Multianalyte LC-MS-based methods in doping control: what are the implications for doping athletes?

Over the last 50 years, the list of doping substances and methods has been progressively expanding, being regularly reviewed by the international antidoping authorities (formerly the Medical Commission of the International Olympic Committee, and afterward, following its constitution in 1999, the World Anti-Doping Agency [WADA]). New substances/classes of substances have been periodically included in the list, keeping the pace with more advanced and sophisticated doping trends. At present, and apart from the prohibited performance enhancing and masking methods (e.g., blood transfusions and tampering strategies), the list comprises several hundreds of biologically active substances, with broad differences in their physicochemical properties (i.e., molecular weight, polarity and acid-basic properties) [1]. As a consequence, the ‘one class – one procedure’ approach, which had been followed by nearly all accredited antidoping laboratories worldwide until the turn of the millennium, is no longer sustainable. The need to minimize the overall number of independent analytical procedures, and, in parallel, to reduce the analytical costs, stimulated the development of multitargeted methods, aimed to increase the overall ratio of ‘target analytes: procedure’ [2–6]. The above evolution has not always been a straight forward process. The need to comply with the WADA technical requirements (both in terms of identification criteria and of minimum required performance limits [7,8]) and with the reduction of the reporting time (a constraint that becomes even more critical during international sport events, where the daily workload also drastically increases) has imposed a thorough re-planning of the analytical procedures. The development of an antidoping analytical method requires the appropriate knowledge not only of the biophysicochemical properties of the target analyte, but also of its PK profile. Historically, immunological methods and GC-based techniques were applied in antidoping science, as preferential screening methods for the detection of prohibited substances, which were originally limited to nonendogenous stimulants and narcotics. In the 1980s, GC–MS became the reference analytical platform for the detection and quantification of the majority of the low molecular weight doping substances [3–6]. In the following two decades, with the inclusion in the Prohibited List of new classes of low molecular weight, hydrophilic, thermolabile, nonvolatile analytes (including, but not limited to, glucocorticoids and designer steroids) and simultaneously of peptide hormones, scientists were obliged to design, develop, validate and apply techniques based on LC–MS/MS

Non-targeted LC-MS based metabolomics analysis of the urinary steroidal profile

The urinary steroidal fraction has been extensively explored as non-invasive alternative to monitor pathological conditions as well as to unveil the illicit intake of pseudo-endogenous anabolic steroids in sport. However, the majority of previous approaches involved the a priori selection of potentially relevant target analytes. Here we describe the non-targeted analysis of the urinary steroidal profiles. The workflow includes minimal sample pretreatment and normalization according to the specific gravity of urine, a 20 min reverse phase ultra-performance liquid chromatographic separation hyphenated to electrospray time-of-flight mass spectrometry. As initial validation, we analyzed a set of quality control urines spiked with glucurono- and sulfo-conjugated steroids at physiological ranges. We then applied the method for the analysis of samples collected after single transdermal administration of testosterone in hypogonadal men. The method allowed profiling of approximately three thousand metabolic features, including steroids of clinical and forensic relevance. It successfully identified metabolic pathways mostly responsible for groups clustering even in the context of high inter-individual variability and allowed the detection of currently unknown metabolic features correlating with testosterone administration. These outcomes set the stage for future studies aimed at implementing currently monitored urinary steroidal markers both in clinical and forensic analysis

Experimental and numerical analysis of directional added mass effects in partially liquid-filled horizontal pipes

The change of pipe natural frequencies due to added mass effects has been investigated in two cylindrical horizontal pipes from empty to completely water filled cases with various intermediate partially-filled conditions. The added mass coefficients of the three first vertical and horizontal modes of vibration have been determined with both experimental modal analysis and finite element analysis (FEA) acoustic-structural numerical simulations, which showed good agreement. The vertical and horizontal added mass coefficients present different behaviors as a function of the water level. Moreover, the pipe cross sectional dimensions determine the magnitude of these effects. For generalization to any pipe size, dependency of the directional added mass coefficients with new vertical and horizontal added mass estimators has been found. These estimators can be used in practical situations with horizontally mounted cylindrical pipes as a reference to predict and quantify air content.Postprint (author's final draft

Why Cohen's Kappa should be avoided as performance measure in classification

We show that Cohen's Kappa and Matthews Correlation Coefficient (MCC), both extended and contrasted measures of performance in multi-class classification, are correlated in most situations, albeit can differ in others. Indeed, although in the symmetric case both match, we consider different unbalanced situations in which Kappa exhibits an undesired behaviour, i.e. a worse classifier gets higher Kappa score, differing qualitatively from that of MCC. The debate about the incoherence in the behaviour of Kappa revolves around the convenience, or not, of using a relative metric, which makes the interpretation of its values difficult. We extend these concerns by showing that its pitfalls can go even further. Through experimentation, we present a novel approach to this topic. We carry on a comprehensive study that identifies an scenario in which the contradictory behaviour among MCC and Kappa emerges. Specifically, we find out that when there is a decrease to zero of the entropy of the elements out of the diagonal of the confusion matrix associated to a classifier, the discrepancy between Kappa and MCC rise, pointing to an anomalous performance of the former. We believe that this finding disables Kappa to be used in general as a performance measure to compare classifiers

