTEN in the horse, revisiting the metabolic reactions in humans appeared warranted. Therefore, deuterated MSTEN together with hydrogen isotope ratio mass spectrometry (IRMS) in combination with high accuracy/high resolution mass spectrometry were employed. After oral administration of a single dose of 10 mg of doubly labeled MSTEN, urine samples were collected for 29 days. Up to 40 different deuterated MSTEN metabolites were detected in post‐administration samples, predominantly as glucuronic acid conjugates, and all were investigated regarding their potential to prolong the detection window for doping controls. Besides MSTEN excreted glucuronidated, three additional metabolites were still detectable at the end of the study on day 29. The most promising candidates for inclusion into routine sports drug testing methods (2α,17α‐dimethyl‐5α‐androst‐1‐ene‐3β,17β‐diol and 2α,17α‐dimethyl‐5α‐androst‐1‐ene‐3α,17β‐diol) were synthesized and characterized by NMR.


SARCOSYL—PAGE: Optimized Protocols for the Separation and Immunological Detection of PEGylated Proteins: Methods and Protocols

January 2019

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77 Reads

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12 Citations

Methods in molecular biology (Clifton, N.J.)

PEGylation of recombinant proteins and synthetic peptides aims to generate biopharmaceuticals with altered physical properties. The modification may lead to a prolonged serum half-life caused by decreased receptor-mediated endocytosis and/or delay in renal clearance caused by the increased hydrodynamic volume of the pharmaceutical. MIRCERA, a PEGylated recombinant erythropoietin (rhEPO) used in the treatment of anemia due to chronic kidney disease, has also been abused by athletes as performance-enhancing drug. While it can be detected by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, the sensitivity of the test is significantly lower compared to other epoetins. By replacing SDS with sarcosyl in the sample and running buffers, the interaction between SDS and the PEG group of the protein no longer reduces the affinity of the monoclonal anti-EPO antibody (clone AE7A5) to the protein chain. Contrary to SDS, sarcosyl only binds to the amino acid chain of the PEGylated protein and thus leads to a sharper electrophoretic band and enhanced antibody binding. While the method was originally developed for anti-doping purposes, it may also be useful for the electrophoretic separation and immunological detection of other PEGylated proteins. Protocols for urine and serum are presented. They are also applicable for the general detection of EPO-based erythropoiesis-stimulating agents (ESA) in these matrices.


Studies on the in vivo metabolism of the SARM YK11: Identification and characterization of metabolites potentially useful for doping controls

October 2018

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1,557 Reads

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18 Citations

Drug Testing and Analysis

A steroidal compound was recently detected in a seized black market product and was identified as (17α,20E)‐17,20‐[(1‐methoxyethylidene) bis (oxy)]‐3‐oxo‐19‐norpregna‐4,20‐diene‐21‐carboxylic acid methyl ester (YK11). This compound is described to possess selective androgen receptor modulator‐ and myostatin inhibitor‐like properties. As YK11 is an experimental drug candidate and a non‐approved substance for humans, scientific data on its metabolism is scarce. Due to its steroidal backbone and the arguably labile orthoester‐derived moiety positioned at the D‐ring, substantial metabolic conversion in vivo was anticipated. In order to unambiguously detect urinary metabolites of YK11, an elimination study with six‐fold deuterated YK11 was conducted. Post administration specimens were analyzed using hydrogen isotope ratio mass spectrometry coupled to single quadrupole mass spectrometry to identify metabolites alongside basic mass spectrometric data. Further characterization of those metabolites relevant to sports drug testing was accomplished using gas chromatography/high resolution‐high accuracy mass spectrometry. Fourteen deuterated urinary metabolites were detected comprising unconjugated, glucuronidated and sulfoconjugated metabolites. As expected, no intact YK11 was observed in the elimination study urine samples. While the unconjugated metabolites disappeared within 24 h post‐administration, both glucuronidated and sulfated metabolites were traceable for more than 48 h. The chemical structures of the two most promising glucuronidated metabolites (5β‐19‐nor‐pregnane‐3α,17β,20‐triol and 5β‐19‐nor‐pregnane‐3α,17β‐diol‐20‐one) were verified by in‐house synthesis of both metabolites and verified by NMR analysis. In order to elucidate their potential in sports drug testing, both were successfully implemented into the currently applied analytical method for the detection of anabolic agents.


