First Steps toward Uncovering Gene Doping with CRISPR/Cas by Identifying SpCas9 in Plasma via HPLC-HRMS/MS

25 Nov 2020

First Steps toward Uncovering Gene Doping with CRISPR/Cas by Identifying SpCas9 in Plasma via HPLC-HRMS/MS / Alina Paßreiter, Andreas Thomas, Nicolas Grogna, Philippe Delahaut, Mario Thevis. - (Analytical Chemistry 92 (2020) 24 (15 December); p. 16322-16328)

  • PMID: 33237723
  • DOI: 10.1021/acs.analchem.0c04445


The discovery of the clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas) system as a programmable, RNA-guided endonuclease has revolutionized the utilization of gene technology. Because it enables the precise modification of any desired DNA sequence and surpasses all hitherto existing alternatives for gene editing in many ways, it is one of the most frequently used tools for genome editing. However, these advantages also potentially facilitate the illicit use of the CRISPR/Cas system in order to achieve performance-enhancing effects in sporting competitions. This abuse is classified as gene doping, which is banned in sports according to the Prohibited List of the World Anti-Doping Agency (WADA). Therefore, there is a pressing need for an adequate analytical method to detect the misuse of the CRISPR/Cas system by athletes. Hence, the first aim accomplished with this study was the identification of the exogenous protein Cas9 from the bacterium Streptococcus pyogenes (SpCas9) in plasma samples by means of a bottom-up analytical approach via immunoaffinity purification, tryptic digestion, and subsequent detection by HPLC-HRMS/MS. A qualitative method validation was conducted with three specific peptides allowing for a limit of detection of 25 ng/mL. Additionally, it was shown that the developed method is also applicable to the detection of (illicit) gene regulation through the identification of catalytically inactive Cas9. A proof-of-concept administration study employing an in vivo mouse model revealed a detection window of SpCas9 for up to 8 h post administration, confirming the suitability of the test strategy for the analysis of authentic doping control samples.

Detecting the misuse of 7-oxo-DHEA by means of carbon isotope ratio mass spectrometry in doping control analysis

6 Mar 2020

Detecting the misuse of 7-oxo-DHEA by means of carbon isotope ratio mass spectrometry in doping control analysis / Thomas Piper, Gregor Fusshöller, Hans Geyer, Ani Toboc, Mădălin-George Dănilă, Mario Thevis. - (Rapid Communications in Mass Spectrometry 34 (2019) 12 (30 June); p. 1-2)

  • PMID: 32143236
  • DOI: 10.1002/rcm.8776


Rationale: The misuse of 7-oxo-DHEA (3β-hydroxyandrost-5-ene-7,17-dione) is prohibited according to the World Anti-Doping Agency (WADA) code. Nevertheless, it is easily available as a dietary supplement and from black market sources. In two recent doping control samples, significant amounts of its main metabolite 7β-OH-DHEA were identified, necessitating further investigations.

Methods: As both 7-oxo-DHEA and 7β-OH-DHEA are endogenously produced steroids and no concentration thresholds applicable to routine doping controls exist, the development and validation of a carbon isotope ratio (CIR) mass spectrometry method ha been desirable. Excretion studies encompassing 7-oxo-DHEA, 7-oxo-DHEA-acetate, and in-house deuterated 7-oxo-DHEA were conducted and evaluated with regard to urinary CIR and potential new metabolites of 7-oxo-DHEA.

Results: Numerous urinary metabolites were identified, some of which have not been reported before, while others corroborate earlier findings on the metabolism of 7-oxo-DHEA. The CIRs of both 7-oxo-DHEA and 7β-OH-DHEA were significantly influenced for more than 50 h after a single oral dose of 100 mg, and a novel metabolite (5α-androstane-3β,7β-diol-17-one) was found to prolong this detection time window by approximately 25 h. Applying the validated method to routine doping control specimens presenting atypically high urinary 7β-OH-DHEA levels clearly demonstrated the exogenous origin of 7-oxo-DHEA and 7β-OH-DHEA.

Conclusions: As established for other endogenously produced steroids such as testosterone, the CIR allows for a clear differentiation between endo- and exogenous sources of 7-oxo-DHEA and 7β-OH-DHEA. The novel metabolites detected after administration may help to improve the detection of 7-oxo-DHEA misuse and simplify its detection in doping control specimens.

