Nana Naumann’s research while affiliated with Deutsche Sporthochschule Köln and other places

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Publications (7)


Bioanalytical methods in doping controls: a review
  • Literature Review

February 2025

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16 Reads

Andreas Thomas

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Katja Walpurgis

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Nana Naumann

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[...]

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Mario Thevis


Detection of doping control sample substitutions via single nucleotide polymorphism-based ID typing

November 2023

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18 Reads

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3 Citations

Drug Testing and Analysis

The authenticity of a doping control sample is a key element of sports drug testing programmes. Doping control sample manipulation by providing another individual's urine or blood (instead of the tested athlete's sample) has been observed in the past and is an unequivocal violation of the World Anti‐Doping Agency anti‐doping rules. To determine attempts of manipulations by sample swapping, the utility of a single nucleotide polymorphism (SNP)‐based sample authentication with a multi‐target SNP panel was assessed. The panel comprises detection assays for 44 different SNPs, 3 gender markers and 5 quality control markers for DNA‐profile determination. Sample analysis is based on a multiplex polymerase chain reaction step followed by a multiplex single base extension (SBE) reaction and subsequent SBE‐product detection by MALDI‐TOF MS. Panel performance was evaluated for urine and dried blood spot (DBS) samples. Urine (8 ml) and DBS (20 μl) test samples were reliably typed and matched to whole blood reference samples, while efficient typing of urine samples correlated with sample quality and input amounts. Robust profiling of urine doping control specimens was confirmed with an assay input of 12 ml. Samples can be processed in a high‐throughput format with an overall assay turnaround time of approximately 11 h. SNP‐based DNA typing via MALDI‐TOF MS thus represents a high throughput‐capable possibility for doping control sample authentication. SNP profiling of samples could offer the opportunity to complement existing steroid profile analytics to substantiate sample manipulations and to support quality control processes in high throughput routine settings.


Analysis of Potential Gene Doping Preparations for Transgenic DNA in the Context of Sports Drug Testing Programs
  • Article
  • Full-text available

October 2023

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56 Reads

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10 Citations

International Journal of Molecular Sciences

Gene doping has been classified as a prohibited method by the World Anti-Doping Agency (WADA) and the International Olympic Committee (IOC) for over two decades. As gene therapeutic approaches improve and, concomitantly, safety concerns regarding clinical applications decline, apprehensions about their illicit use in elite sports continue to grow. Two products available via Internet-based providers and advertised as EPO-gene- and IGF1-gene-containing materials were analyzed for the presence of potential gene doping agents using a newly developed analytical approach, allowing for the detection of transgenic DNA corresponding to seven potential targets (EPO, FST, GH1, MSTN (Propeptide), IGF1, VEGFA, and VEGFD). Panel detection was based on a 20-plex polymerase chain reaction (PCR) followed by a single base extension (SBE) reaction and subsequent SBE product analyses via matrix-assisted time-of-flight laser desorption/ionization mass spectrometry (MALDI-TOF MS). Extracts of both products were found to contain transgenic EPO-DNA, while transgenic DNA for IGF-1 was not detected. The results were confirmed using SYBR Green qPCR with primer sets directed against EPO and IGF1 cDNA, and the CMV promotor sequence. In this case study, the detection of authentic (whilst low concentrated) transgenes, potentially intended for gene doping practices in readily available products, is reported for the first time.

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Scheme 2. Proposed dissociation pathway of SARM 2f: (a) in negative ionization mode; (b) in positive ionization mode.
Scheme 4. Proposed dissociation pathway of the deprotonated metabolite M3. Scheme 4. Proposed dissociation pathway of the deprotonated metabolite M3.
Identification and Synthesis of Selected In Vitro Generated Metabolites of the Novel Selective Androgen Receptor Modulator (SARM) 2f

July 2023

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86 Reads

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4 Citations

Molecules

Among anabolic agents, selective androgen receptor modulators (SARMs) represent a new class of potential drugs that can exhibit anabolic effects on muscle and bone with reduced side effects due to a tissue-selective mode of action. Besides possible medical applications, SARMs are used as performance-enhancing agents in sports. Therefore, they are prohibited by the World Anti-Doping Agency (WADA) in and out of competition. Since their inclusion into the WADA Prohibited List in 2008, there has been an increase in not only the number of adverse analytical findings, but also the total number of SARMs, making continuous research into SARMs an ongoing topic in the field of doping controls. 4-((2R,3R)-2-Ethyl-3-hydroxy-5-oxopyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile (SARM 2f) is a novel SARM candidate and is therefore of particular interest for sports drug testing. This study describes the synthesis of SARM 2f using a multi-step approach, followed by full characterization using liquid chromatography–high-resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance spectroscopy (NMR). To provide the first insights into its biotransformation in humans, SARM 2f was metabolized using human liver microsomes and the microsomal S9 fraction. A total of seven metabolites, including phase I and phase II metabolites, were found, of which three metabolites were chemically synthesized in order to confirm their structure. Those can be employed in testing procedures for routine doping controls, further improving anti-doping efforts.