Androgen- and estrogen-receptor mediated activities of 4-hydroxytestosterone, 4-hydroxyandrostenedione and their human metabolites in yeast based assays

April 2018

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132 Reads

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8 Citations

Toxicology Letters

4-Hydroxyandrost-4-ene-3,17-dione, also named formestane, is an irreversible aromatase inhibitor and therapeutically used as anti-breast cancer medication in post-menopausal women. Currently, no therapeutical indication led to approval of its 17-hydroxylated analog 4-hydroxytestosterone, an anabolic steroid. However, it is currently investigated in a clinical trial for breast cancer. In context with sports doping, aromatase inhibitors are administered to reduce estrogenic side effects of misused anabolic substances or their metabolites. Therefore, both substances are prohibited in sports by the World Anti-Doping Agency (WADA). Analysis of urinary phase I and phase II metabolites showed similar results for both compounds. In the current investigation, 4-hydroxyandrost-4-ene-3,17-dione, 4-hydroxytestosterone and seven of their described urinary metabolites as well as 2α-hydroxyandrostenedione were tested in the yeast androgen screen and the yeast estrogen screen. Androgenic effects were observed for all tested substances, except for one, which showed anti-androgenic properties. With regard to the yeast estrogen screen, estrogenic effects were observed for only two metabolites at rather high concentrations, while six out of the ten substances tested showed anti-estrogenic properties. In terms of the strong androgenic effect observed for 4-hydroxytestosterone (10-8 M), 4-hydroxyandrost-4-ene-3,17-dione (10-8 M) and two more urinary metabolites, the yeast androgen assay may also be used to trace abuse in urine samples.


Citations (94)


... These drugs may have an influence on fertility, as around 30-50 % of all cases of infertility can be attributed to men (Ajayi and Akhigbe, 2020), and it is speculated that these substances have an influence on the conceptus during seminal fluid transfer through sexual intercourse (Klemmt and Scialli, 2005). In addition to these clinically relevant aspects in terms of reproduction and fertility medicine, a contamination scenario through seminal fluid has also been discussed in the doping control context (Breuer et al., 2022;Handelsman et al., 2022;Breuer et al., 2023;Kintz, 2024;Kintz and Gheddar, 2024). ...

Reference:

Investigations into the Concentrations and Metabolite Profiles of Doping Agents and Antidepressants in Human Seminal Fluid Using Liquid Chromatography–Mass Spectrometry
Investigations into the concentration and metabolite profiles of stanozolol and LGD-4033 in blood plasma and seminal fluid using liquid chromatography high-resolution mass spectrometry

Analytical and Bioanalytical Chemistry

... Red blood cells carry oxygen to cells, including muscle cells, enabling them to function more efficiently. Physiological effect: Agonist of the androgen receptor [41]. It is used to increase muscle mass and strength. ...

Stanozolol‐N‐glucuronide metabolites in human urine samples as suitable targets in terms of routine anti‐doping analysis
  • Citing Article
  • June 2021

Drug Testing and Analysis

... We demonstrate the effectiveness of our approach in doping detection in sports, where it is applied to detect suspicious urine samples within athletes' longitudinal profiles. These profiles include the concentrations of various metabolites, reflecting the steroid metabolism as shown in Fig.1, and are important for identifying potential doping activities [12]. The key contributions of our paper can be summarised as follows: ...