Elevated urinary cobalt concentrations identified in routine doping controls can originate from vitamin B12

12 Nov 2019

Elevated urinary cobalt concentrations identified in routine doping controls can originate from vitamin B12 / Andre Knoop, Christian Görgens, Hans Geyer, Mario Thevis. - (Rapid Communications in Mass Spectrometry 34 (2019) 7 (15 April); p. 1-3)

  • DOI: 10.1002/rcm.8649

Cyanocobalamin was unequivocally identified in the doping control urine sample by HRMS. The intact molecular ion was observed in full‐MS measurements, and diagnostic product ions obtained by collision‐induced dissociation were detected and matched previous publications.21 It is noteworthy that a concentration of 565 ng/mL was calculated using the prepared calibration curve, computed by signal area ratios of the target analyte and the employed internal standard (fivefold deuterated isoxsuprine, ISTD).

The presented data indicate the importance of the careful assessment of total urinary cobalt concentrations in the context of routine sports drug testing programs and, potentially, also in other clinical applications. The determination of inorganic cobalt and the differentiation from cyanocobalamin can be accommodated by simple SPE‐based depletion of the cobalt‐containing organic molecule and subsequent analysis of the extract's flow‐through by ICP‐MS. Alternatively, routine tests by means of LC‐ICP‐MS and/or concomitant analyses of urine samples for vitamin B12 are recommended for adequate result interpretation and management.

An in vitro assay approach to investigate the potential impact of different doping agents on the steroid profile

7 Dec 2020

An in vitro assay approach to investigate the potential impact of different doping agents on the steroid profile / Thomas Piper, Sonja Heimbach, Martin Adamczewski, Mario Thevis. - (Drug Testing and Analysis (2020) 7 December; p. 1-13)

  • PMID: 33283964
  • DOI: 10.1002/dta.2991


The steroid profile, that is, the urinary concentrations and concentration ratios of selected steroids, is used in sports drug testing to detect the misuse of endogenous steroids such as testosterone. Since several years, not only population-based thresholds are applied but also the steroid profile is monitored via the Athlete Biological Passport whereby the individual reference ranges derived from multiple test results of the same athlete are compared to population-based thresholds. In order to maintain a high probative force of the passport, samples collected or analyzed under suboptimal conditions should not be included in the longitudinal review. This applies to biologically affected or degraded samples and to samples excluded owing to the presence of other substances potentially (or evidently) altering the steroid profile. Nineteen different doping agents comprising anabolic steroids, selective androgen receptor modulators, selective estrogen receptor modulators, ibutamoren, and tibolone were investigated for their effect on the steroid profile using an androgen receptor activation test, an androgen receptor binding assay, an aromatase assay, and a steroidogenesis assay. The in vitro tests were coupled with well-established liquid chromatography/mass spectrometry-based analytical approaches and for a subset of steroidal analytes by gas chromatography/mass spectrometry. The variety of tests employed should produce a comprehensive data set to better understand how a compound under investigation may impact the steroid profile. Although our data set may allow an estimate of whether or not a substance will have an impact on the overall steroid metabolism, predicting which parameter in particular may be influenced remains difficult.

Mass spectrometric identification and characterization of urinary metabolites of isopropylnorsynephrine for doping control purposes

5 Feb 2021

Mass spectrometric identification and characterization of urinary metabolites of isopropylnorsynephrine for doping control purposes / Oliver Krug, Andreas Thomas, Mario Thevis. - (Analytical Science Advances (2021) 5 February;  p. 1-8)

  • DOI: 10.1002/ansa.202100004


Isopropylnorsynephrine (isopropyloctopamine, deterenol, 4‐(1‐hydroxy‐2‐(isopropylamino)ethyl)phenol), a beta‐selective and direct‐acting adrenergic agonist, has been reported in the past as declared as well as non‐declared ingredient of dietary supplements. The proven biological activity and the structural similarity of isopropylnorsynephrine to substances classified as prohibited compounds according to the World Anti‐Doping Agency's (WADA's) regulations could necessitate the inclusion of this sympathomimetic amine into routine doping control analytical assays. Therefore, information on urinary metabolites is desirable in order to allow for an efficient implementation of target compounds into existing multi‐analyte testing procedures, enabling the unequivocal identification of the administration of isopropylnorsynephrine by an athlete. In a pilot study setting, urine samples were collected prior to and after the oral application of ca. 8.7 mg of isopropylnorsynephrine, which were subjected to liquid chromatography‐high resolution/high accuracy (tandem) mass spectrometry. The intact drug, hydroxylated and/or glucurono‐ or sulfo‐conjugated isopropylnorsynephrine were detected up to 48 h post‐administration, with isopropylnorsynephrine sulfate representing the most abundant urinary target analyte. No relevant amounts of the dealkylation product (octopamine) were observed, indicating that merely moderate adaptations of existing test methods (or data evaluation strategies) are required to include isporpoylnorsynephrine in antidoping analytics, if required.

