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Antidoping detection strategies to combat emerging threats
Robust doping control measures are essential to protect the belief that victory is earned through hard work, talent, and dedication, rather than unethical shortcuts. Utilizing highly sensitive and high-resolution analytical technologies is paramount in the fight against doping in sports. These advanced analytical tools help enable detection of even minuscule traces of prohibited substances, leaving minimal opportunity for undetected doping.
Prohibited substance screening
Initial testing procedures (ITPs) efficiently screen urinary or blood samples for numerous prohibited substances, at times requiring high-resolution mass spectrometry with resolution over 60,000 FWHM and mass errors under 2 ppm. These assays for direct urine analysis target substances such as steroids, stimulants, corticoids, metabolic modulators, diuretics, narcotics, cannabinoids, and low molecular mass peptides.
The table below provides a general overview of doping control analysis including the classes of prohibited compounds and the mainly applied methodology.
| LC-MS, LC-MS/MS, LC-HRAM MS | GC-MS or GC-MS/MS | Alternative analysis |
|---|---|---|
Glucocorticoids Stimulants Anabolic agents β2-agonists β-blocker Narcotics Metabolic modulators Diuretics Peptides (small & large) Proteases siRNA |
Exogenous steroids Endogenous steroids Stimulants Narcotics β2-agonists Cannabinoids Anabolic agents Xenon |
Erythropoiesis stimulating agents Growth hormone (GH) Chorionic gonadotrophin (CG) and luteinizing hormone (LH) Hematological parameters Blood transfusion Gene doping |
| Initial testing procedures (ITP) Confirmation procedures (CP) |
Initial testing procedures (ITP) Confirmation procedures (CP) |
Initial testing procedures (ITP) Confirmation procedures (CP) |
High-Resolution MS: Orbitraps, time-of-flight MS LC-MS/MS: triple-quadrupoles, linear ion traps Ionization: ESI, APCI |
GC-MS: single quadrupoles GC-MS/MS: triple quadrupoles GC-C-IRMS: single magnetic sector analyzer Ionization: EI |
ECLIA IEF, SDS-SAR-PAGE Flowcytometry ILMA PCR |
*Abbreviations: GC-MS: gas chromatography mass spectrometry, LC-MS: liquid chromatography mass spectrometry, HRMS: high-resolution mass spectrometry, IRMS: isotope-ratio mass spectrometry, C: combustion, EI: electron ionization, ESI: electro spray ionization, APCI: atmospheric pressure chemical ionization, SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis, ECLIA: electrochemiluminescence immunoassay, IEF: isoelectric focusing, SAR: sarcosyl, ILMA: immunoluminometric assay, PCR: polymerase chain reaction.
Typical prohibited substance screening workflows
- Sample preparation
- Targeted screening and confirmation
- Data analysis
Solid phase extraction (SPE) is an effective and versatile extraction technique for small peptides and other prohibited substances associated with the field of doping. Starting with urine-based samples, laboratories will need to utilize a multi-step preparation to maximize system sensitivity.
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Targeted screening focuses on identifying specific known substances, such as anabolic steroids, stimulants, or erythropoiesis-stimulating agents, using pre-defined analytical methods like liquid chromatography-mass spectrometry (LC-MS) or gas chromatography-mass spectrometry (GC-MS). Over the past decade, the majority of targeted screening of the initial testing procedures (ITP) utilized in testing labs have employed high-resolution analytical techniques to capture a wide range of compounds, including new designer drugs or unusual biomarkers of doping. An independent, confirmatory analysis of the suspicious sample follows and is focused solely on the prohibited substances and its metabolite.
 Thermo Scientific TSQ 9610 Triple Quadrupole GC-MS |
 Thermo Scientific TSQ Altis Plus Triple Quadrupole Mass Spectrometer |
 Thermo Scientific Orbitrap Exploris 120 Mass Spectrometer* |
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*The Thermo Scientific Orbitrap Exploris 240 may also be used for prohibited substance screening.
