Analytical Approaches in Sports Drug Testing

The guidelines and implementations regarding anti-doping policies and drug testing were first brought about by the World Anti-Doping Agency (WADA) in 1999, which took the form of the World Anti-Doping Code.

Doping in Sport Concept

Image Credit: ADragan/

The implementation and support of this policy have not faltered, as those who are part of this agency ardently work to preserve the integrity of many sports. This code has aided organizations such as UEFA, FIFA, the NFL, and many others by hindering “performance enactment deceits.” This is realized by the code through its many technical aspects, enforced through a list of prohibited substances and methods, laboratories, testing and investigations, therapeutic use exemptions, and protection of privacy and personal information.

Lab work conducted through the (WADA)

Within our current paradigm of drug testing, apparatus such as gas chromatography (GC) and liquid chromatography (LC) are fit with mass spectrometry (MS) and are found at the forefront of molecular assays. They are used to report a surfeit of endogenous anabolic agents. This is all done to construct an Athlete Biological Passport (ABP), an electronic report that functions as an ethical code for performers and athletes alike.

The key analytes and compounds that are found within WADA’s list of prohibited substances are classified into ten (S0-S9) and three (M1-M3) differing categories. Anabolic steroids, amphetamines, and other stimulants are regularly screened for, while other drugs such as alcohol and β-blocker are substrates barred from some sports, but not others.

To garner homogeneous results between all sports and practices, a Minimum Required Performance Levels (MRPL) for analytical methods was created by the WADA. This establishes a minimum capability for the detection of non-threshold substances such as Molidustat, Clenbuterol, and Stanozolol. Threshold compounds such as Salbutamol and Formoterol are instead covered by other dedicated documents.

Common Testing performed by the (WADA)

The biological media that are most scrupulously assayed are urine and blood (which pertains to whole blood, serum, and plasma. These are the matrices of choice when analyzing for the roughly 250 compounds the WADA list for, conferring a diverse range of physicochemical properties. Narrowing our scope, these practices concern direct detection methods of small molecules, proteins, and peptides, requiring high sensitivity and selectivity.

Small molecules vary in polarity, molecular weight, acido-basic properties, etc. These caveats come with their own set of challenges and will thus require different methodologies to cover all categories- ensuring the quality of all analytical results.

Chromatographic methods are the golden standard for testing biological media. Sample preparation is ultimately the most curtail step in this process, on account of the heterogenic nature of analytes like lipids, proteins, and other particles.

Three of the most common “sample preparation practices” include solid-phase extraction (SPE), liquid-liquid extraction (LLE), or supported-liquid extraction (SLE), each one harboring its own balance between percent recovery, and purity of extract. The practice of choice is dependent on both the physicochemical property of the drug and the analytical instrumentation employed.

Liquid-Liquid extraction LLE

Liquid-liquid extraction encompasses an alternative for separation, geared towards compounds that cannot be separated via distillation. The low-temperature environment in this methodology is ideal when separating heat-sensitive compounds. These include anabolic androgenic steroids, which are monitored at trace levels.

In addition, LLE procedures deliver a higher sensitivity when incorporated with LC-MS and GC-MS platforms. Yet, cons are apparent in this procedure, as the technique itself is not apt when assaying substrates with polar functional groups. Furthermore, a greater amount of both sample and solvent is needed, and two extractions in acidic and basic conditions are often required.

Supported-Liquid Extraction (SLE)

This form of sample preparation is thought of as a streamlined, automated version of LLE, where an aqueous biological mobile phase is adsorbed through a diatomaceous earth stationary phase. When a non-miscible solvent is pulled through, accompanying the mobile phase, the extraction and elution of analytes are carried out, which can later be quantified via spectrometry.

SLE bypasses the requirements for phase separation that is fixed to LLE, circumventing issues with emulsion, and ultimately yielding a faster sample preparation altogether. This sample is most suitable for glucocorticoids, because of their low polarity.

Solid-Phase Extraction (SPE)

This variant of sample preparation was first employed during the 2012 Olympic games in London and embodies a good alternative to both LLE and SLE. This technique is vastly different from those prior and is used in the analysis of anabolic agents, β2-agonists, hormone antagonists, narcotics, glucocorticoids, diuretics, and β- blockers. This method of sample preparation constitutes less solvent consumption, and ease of automation.

Contrary to the name, this approach is most often incorporated into urine samples- used to analyze the ionizable compounds above that have a limited metabolism. The minimum levels of detection within the urine matrix, range from tens to hundreds of ng/mL, allowing for the retention of almost all compounds.

This sample preparation approach is used for the screening of many illicit substrates and incorporates the nonselective “dilute-and-shoot” procedure to limit matrix effects. This is where samples are diluted using an isotope-labeled internal standard (IL-ISTD), followed by a direct injection onto LC-MS systems, often used in forensic toxicology.

When determining the origin of any analyte, whether it be endogenously produced, or derived from an exogenous source, these techniques above have aided the WADA over the past 2 decades, applying the very best of isotope ratio mass spectrometry (IRMS).

By preparing samples through the three practices above, the distinctions in carbon and hydrogen isotopes between endogenous biosynthesized molecules, and exogenous analogs can be accomplished, enforcing the veracity of results, and the integrity of sports.


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Further Reading

Last Updated: Sep 27, 2021

Vasco Medeiros

Written by

Vasco Medeiros

Obtaining an International Baccalaureate Degree at Oeiras International School, with higher levels in Chemistry, Biology, and Portuguese, Vasco Medeiros has just graduated from the University of Providence College with a Bachelor of Science. Before his work as an undergraduate, he first began his vocational training at the HIKMA Pharmaceuticals PLC plant in Ribeiro Novo. Here he worked as a validation specialist, tasked with monitoring the gauging and pressure equipment of the plant, as well as the inspection of weights and products.


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