Subspace procrustes analysis

Postprint (author's final draft

Rapid screening of beta-adrenergic agents and related compounds in human urine for anti-doping purpose using capillary electrophoresis with dynamic coating

This paper presents a capillary electrophoresis method, developed for the detection, in human urine, of beta-adrenergic agents and phenolalkylamines. The electrophoretic separation is achieved in less than 10 min and is based on the use of CEofix kit, for the dynamic capillary coating. The effects of accelerator buffer pH and separation voltage were investigated. The optimum buffer pH was found to be 2.5 for beta2-agonists and 6.2 for beta-blockers and phenoalkylamines with a separation voltage of 15 IN. Urine samples spiked with the compounds here studied were treated according to the standard procedure (SPE and evaporation to dryness) and analyzed by CE interfaced with an UV diode-array, set at 195 and 210 nm. The quantitative validation results, obtained analyzing samples at three different concentrations, show a good precision of peak areas that do not exceed 5% for intra-day assays and 10% for inter-day assays. Good linearity (r(2) > 0.995) was obtained within the 50-500 ng/mL concentration range. The qualitative validation data show a relative migration times (MTs) variation lower than 1%. The analytes were clearly distinguishable in urine, with LOD and LOQ in the range of 10-80 and 40-100 ng/mL, respectively

Experimental and numerical study on the detection of fatigue failure in hydraulic turbines

Detecting fatigue cracks in hydraulic turbine runners is costly, as it requires to stop the unit, empty it of water and access the runner for inspection. Thus, an alternative way based on monitoring the changes of the structural modal response induced by the formation and growth of a crack has been investigated. To do so, the crack propagation induced by a resonance has been numerically predicted and experimentally machined on a disk-like structure that resembles a Kaplan turbine runner. The analysis of the results shows how the different stages of the fatigue crack growth can be monitored based on the change of the natural frequencies and mode shapes of several specific modes. Based on the obtained results, a Structural Health Monitoring system is going to be designed to monitor the turbine runner modes of vibration without the need to stop and inspect the unit.This work was supported by the European Union’s Horizon 2020 research and innovation program under grant agreement number 814958.Peer ReviewedPreprin

In-flight cost index optimisation upon weather forecast updates

This paper presents an optimisation framework to compute the altitude and speed profiles of a trajectory in the execution phase of the flight, such that the expected total cost (ETC) of the operation is minimised (i.e. modelling the expected cost of delay and fuel – including arrival uncertainties – at the arrival gate). This is achieved with a two-stage optimisation strategy: a trajectory optimiser that minimises a generalised direct operating cost function, for a given cost index; and an upper-level optimiser, which obtains the best cost index that minimises the ETC. Several case studies are presented for different departure delays, while considering the impact of two different weather forecast updates too: a region with relative high head-winds appearing half way across teh flight; and a cold atmosphere scenario, with a tropopause altitude lower than standard conditions. ETC savings with respect to following the operational flight plan increase with departure delay, as expected. Due to the non-linearity of the cost function, however, the benefits of considering the weather update depend on the actual value of the departure delay, showing the convenience of integrating the proposed approach into a crew decision support tool in order to avoid sub-optimal decisions.This work has been performed as part of Pilot3 innovation action which has received funding from the Clean Sky 2Joint Undertaking (JU) under grant agreements No 863802. The JU receives support from the European Union’s Horizon 2020 research and innovation programme and the Clean Sky 2 JU members other than the Union.Peer ReviewedPostprint (author's final draft

Assessment of turbulence models for the prediction of Bénard- Von Kármán vortex shedding behind a truncated hydrofoil in cavitation conditions

The transient cavitating flow in the wake of a hydrofoil at zero incidence angle has been simulated using a homogeneous mixture cavitation mass transfer model combined with both Reynolds Average Navier-Stokes (RANS) and Scale Resolving Simulation (SRS) turbulence models. The hydrofoil geometry corresponds to a 2D NACA 0009 with a truncated trailing edge which has already been extensively investigated in the High-Speed Cavitation Tunnel of the EPFL. The hydrodynamic conditions of interest correspond to a free stream velocity of 20 m/s (Re = 2 · 106) without cavitation and with two different degrees of cavitation. To improve the prediction of the vortex shedding behing the hydrofoil, the ¿ - R¿t transitional boundary layer model has been coupled with the turbulence models. At cavitation-free regime, all the turbulence models with the exception of the SST and LES WALE ones have the ability to predict the experimentally measured vortex shedding frequency. Nevertheless, the results indicate that, neither the SST nor the DES-SST ¿ - R¿t, can predict the vortex shedding frequency increase which has been experimentally observed when cavitation occurs. In contrast, the numerical results provided by the SST ¿ - R¿t and the SSTCC ¿ - R¿t show the capability to predict the expected shedding frequencies for both non cavitation and cavitation conditions. Beyond all expectation, the results provided by the LES WALE seem not only to overestimate the vortex shedding frequency at cavitation free conditions but also to underestimate the frequency when the cavitation number is significantly reduced.Peer ReviewedPostprint (published version