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Thermo Scientific TraceFinder data acquisition and processing software is widely used for the analysis of doping compounds, offering streamlined data processing and high-throughput screening capabilities. It helps enable targeted detection by integrating LC-MS data with automated peak identification, quantification, and reporting. As an alternative, Thermo Scientific Chromeleon Chromatography Data System Software may also be employed.
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Doping control with retrospective analysis
High-resolution accurate mass (HRAM) mass spectrometry (MS) has become the technology of choice for precise mass measurements and the ability to detect new doping markers. Widely used in doping analysis, laboratories trust Thermo Scientific proprietary Orbitrap mass spectrometers, which are capable of running thousands of sample injections for the comprehensive screening of both known and unknown performance-enhancing substances.
High-resolution Orbitrap MS enables retrospective analysis of stored samples to confirm new illicit substances. The International Testing Agency (ITA) stores athlete samples for 10 years, deterring misuse of performance-enhancing substances. Combined with retrospective analysis, it helps detect new metabolites, extend detection windows, and monitor an athlete's biological passport (ABP) for longitudinal profiling.
Typical doping control with retrospective analysis workflows
- Sample preparation
- Screen, confirm, and quantify
- Data analysis
Solid phase extraction (SPE) is an effective and versatile extraction technique for small peptides and other prohibited substances associated with the field of doping. Starting with urine-based samples, laboratories will need to utilize a multi-step preparation to maximize system sensitivity.
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Steroid quantitation and confirmation
Steroid quantitation and confirmation are critical in doping control to detect the misuse of anabolic steroids, corticosteroids, and other performance-enhancing substances. Analytical instrumentation such as mass spectrometers play a vital role in helping to ensure the accuracy, sensitivity, and reliability of these detections.
GC-triple quadrupole (QQQ) MS and GC-high resolution accurate mass MS continue to play a crucial role in antidoping laboratories, especially for analyzing (naturally occurring) androgens in steroid profile studies and detecting anabolic androgenic steroids (AAS) and their metabolites. Additionally, GC coupled with isotope ratio or combustion isotope ratio mass spectrometry (GC/C/IRMS) enhances sensitivity, distinguishing natural from synthetic steroids.
Athlete Biological Passport testing
Antidoping laboratories use two main strategies. The first involves analyzing a single sample, typically at competitive events, to detect prohibited substances. The second is longitudinal testing, where results are part of a broader performance profiling framework. This approach forms the basis of the Athlete Biological Passport (ABP), which monitors the effects of doping on the body over time, which allows anti-doping organizations to establish individual baseline profiles for each athlete. By tracking fluctuations outside an athlete's normal range, the ABP helps identify potential doping practices, including steroid abuse.
Endogenous anabolic androgenic steroids testing workflow
- Sample preparation
- Screen, quantify and profile
- Data analysis
- Isotopic confirmation
Screening for anabolic steroid misuse and its metabolites in athletes is essential to maintaining integrity in sports. Accurate quantification of endogenous versus exogenous steroids requires consistent sample preparation. Thermo Fisher Scientific provides advanced sample preparation solutions for steroid analysis using highly sensitive techniques like mass spectrometry.
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Anabolic-androgenic steroids (AAS) remain prevalent in doping, despite known health risks, including hormonal, cardiovascular, and psychological effects. In 2022, AAS accounted for over 86% of anabolic agent-related Adverse Analytical Findings (AAFs), highlighting the need for improved antidoping testing. Mass spectrometers enable the identification of adverse analytical findings (AAF), which occur when prohibited substances or their markers are found in an athlete's sample, indicating a potential violation of antidoping regulations.