Current Insights into the Steroidal Module of the Athlete Biological Passport

International Journal of Sports Medicine

... Very recently, in 2020, a series of publications (12), presenting investigations performed by WADA-accredited laboratories, have demonstrated that the forensic approach can be useful to document unusual adverse finding. For example, it was retrospectively demonstrated that the AAF of Dieter Baumann, in 1999, could be the consequence of a contaminated toothpaste with norandrostenedione, a prohormone of the anabolic drug nandrolone (13). This clearly indicates that there is room for additional proofs in sports, despite a very strong regulation of evidence admissibility. ...

Tainted toothpaste – Analytical investigation into an unusual adverse finding

Drug Testing and Analysis

... The in vivo metabolism of the steroidal SARM YK-11 and the anabolic steroids methylstenbolone and trenbolone was investigated by Piper et al. by analysing post-administration urine specimens. Several metabolites, relevant to sports drug testing, were characterised using GC-HRMS (as trimethylsilyl derivatives) and/or LC-HRMS instruments [18,161,162]. Moreover, GC-HRMS and GC-MS/MS techniques were recently employed for detecting novel A-Ring reduced metabolites of the anabolic steroids methyltestosterone and metandienone [163]. ...

Studies on the in vivo metabolism of Methylstenbolone and detection of novel long term metabolites for doping control analysis

Drug Testing and Analysis

... Antidoping testing for recombinant human erythropoietin (EPO) is routinely performed by gel electrophoresis followed by western blot analysis [1][2][3]. Sample preparation consists of a mandatory immunopurification with a different antibody than the one used for the western blot. There are different immunopurification procedures which are applied in antidoping laboratories. ...

SARCOSYL—PAGE: Optimized Protocols for the Separation and Immunological Detection of PEGylated Proteins: Methods and Protocols
  • Citing Chapter
  • January 2019

Methods in molecular biology (Clifton, N.J.)

... The in vivo metabolism of the steroidal SARM YK-11 and the anabolic steroids methylstenbolone and trenbolone was investigated by Piper et al. by analysing post-administration urine specimens. Several metabolites, relevant to sports drug testing, were characterised using GC-HRMS (as trimethylsilyl derivatives) and/or LC-HRMS instruments [18,161,162]. Moreover, GC-HRMS and GC-MS/MS techniques were recently employed for detecting novel A-Ring reduced metabolites of the anabolic steroids methyltestosterone and metandienone [163]. ...

Studies on the in vivo metabolism of the SARM YK11: Identification and characterization of metabolites potentially useful for doping controls
  • Citing Article
  • October 2018

Drug Testing and Analysis

... For evaluating the ability of DIO to interact with the androgen or estrogen receptors in vitro, the yeast transactivation system was used. Yeast assay was performed as described by Keiler et al. (Keiler et al. 2018). ...

Androgen- and estrogen-receptor mediated activities of 4-hydroxytestosterone, 4-hydroxyandrostenedione and their human metabolites in yeast based assays
  • Citing Article
  • April 2018

Toxicology Letters

... The determination of the protein drugs at the amino acid level is achieved by HRMS. This technique was implemented in the quantitation of anti-ActRII antibody, bimagrumab, and the antimyostatin antibody domagrozumab combined with affinity purification, tryptic digestion, and liquid chromatography in doping control serum samples (63,64) . The obtained detection limits by the doping control testing techniques are as follows; (bimagrumab: 20 ng/mL (63) ; domagrozumab: 50 ng/mL (64)) in serum samples. ...

Detection of the Human Anti-ActRII Antibody Bimagrumab in Serum by Means of Affinity Purification, Tryptic Digestion, and LC-HRMS
  • Citing Article
  • December 2017

PROTEOMICS - CLINICAL APPLICATIONS

... The LODs of the method were determined by analyzing DBS samples prepared from spiked blood samples at 0.1, 0.2, 0.5, 1, 2, 5, and 10 ng/mL. The LOD was defined as the lowest concentration with the analyte present and a signalto-noise (S/N) ratio of at least 3 [39]. ...

Screening for Adiponectin Receptor Agonists and their Metabolites in Urine and Dried Blood Spots
  • Citing Article
  • October 2017

Clinical Mass Spectrometry