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This article presents newly developed screening and confirmation analytical procedures to detect the misuse of nine prolyl-hydroxylase inhibitors of the hypoxia-inducible factor: daprodustat, desidustat, FG2216, IOX2, IOX4, JNJ-42041935, molidustat, roxadustat and vadadustat, targeting either the parent drugs and/or their main metabolite(s). For the sample pretreatment, different extraction protocols and technologies were evaluated. The instrumental analysis was performed by ultra-high-performance liquid chromatography coupled to either high- or low-resolution mass spectrometry. The chromatographic separation was performed on a C18 column, employing water and acetonitrile, both containing 0.1% formic acid, as mobile phase. Detection was achieved using as analyzer either a triple quadrupole or an Orbitrap, with positive and negative electrospray ionization and different acquisition modes. Validation of the procedures was performed according to the ISO 17025 and World Anti-Doping Agency guidelines. The methods do not show any significant interference at the retention times of the analytes of interest. The extraction efficiency was estimated to be greater than 75% for all analytes and the matrix effect smaller than 35%. Detection capability was determined in the range of 0.25-2.0 for the screening procedure and in the range of 0.5-2.0 ng/mL for the confirmation procedure, that is, in a range of concentration small enough to reveal the abuse of the compounds considered, in case they are used as performance-enhancing agents. The repeatability of the relative retention times (CV% < 0.5) and of the relative abundances of the selected ion transitions, considered only in the case of triple quadrupole (CV% < 15), was confirmed to be fit for purpose to ensure the unambiguous identification of all the target analytes in human urine. The applicability of the newly developed methods was verified by the analysis of urine samples containing molidustat, roxadustat or daprodustat. The developed procedures enabled to detect the compounds under investigation and their main metabolites., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Anabolic androgenic steroids (AAS) are prohibited as performance-enhancing drugs in sports. Among them, testosterone and its precursors are often referred to as "pseudoendogenous" AAS, that is, endogenous steroids that are prohibited when administered exogenously. To detect their misuse, among other methods, the World Anti-Doping Agency-accredited laboratories monitor the steroid profile (concentrations and concentration ratios of endogenous steroids, precursors and metabolites) in urine samples collected from athletes in and out of competition. Alterations in steroid profile markers are used as indicators for misuse of anabolic steroids in sports. Therefore, especially their metabolic pathways with possible interactions are crucial to elucidate. As steroid metabolism is very complex, and many enzymes are involved, certain non-prohibited drugs may influence steroid metabolite excretion. One important group of steroid-metabolizing enzymes is aldo-keto reductases (AKRs). An inhibition of them by non-steroidal anti-inflammatory drugs (NSAIDs), which are neither prohibited nor monitored, but frequently used drugs in sports, was demonstrated in vitro. Thus, this work aims to investigate the influence of NSAID intake on the urinary steroid profile. Kinetic and inhibitory studies were performed using 5α-dihydrotestosterone as substrate. The results obtained from in vitro experiments show that ibuprofen inhibits AKR1C2 and thus influences steroid biotransformation. For in vivo investigations, urine samples prior, during and postadministration of ibuprofen were analyzed using routine methods to monitor the steroid profile. Changes in markers of the steroid profile of volunteers were observed. The combination of in vitro and in vivo results suggests that monitoring of ibuprofen may be useful in doping control analysis. The presented work illustrates the importance to consider co-administration of (non-prohibited) drugs during antidoping analysis. Intake of multiple substances is likely leading to interfering effects. Divergent results in antidoping analysis may therefore be observed and misinterpretation of analytical data may occur. Similar considerations may be appropriate for other fields of forensic applications., (© The Author(s) 2020. Published by Oxford University Press on behalf of The International Association of Forensic Toxicologists, Inc. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Steroid abuse is a growing problem among amateur and professional athletes. Because of an inundation of newly and illegally synthesized steroids with minor structural modifications and other designer steroid receptor modulators, there is a need to develop new methods of detection which do not require prior knowledge of the abused steroid structure. The number of designer steroids currently being abused is unknown because detection methods in general are only identifying substances with a known structure. The detection of doping is moving away from merely checking for exposure to prohibited substance toward detecting an effect of prohibited substances, as biological assays can do. Cell-based biological assays are the next generation of assays which should be utilized by antidoping laboratories; they can detect androgenic anabolic steroid and other human androgen receptor (hAR) ligand presence without knowledge of their structure and assess the relative biological activity of these compounds. This review summarizes the hAR and its action and discusses its relevance to sports doping and its use in biological assays.

This study was designed to optimize a method for the identification and quantification of ephedrines in oral fluid (OF) and for its application to subjects taking different doses of pseudoephedrine. Ephedrines use by athletes is banned by World Anti-Doping Agency (WADA), only "in competition" if their concentration in urine exceeds the cutoff limit. The study aimed to establish if there is a correlation in terms of times of elimination and of concentration trends of ephedrine in OF and urine after administration of therapeutic doses of pseudoephedrine to various subjects. Results obtained from excretion studies performed on eight subjects showed reproducible times of disappearance of ephedrines from OF. Pseudoephedrine was generally at low concentrations or undetectable in oral fluid samples 12 h after administration, whereas urine samples collected in the same period of time showed higher ephedrine concentrations and exceeding cutoff values generally between 8 and 24 h after administration of the drug. Within- and between-individual variability was observed in terms of concentrations of pseudoephedrine in OF following the administration of the same dose. Only in the case of sustained-release drugs were constant pseudoephedrine concentrations achieved in OF.

This paper describes the application of solid-phase microextraction (SPME) with subsequent injection in a gas chromatograph-mass spectrometer (GC-MS) (electron impact, full scan) for the screening analysis of stimulants and narcotics in urine. Several different kinds of fibers were preliminarily tested and comparatively evaluated considering the influence on the overall analytical performance of the method; other experimental parameters; and, primarily among them, the volume of urine, the pH value, and the time of adsorbtion. The optimal experimental conditions have been recorded using 0.5 mL of urine with the pH value adjusted to 10 with carbonate buffer, and in which is immersed a polydimethylsiloxane/divinylbenzene fiber, with a sampling time of 30 min; the fiber is then directly desorbed in the injection port of the GC-MS equipment. All the analytes show a good linearity (R2 > 0.99 for most substances) and a good reproducibility at the concentration corresponding to the minimum performance requirement limit or at the cut-off value fixed by the World AntiDoping Agency (CV% < 11). The limit of detection of the method is 50 ng/mL for the majority of the substances investigated. Imidazole-based drugs (e.g., naphazoline) and local anesthetics can also be included in this screening method. Whenever necessary, confirmation analyses may also be performed by following the same pre-chromatographic procedure. Integrating the SPME process and the GC-MS analysis with a dedicated autosampler that combines the microextraction and injection capacities maximizes the overall analytical capacity of a single GC-MS system and reduces the human labor necessary for and the environmental impact of screening for stimulants and narcotics excreted free in urine.