Perspectives and Strategies for Anti-Doping Analysis

Bioanalysis in modern anti-doping testingCurrent doping testing in sports provides identification, often followed by quantitation, of banned substances and methods from biological samples collected from athletes in-and out-of-competition.The compounds (approximately 250) and methods included into the World Anti-Doping Agency (WADA) list of prohibited substances and methods [1] as part of the World Anti-Doping Code [2] are classified in the S0-S9 and M1-M3 categories, respectively, while alcohol (P1) and β-blockers (P2) are forbidden in selected sports.Compounds are divided into non-threshold substances, for which identification is sufficient to provide an adverse analytical finding (AAF), and threshold substances prohibited above a certain level.To harmonize the results obtained from WADA-accredited laboratories, minimum required performance levels for analytical methods have been established, defining the minimum sensitivity requirements for the analysis of non-threshold substances [3].Urine, and in some cases also hematic matrices (whole blood, serum, plasma), are considered the biological specimens of choice for routine anti-doping analysis.Advantages of urine samples include non-invasive collection and relatively large volumes available, whereas blood collection is invasive and yields a limited volume.Mainly for these reasons, most anti-doping analyses are still performed in urine, even if the prevalence of blood testing is recently increasing.Thus, investigations on the presence of a banned compound in urine are routinely carried out by means of a common workflow, starting with an initial screening followed by a confirmation procedure.The screening step should be fast, selective and sensitive enough to avoid false-negative and false-positive results.In the case of a positive screening result, it must be confirmed by targeting the identified compound and its metabolites [4].To achieve this workflow on such a wide range of compounds included in the WADA prohibited list, accredited anti-doping laboratories should use multiple analytical methodologies and first of all MS-based ones (GC-MS/MS and LC-MS/MS), considered reference methods due to their higher selectivity and sensitivity.In order to investigate the constantly evolving illicit doping practices in sports, direct screening and confirmation strategies should be continuously updated, by including the monitoring of additional substances with potential doping properties and new metabolites of known compounds with increased detection windows; by taking into account alternative biological matrices to obtain complementary data; by optimizing all the pre-analytical steps to enhance sample stability and maximize result soundness and reliability. Challenges & possible solutions in anti-doping analytical researchFrom these premises, it is clear that the challenges faced by anti-doping activities are manifold, ranging from political to technical, economical and scientific.Analytical methods, biological specimens, collection containers and pretreatment procedures are continually updated and made tighter and more sophisticated in response to ever-increasing sophistication in doping illicit