DRUG ANALYSIS IN SPORTS

Performance enhancement has featured in sports since ancient times. Doping seems to be as old as sports itself, although the word "doping" was introduced in the English dictionary for the first time in 1889. According to the International Olympic Committee (IOC), doping is the administration of or use by a competing athlete of any substance foreign to the body or any physiological substance taken in abnormal quantity or taken by an abnormal route of entry into the body with the sole purpose of increasing in an artificial and unfair manner his/her performance in competition. The drug analysis methods are designed for routine doping control to determine whether a prohibited substance is present in a doping control sample. From an outsider’s perspective, the process of doping control, spanning everything from receiving a urine or blood sample to reporting a violation of anti-doping rules, seems relatively straightforward. Nonetheless, a lot of brainpower goes into developing the process of drug analysis that the athletes’ samples go through.

Generally, urine samples are homogeneous mixtures containing many different substances, including water, urea, salt, and other electrolytes. The presence of other chemicals depends on a person’s diet, medication or supplements taken. The testing methods designed for drug analysis in an athlete’s sample are governed by the World Anti-Doping Agency (WADA). The main activities of WADA include scientific research, education, development of anti-doping capacities and monitoring the compliance with the World Anti-Doping Code. The World Anti-Doping Code includes the prohibited drugs list, which covers all the substances and methods banned from sports inand out of competition. The different compounds are divided in several classes which are forbidden either out-/and in-competition or only during competition.

For athletes, samples are analysed at one of 30 WADA accredited laboratories which covers a list of over 350 prohibited substances and methods in sports — updated every year — that samples are screened for. Doping control relies on the separation and identification of all of the substances present in a sample, as per the WADA prohibited list, whether they might be metabolites of a performance-enhancing drug, or the confounding factors which can affect the results. To ensure reliability, repeatability and clean separations, different screening methods have been developed for each of the substances on the list. Most of the banned substances require qualitative analysis. For some specific compounds, it is difficult to distinguish between the social or therapeutic use and the misuse, and, therefore, threshold concentrations have been established. In other cases, the threshold is used to differentiate between physiological values and exogenous administration of the compound.

The analytics are divided into screening procedures and methods of confirmatory analysis. The aim of screening is to isolate suspicious samples for further analysis. In confirmation, samples are analysed with methods that provide unequivocal identification of the substances. Because of the large array of target compounds, many different analytical methods are used. Most methods are based on chromatography combined with mass spectrometry (MS). Analytical procedures have to be constantly improved and updated in order to keep pace with trends in substance abuse and to fulfill increasing quality requirements.

The work of anti-doping laboratories is regulated by WADA, which ensures global harmonisation of the anti-doping procedures/guidelines. The List of Prohibited Substances and Methods includes hundreds of chemically and pharmacologically diverse compounds from different classes. The List is updated annually, which means that laboratories have to constantly update their methods and to catch up with the ever-growing selection of drugs. This imposes a considerable demand on laboratories to be able to identify suspicious samples in a screening process with only a limited sample amount in a short period of time for a more specific conformation analysis. In the case of in-competition substances, the analyte selection is the broadest and the results from screening sometimes have to be reported in just 24 hours, emphasising the need for high throughput methods. Every presumptive analytical finding in screening has to be confirmed with a more specific method optimised for the target analyte. The results are compared with reference standards, and if WADA ‘s identification criteria, such as diagnostic ions, ions ratios and retention times (RT) are fulfilled, an adverse analytical finding is reported.

The analysis of prohibited substances and methods in the athlete biological samples mainly urine and blood is a highly challenging task. Mostly because of limited sample volumes, faster turn-around time and analysis of compounds with a wide range of physico-chemical properties and molecular weight. Hence, it is always required from an anti-doping analytical method to be highly sensitive to be able to detect at very low levels and highly selective to be able to conclude that the signals are not arising from any interference. There are several issues that have to be considered before establishing an analysis method for a doping agent which involves studying the metabolism of the drug, its physicochemical properties, availability of reference standard, probability for inclusion in the existing test method as an extension etc. The approach for extension of detection windows by inclusion of drug metabolites also increases the number of target compounds. The analysis is predominantly performed on a urine sample, although few of the analytes are tested in blood viz. continuous erythropoietin receptor activator (CERA), haemoglobin-based oxygen carriers (HBOCs), human growth hormone (hGH) or blood transfusions. Other specimens such as hair and saliva have been proposed but are not used as authenticated tests in doping control. However, urine is still the specimen of choice since the collection is noninvasive, the volume available is quite large, the concentrations of drugs are higher than in blood, and since hydrophilic metabolites are also excreted in urine, thus enlarging the detection time window.