Thermo Scientific TSQ 9610 Triple Quadrupole GC-MS |
Thermo Scientific TSQ Altis Plus Triple Quadrupole Mass Spectrometer |
Thermo Scientific Orbitrap Exploris 120 Mass Spectrometer |
 Thermo Scientific Stellar Mass Spectrometer |
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TraceFinder data acquisition software is widely used for the analysis of doping compounds, including steroids, offering streamlined data processing and high-throughput screening capabilities. It facilitates both targeted and untargeted detection by integrating multi-channel LC, single quadrupole, triple quadrupole and high-resolution LC-MS systems, as well as IC and GC systems.
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GC coupled with isotope ratio MS or combustion isotope ratio MS (GC/C/IRMS) allows distinguishing natural and synthetic steroids by measuring carbon isotope ratio. A classic example is testosterone—both an endogenous compound in the body and an exogenous anabolic androgenic steroid. The analytical measurement of the 13 C/12 C ratio can distinguish between the endogenous steroid production versus synthetic administration of the steroid.
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Equine sports testing
With an increase in anabolic androgenic steroids (e.g.: hemapolin and oxandrolone) in positive case reports, the most dominant analytical technique within equine sports testing remains LC-MS, which includes broad screening methods to include horsehair matrices. Additionally, while primarily focused on the human doping field, the role of peptides and macromolecules such as erythropoietin (EPO), recombinant human EOP (rhuEPO) and biomarker identification (e.g. testosterone esters) are equally applicable to equine doping. Finally, the use of ion chromatography mass spectrometry (IC-MS) has emerged as a more streamlined technique for identification of bisphosphonates, a compound generally used to manage bone issues in horses.
Equine sports testing workflow
- Sample preparation
- Screen, quantify and identify
- Data analysis
Equine sport testing laboratories may run thousands of samples each year, putting their reputations on the line. The volume of samples, complex sample matrices, variety of compounds, and unique small molecules mean sample prep techniques must require minimal time while offering fast throughput. Thermo Fisher Scientific offers solutions to help maximize sample clean up and improve your analytical sensitivity.
Multiple governing bodies control doping in racehorses. These include the Fédération Equestre Internationale (FEI), Horseracing Integrity and Safety Authority (HISA), United States Equestrian Federation (USEF), Horseracing Integrity & Welfare Unit (HIWU), to name a few.
As a result, antidoping regulations can vary, sometimes significantly, from one country to another. Additionally, there is no official standardization on thresholds, screening methods, screening limits, or what to do with the B sample—the “verification” sample and how to test it. Working with industry leaders, we continue to support leading edge equine testing.
 Thermo Scientific Transcend TurboFlow System |
 Thermo Scientific Vanquish Flex with Thermo Scientific Altis Plus MS |
 Thermo Scientific Dionex ICS-6000 HPIC IC System |
 Thermo Scientific Orbitrap Exploris 240 Mass Spectrometer with Vanquish UHPLC System |
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Learn more:
TraceFinder data acquisition software is widely used for the analysis of doping compounds, including steroids, offering streamlined data processing and high-throughput screening capabilities. It facilitates both targeted and untargeted detection by integrating multi-channel LC, single quadrupole, triple quadrupole and high-resolution LC-MS systems, as well as IC and GC systems.
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Research-based analysis
- Untargeted analysis
- Data analysis
Healthcare today has witnessed the rapid development of new therapeutic approaches for various health-related diseases and disorders and offers more efficient and patient-oriented treatment options. However, these new drugs and methods can sometimes be misused to enhance athletic performance, posing challenges for antidoping programs. Additionally, the enhanced analytical power and inevitable exposure of elite athletes to environmental factors, chemicals and drugs, necessitates dedicated research in order to distinguish between unintentional contamination and intentional doping crucial for analytical testing laboratories.
 Thermo Scientific Orbitrap Exploris 240 Mass Spectrometer |
 Thermo Scientific Orbitrap Excedion Pro Mass Spectrometer |
 Thermo Scientific Orbitrap IQ-X Tribrid Mass Spectrometer |
 Thermo Scientific Orbitrap Ascend Editions Tribrid Mass Spectrometer |
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For Research Use Only. Not for use in diagnostic procedures.