The potential role of oral fluid in antidoping testing

1 Feb 2014

The potential role of oral fluid in antidoping testing / Sebastien Anizan, Marilyn A. Huestis. - (Clinical Chemistry 60 (2014) 2 (1 February); p. 307-322)

  • PMID: 24153253
  • PMCID: PMC4545493
  • DOI: 10.1373/clinchem.2013.209676


Background: Currently, urine and blood are the only matrices authorized for antidoping testing by the World Anti-Doping Agency (WADA). Although the usefulness of urine and blood is proven, issues remain for monitoring some drug classes and for drugs prohibited only in competition. The alternative matrix oral fluid (OF) may offer solutions to some of these issues. OF collection is easy, noninvasive, and sex neutral and is directly observed, limiting potential adulteration, a major problem for urine testing. OF is used to monitor drug intake in workplace, clinical toxicology, criminal justice, and driving under the influence of drugs programs and potentially could complement urine and blood for antidoping testing in sports.

Content: This review outlines the present state of knowledge and the advantages and limitations of OF testing for each of the WADA drug classes and the research needed to advance OF testing as a viable alternative for antidoping testing.

Summary: Doping agents are either prohibited at all times or prohibited in competition only. Few OF data from controlled drug administration studies are available for substances banned at all times, whereas for some agents prohibited only in competition, sufficient data may be available to suggest appropriate analytes and cutoffs (analytical threshold concentrations) to identify recent drug use. Additional research is needed to characterize the disposition of many banned substances into OF; OF collection methods and doping agent stability in OF also require investigation to allow the accurate interpretation of OF tests for antidoping monitoring.

Subject-based steroid profiling and the determination of novel biomarkers for DHT and DHEA misuse in sports

1 Dec 2010

Subject-based steroid profiling and the determination of novel biomarkers for DHT and DHEA misuse in sports / Pieter Van Renterghem, Peter Van Eenoo, Pierre-Edouard Sottas, Martial Saugy, Frans Delbeke. - (Drug Testing and Analysis 2 (2010) 11-12 (November); p. 582-588)

  • PMID: 21204290
  • DOI: 10.1002/dta.206
  • Special Issue: 28th Cologne Workshop: Advances in Sports Drug Testing


Doping with natural steroids can be detected by evaluating the urinary concentrations and ratios of several endogenous steroids. Since these biomarkers of steroid doping are known to present large inter-individual variations, monitoring of individual steroid profiles over time allows switching from population-based towards subject-based reference ranges for improved detection. In an Athlete Biological Passport (ABP), biomarkers data are collated throughout the athlete's sporting career and individual thresholds defined adaptively. For now, this approach has been validated on a limited number of markers of steroid doping, such as the testosterone (T) over epitestosterone (E) ratio to detect T misuse in athletes. Additional markers are required for other endogenous steroids like dihydrotestosterone (DHT) and dehydroepiandrosterone (DHEA). By combining comprehensive steroid profiles composed of 24 steroid concentrations with Bayesian inference techniques for longitudinal profiling, a selection was made for the detection of DHT and DHEA misuse. The biomarkers found were rated according to relative response, parameter stability, discriminative power, and maximal detection time. This analysis revealed DHT/E, DHT/5β-androstane-3α,17β-diol and 5α-androstane-3α,17β-diol/5β-androstane-3α,17β-diol as best biomarkers for DHT administration and DHEA/E, 16α-hydroxydehydroepiandrosterone/E, 7β-hydroxydehydroepiandrosterone/E and 5β-androstane-3α,17β-diol/5α-androstane-3α,17β-diol for DHEA. The selected biomarkers were found suitable for individual referencing. A drastic overall increase in sensitivity was obtained. The use of multiple markers as formalized in an Athlete Steroidal Passport (ASP) can provide firm evidence of doping with endogenous steroids.

Detection of testosterone administration based on the carbon isotope ratio profiling of endogenous steroids: international reference populations of professional soccer players

22 Jun 2009

Detection of testosterone administration based on the carbon isotope ratio profiling of endogenous steroids : international reference populations of professional soccer players / E. Strahm, C. Emery, M. Saugy, J. Dvorak, C. Saudan. - (International Journal of Sports Medicine 25 (2004) 13 (December); p. 528-543)

  • PMID: 19549614
  • PMCID: PMC2784500
  • DOI: 10.1136/bjsm.2009.058669


Background and objectives: The determination of the carbon isotope ratio in androgen metabolites has been previously shown to be a reliable, direct method to detect testosterone misuse in the context of antidoping testing. Here, the variability in the 13C/12C ratios in urinary steroids in a widely heterogeneous cohort of professional soccer players residing in different countries (Argentina, Italy, Japan, South Africa, Switzerland and Uganda) is examined.

Methods: Carbon isotope ratios of selected androgens in urine specimens were determined using gas chromatography/combustion/isotope ratio mass spectrometry (GC-C-IRMS).

