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3. Analytical advances in drug monitoring in sport


Anabolic Agents. Stanozolol was the highest reported compound among the anabolic agents in 2017 [1]. Before 2014, the trend for this drug class showed a higher occurrence for “testosterone/epitestosterone –T/E- ratio > 4”. While numbers relating to T/E findings have probably decreased due to the adaptation of the athlete biological passport (ABP), the increased frequency in stanozolol findings is a product of the use of improved liquid chromatography mass spectrometry (LC-MS) protocols, which facilitate the detection of more polar steroids, together with the implementation of data interpretation on their metabolism through the usage of high-resolution mass spectrometry (HRMS) [2]. Progress has also been made recently on the increasing effective use of gas chromatography combustion isotope ratio mass spectrometry (GC/C/IRMS), a technique that is fundamental in distinguishing between the exogenous and endogenous origin of steroids such as testosterone [3]. In this regard, GC/C/IRMS is the gold standard technique particularly when used in combination with the ABP, as IRMS can effectively distinguish between atypical steroid profiles that result from doping,


and those that result from other factors such as alcohol consumption. Despite a few limitations in the evaluation of ABP, such as in some female athlete cases where steroid concentrations may be very low, transdermal testosterone application and DHEA [4], its adoption has been successful. As the ABP requires numerous data points to be collected, the number of steroidal ABP tests increased by 13% in 2017 [1] and will increase further. Moreover, steroidal ABP would benefit from the inclusion of other steroid markers to make it more effective.


Stimulants. Methylphenidate (19 %), amphetamine (18%) and cocaine (12 %) are compounds that have seen the highest occurrence in AAFs [1]. The WADA prohibited list has been modified several times in order to comply with new emerging trends in misused stimulants, as in the case of the introduction of synthetic cathinones and the re-classification of 3,4-Methylenedioxymethamphetamine (MDMA) and


3,4-Methylenedioxyamphetamine (MDA) as doping agents. This list has also been updated to re-classify substances for which metabolism studies have clarified their involvement in the production of the banned amphetamine and methamphetamine [1]. Ad-hoc analytical methods are often used


for their screening (e.g. those using HRMS) and confirmation is performed by LC-MS/MS (i.e. triple quadrupole) or GC-MS.


Peptide hormones, Growth Factors and Related Substances. In 2017, within this drug class, the detection of erythropoietin (EPO) was high in samples analysed (48%), followed by the analogues and human chorionic gonadotropin (hCG) at 12% [1]. The latter is a heterodimeric glycoprotein, used to stimulate natural production of steroids after the intake of synthetic ones, and is analysed mainly by immunoassays. However, recently one of the first confirmation methods by LC-MS/MS for proteins was applied to hCG in urine. An increased number of tests for Erythropoiesis Stimulating Agents (ESAs), human Growth Hormone (hGH) and GH Releasing Factors (GHRF) has been performed in recent years despite the relative low number in AAFs for monitoring purposes. Major research on this drug class has been undertaken in London to investigate more sensitive analytical methods suitable to screen and confirm with low detection limits, and to explore the excretion profile of these substances. After hCG, ibutamoren, a GH secretagogue that mimics the endogenous GH ghrelin, is the next most prevalent substance and has been reported with an occurrence of 8% [1].


AAFS PER DRUG CLASS AS REPORTED IN ADAMS IN 2017


Figure 1: AAFs per drug class as reported in ADAMS in 2017 by (a) the WADA-accredited Laboratory in London (UK) (n=78) (b) compared to total AAFs reported by all WADA-accredited laboratories (n=4076). Data from the pie chart have been extrapolated by Table reported in ‘2017 Anti-Doping Testing Figures’ report and re-arranged.


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