Fig. 1 Structure of RAD140 and proposed structures of the newly described metabolites of RAD140, M2c, M8, M9, M10a, M10b, and M11. The HRMS/MS spectra can be found in Electronic supplemen-
Fig. 4 All metabolites that were detected in samples from the incubation with subcellular liver fraction experiments are shown as added analyte to IS area ratios. a Samples after phase I incubation are shown in dark blue and light blue, and the corresponding samples lacking the subcellular fractions are shown in dark green and light
Comparison of in vitro approaches for predicting the metabolism of the selective androgen receptor modulator RAD140

July 2023

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131 Reads

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5 Citations

Analytical and Bioanalytical Chemistry

The identification of metabolites allows for the expansion of possible targets for anti-doping analysis. Especially for novel substances such as selective androgen receptor modulators (SARMs), information on metabolic fate is scarce. Novel approaches such as the organ on a chip technology may provide a metabolic profile that resembles human in vivo samples more closely than approaches that rely on human liver fractions only. In this study, the SARM RAD140 was metabolized by means of subcellular human liver fractions, human liver spheroids in an organ on a chip platform, and electrochemical (EC) conversion. The resulting metabolites were analyzed with LC-HRMS/MS and compared to a human doping control urine sample that yielded an adverse analytical finding for RAD140. A total of 16 metabolites were detected in urine, while 14, 13, and 7 metabolites were detected in samples obtained from the organ on a chip experiment, the subcellular liver fraction, and EC experiments, respectively. All tested techniques resulted in the detection of RAD140 metabolites. In the organ on a chip samples, the highest number of metabolites were detected. The subcellular liver fractions and organ on a chip techniques are deemed complementary to predict metabolites of RAD140, as both techniques produce distinct metabolites that are also found in an anonymized human in vivo urine sample. Graphical abstract


How to detect CRISPR with CRISPR - employing SHERLOCK for doping control purposes

November 2022

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65 Reads

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9 Citations

The Analyst

The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) tool kit constitutes one of today's most frequently used gene editing techniques. Editing of virtually any DNA sequence can be realised, due to the quickly progressing research into different Cas effectors and their ever-expanding range of targets. Moreover, the simplicity and cost-effectiveness of those CRISPR tools can, unfortunately, also facilitate the illicit utilisation of CRISPR/Cas in order to achieve performance enhancements amongst athletes. Consequently, there is an urgent need for the direct detection of illegally applied CRISPR/Cas methods in doping control samples, for which a promising strategy is presented herein employing Specific High Sensitive Enzymatic Reporter UnLOCKing (SHERLOCK) for targeted nucleic acid detection. An analytical method was developed that enables the detection of sgRNA associated with Cas9 from Streptococcus pyogenes (SpCas9) in serum samples by means of reverse transcriptase-recombinase polymerase amplification (RT-RPA) and subsequent qualitative nucleic acid detection via SHERLOCK in combination with a complementary gel-based screening procedure in order to uncover doping attempts with lipid mediated CRISPR ribonucleoprotein (RNP) complexes. Initial qualitative method characterisation confirmed the specificity of both procedures and established a detection sensitivity of 10 nM uncomplexed target sequence and 100 pM sgRNA in the form of RNP complexes. Furthermore, a proof-of-concept in vivo adimistration study simulating a hypothetical gene doping scenario employing a mouse model revealed a detection window of 8 h after intravenous injection, supporting the principal applicability of the test strategy to authentic doping control samples in the future.

Citations (4)


... Therefore, automating most processes in doping testing is desirable as well. There are previous studies on the application of DBSs in doping testing, such as the targeting of steroids and other compounds [17][18][19][20], genetic polymorphisms [9,21,22], plasmids via the spike-in test [23], hEPO proteins with mutations [24], and mRNAs [25]. Although these previous studies described the affinity and future promise of DBSs in doping testing, there was only one report of an automated testing process [18]. ...

Reference:

Detection of Gene Doping Using Dried Blood Spots from a Mouse Model with rAAV9 Vector-Mediated Human Erythropoietin Expression as a Pilot Study
Detection of doping control sample substitutions via single nucleotide polymorphism-based ID typing
  • Citing Article
  • November 2023

Drug Testing and Analysis

... Psychological interventions such as mental skills training and biofeedback enhance focus and stress management (Lindsay et al., 2023). Recovery technologies like cryotherapy (Poignard et al., 2023) and Normatec systems (Gras, 2023) accelerate recovery, and genetic research explores the potential of gene editing and epigenetics in sports performance (Naumann et al., 2023). Data analytics and machine learning aid in performance analysis and injury prevention, while VR (Virtual Reality) (Richlan et al., 2023b) and AR (Augmented Reality) (Cossich et al., 2023) technologies create immersive training environments. ...

Analysis of Potential Gene Doping Preparations for Transgenic DNA in the Context of Sports Drug Testing Programs

International Journal of Molecular Sciences

... Among them, studies with subcellular fractions, such as microsomes and the S9 fraction, usually provide a high number of metabolites and are useful to quickly generate many potential metabolites and obtain retention times (RTs) and mass spectra to be matched with in vivo samples. Combined with advanced analytical techniques, especially UHPLC-HRMS-based approaches, remarkable progress has been made in the investigation of doping metabolism based on in vitro experiments [12,13]. However, some issues remain challenging. ...

Identification and Synthesis of Selected In Vitro Generated Metabolites of the Novel Selective Androgen Receptor Modulator (SARM) 2f

Molecules

... [117] The authors achieved a quantitative recovery that was independent of the samples' hematocrit with an optimized impact-assisted extraction Leuenberger et al. [118] mentioned DBS for RNA analysis, however, discussed them as biomarkers for substance misuse rather than as and amplified RNA. [119] Only few methods are reported for targeting oligonucleotide therapeutics in urine samples. The analysis of rat urine samples after treatment with two potential myostatin-inhibiting siRNAs was reported by Thomas et al. [120] These ...

How to detect CRISPR with CRISPR - employing SHERLOCK for doping control purposes
  • Citing Article
  • November 2022

The Analyst