The main tools for anti-doping analysis have always been chromatography and mass spectrometry. Gas Chromatography Mass Spectrometry (GC-MS) was the gold standard technique until early 2000. The combination of GC with MS was first reported in 1958. Since then, it has become increasingly utilised in doping control. The GC-MS can separate the volatile components of complex mixtures and can record a mass spectrum of each component. This hybrid instrument provides two separate dimensions of information about the components in the sample, GC retention times and electron ionisation (EI) mass spectra. GC retention time is related to specific chemical properties of the molecules in question (e.g. volatility, polarity, presence of specific functional groups) while molecular weight (derived from the mass spectrum) is indicative of atomic composition. The limitation of GC-MS includes that only volatile substances can be measured and extensive derivatisation steps. In GC-MS techniques, sensitivity may be lost due to chemical oxidation or derivatisation and are limited to volatile, non-polar and thermally stable compounds.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) is a well-established technique for quantitative and qualitative analyses in the field of doping control. Availability of new generation LC-MS instruments have provided significant advancement in the detection of prohibited substances. The technique of liquid chromatography used in concert with (tandem) mass spectrometry has complemented sports drug testing strategies ever since soft ionisation interfaces such as ESI or APCI became commercially available. Commonly no or little derivatisation is required while preparing samples for LC-MS analysis. Numerous applications have been developed that allow the determination of prohibited therapeutics that are barely detectable or undetectable with conventional GC-MS and comprehensive summaries of the methods commonly employed in doping controls have been published in the past. Due to the progressive nature of doping controls, numerous new applications and drug-testing strategies based on LC–MS/MS are frequently developed in order to improve the portfolios of drugtesting laboratories. With the advancements in the testing technology and invention of the LC-High Resolution Mass Spectrometers (HRMS), the identification of analytes and unknown structure elucidation can be facilitated. The untargeted approach has become a promising tool in doping control.

However, for some prohibited compounds and methods neither GC– nor LC–MS(/MS) techniques are suitable to report a positive finding. This led to enforcement of few alternative strategies for detection in doping control which includes immunoassay techniques, setting up of thresholds, inclusion of Isotope Ratio Mass Spectrometry (IRMS) to differentiate between endogenous and exogenous analogs of steroids. Immunoassays or electrophoretic methods are usually employed for macromolecules. In recent years, few indirect approaches have also been established as an advancement in the doping control detection methods. For example, the monitoring of biomarkers in case of human growth hormone (hGH) to discriminate between its endogenous and exogenous administration. The monitoring of an athlete's biological passport for monitoring intra-individual variation can be utilised as an indirect marker to conclude to administration of a banned substance. For example, erythropoietin (EPO) misuse is indirectly monitored by the measurement of blood parameters, such as haemoglobin, hematocrit, ferritin, soluble transferrin receptor or reticulocyte.

Sample preparation remains the backbone of any analytical test procedures and has evolved in recent years from liquid-liquid extraction to solid phase extraction involving various chemistries. Various methods have been developed in recent years with use of reduced sample and solvent volume. The automation in sample preparation has also helped doping control laboratories in reducing their turn-around-times.

Recently, the Dried Blood Sample (DBS) methodology is also being explored in various anti-doping laboratories to enhance their technical capabilities. The literature suggests that the use of dried blood spots as a method for sample collection for anti-doping is a highly promising avenue of research, offering several advantages over existing methodology such as urine collection. which include reduced costs and ease of sample collection. The DBSs testing refers to a biosampling technique where a small volume of whole blood, typically in the 5-100 µL range (i.e. from a finger “prick”) is “spotted” and dried onto a piece of filter paper. This technique has recently been adopted to test samples from approximately 70 athletes during the Tokyo Olympic Games 2021.

Anti-doping research is ever-evolving, as new performance-enhancing drugs and derivatives keep on emerging during the process of drug discovery. Following the inclusion of a new compound on the WADA prohibited list, a screening method for it needs to be developed and validated alongside the over 350 other compounds while keeping up with the turn-around times for reporting samples. This can be especially tricky at huge sporting events, which leaves the Drug Control Centre with upward of 200 samples a day to analyse within a 24-hour turn-around.

My take on it: “The job of an antidoping scientist is for a lifetime; there are constantly new things to learn and explore. My quest for knowledge always finds its way because of the ever-improving scientific progress in the field of anti-doping, and that’s what I love about my work. This passion is reflected in my ongoing research projects: investigating flagged, suspicious peaks revealing possibly new drugs, finding analysis methods with lower limits of detection and more efficient sample preparation, and giving inspiring talks to aspiring chemists.

--Issue 54--