Results: Urinary steroids in Italian and Swiss populations were found to be enriched in 13C relative to other groups, reflecting higher consumption of C3 plants in these two countries. Importantly, detection criteria based on the difference in the carbon isotope ratio of androsterone and pregnanediol for each population were found to be well below the established threshold value for positive cases.

Conclusions: The results obtained with the tested diet groups highlight the importance of adapting the criteria if one wishes to increase the sensitivity of exogenous testosterone detection. In addition, confirmatory tests might be rendered more efficient by combining isotope ratio mass spectrometry with refined interpretation criteria for positivity and subject-based profiling of steroids.

Norandrosterone and noretiocholanolone concentration before and after submaximal standardized exercise

25 Oct 2004

Norandrosterone and noretiocholanolone concentration before and after submaximal standardized exercise / B. de Geus, F. Delbeke, R. Meeusen, P. Van Eenoo, K. De Meirleir, B. Busschaert. - (International Jurnal of Sports Medicine 25 (2004) 7; p. 528-543)

  • PMID: 15459834
  • DOI: 10.1055/s-2004-820954


19-Norandrosterone (19-NA) and 19-noretiocholanolone (19-NE) are the two main urinary indicators used to detect illegal use of nandrolone. Recent studies showed that 19-NA and 19-NE can be endogenously produced in non-treated humans. The concentrations were close to the threshold of the International Olympic Committee (IOC), i.e. 2 ng/ml for men and seem to increase after prolonged intense effort. Androgens are involved in the biosynthesis of estrogens and estrogen has a protective effect against skeletal muscle damage following eccentric exercise. Furthermore, the testicular tissue can synthesize 19-norandrogens from androgens, we hypothetisize that the 19-norandrogen production might be influenced by muscle damage following eccentric exercise. Therefore the purpose of this study is to examine if three different exercise methods will influence the urinary concentration of 19-NA and 19-NE in healthy young subjects. Fifteen amateur hockey players undertook a 30 min submaximal standardized exercise protocol. They were randomised for three different types of exercise, namely a cycle ergometer test (cyclic muscle activity), a treadmill test (concentric muscle activity), or a bench-steptest (eccentric muscle activity) at a target heart rate corresponding to 65 % (+/- 5 %) of Karvonen heart rate. Urine samples were obtained before the test and 60 min and 120 min after the end of exercise. Subjects completed a Likert scale of muscle soreness before and 12 h after exercise. 19-NA and 19-NE were determined by gas chromatography-tandem mass spectrometry (GC-MS-MS). Baseline urinary 19-NA and 19-NE concentrations were under limit of detection of 0.05 ng/ml, except for one sample (0.13 ng/ml). No 19-NA or 19-NE could be detected post exercise. In our experimental conditions, the exercise mode (eccentric or concentric) had no impact on 19-NA or 19-NE excretion. Our findings confirm that the current International Olympic Committee threshold level for nandrolone metabolites is sufficiently high to avoid false positive cases.

Profiling of 19-norandrosterone sulfate and glucuronide in human urine: implications in athlete's drug testing

1 Mar 2021

Profiling of 19-norandrosterone sulfate and glucuronide in human urine : implications in athlete's drug testing / Emmanuel Strahm, Norbert Baume, Patrice Mangin, Martial Saugy, Christiane Ayotte, Christophe Saudan. - (Steroids 74 (2009) 3 (March); p. 359-364)

  • PMID: 19056413
  • DOI: 10.1016/j.steroids.2008.11.005


19-Norandrosterone (19-NA) as its glucuronide derivative is the target metabolite in anti-doping testing to reveal an abuse of nandrolone or nandrolone prohormone. To provide further evidence of a doping with these steroids, the sulfoconjugate form of 19-norandrosterone in human urine might be monitored as well. In the present study, the profiling of sulfate and glucuronide derivatives of 19-norandrosterone together with 19-noretiocholanolone (19-NE) were assessed in the spot urines of 8 male subjects, collected after administration of 19-nor-4-androstenedione (100mg). An LC/MS/MS assay was employed for the direct quantification of sulfoconjugates, whereas a standard GC/MS method was applied for the assessment of glucuroconjugates in urine specimens. Although the 19-NA glucuronide derivative was always the most prominent at the excretion peak, inter-individual variability of the excretion patterns was observed for both conjugate forms of 19-NA and 19-NE. The ratio between the glucuro- and sulfoconjugate derivatives of 19-NA and 19-NE could not discriminate the endogenous versus the exogenous origin of the parent compound. However, after ingestion of 100mg 19-nor-4-androstenedione, it was observed in the urine specimens that the sulfate conjugates of 19-NA was detectable over a longer period of time with respect to the other metabolites. These findings indicate that more interest shall be given to this type of conjugation to deter a potential doping with norsteroids.

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