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We considered in this study the possibility of developing an indirect procedure for detecting myostatin inhibition/suppression, a practice that is prohibited as doping in sport. We have specifically considered the potential diagnostic utility of human serum myokines as indirect markers of myostatin inhibition. Myostatin, its main antagonist follistatin, and other myokines (follistatin-like 1, musclin, oncostatin, osteonectin, irisin, brain derived neurotrophic factor, and insulin-like growth factor-1) were selected as a panel of potential biomarkers whose levels may be altered following myostatine suppression. The serum levels of myostatin and of the nine myokines were measured in elite athletes of different age, sex, and sport discipline, and their cross correlation assessed by multivariate analysis. All myokines resulted to be measurable in human serum, except for musclin and irisine, whose levels were below the limits of quantitation in a reduced number of samples. Serum concentrations varied of different orders in magnitude (musclin and osteonectin < 1 ng/mL; follistatin, myostatine and irisine 1-5 ng/mL; brainderived neurotrophic factor, follistatin-like 1 and iinsulin-like growth factor-1 > 10 ng/mL), while no significant differences were found between female and male subjects, with the exceptions of follistatin-like 1 and musclin, showing a higher concentrations in females (p < 0.05). Levels of insulin-like growth factor 1 and brain derived neurotrophic factor were significantly higher in power athletes than in endurance ones. Multivariate statistics showed that musclin, follistatin-like 1 and oncostatin are more clustered and correlated to myostatin than other myokines, suggesting they could be considered as potential biomarkers of doping by myostatin inhibitors., Competing Interests: The authors declare no conflict of interest., (Copyright © Biology of Sport 2024.)

In comparison to well-known drug-metabolizing organs such as the liver, the metabolic capacity of human skin is still not well elucidated despite the widespread use of topical drug application. To gain a comprehensive insight into anabolic steroid metabolism in the skin, six structurally related anabolic androgenic steroids, testosterone, metandienone, methyltestosterone, clostebol, dehydrochloromethyltestosterone, and methylclostebol, were applied to human keratinocytes and fibroblasts derived from the juvenile foreskin. Phase I metabolites obtained from incubation media were analyzed by gas chromatography-mass spectrometry. The 5α-reductase activity was predominant in the metabolic pathways as supported by the detection of 5α-reduced metabolites after incubation of testosterone, methyltestosterone, clostebol, and methylclostebol. Additionally, the stereochemistry structures of fully reduced metabolites (4α,5α-isomers) of clostebol and methylclostebol were newly confirmed in this study by the help of inhouse synthesized reference materials. The results provide insights into the steroid metabolism in human skin cells with respect to the characteristics of the chemical structures., Competing Interests: Declaration of Competing Interest All authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The detection of blood doping represents a current major issue in sports and an ongoing challenge for antidoping research. Initially focusing on direct detection methods to identify a banned substance or its metabolites, the antidoping effort has been progressively complemented by indirect approaches. The longitudinal and individual monitoring of specific biomarkers aims to identify nonphysiological variations that may be related to doping practices. From this perspective, the identification of markers sensitive to erythropoiesis alteration is key in the screening of blood doping. The current Athlete Biological Passport implemented since 2009 is composed of 14 variables (including two primary markers, i.e., hemoglobin concentration and OFF score) for the hematological module to be used for indirect detection of blood doping. Nevertheless, research has continually proposed and investigated new markers sensitive to an alteration of the erythropoietic cascade and specific to blood doping. If multiple early markers have been identified (at the transcriptomic level) or developed directly in a diagnostics' kit (at a proteomic level), other target variables at the end of the erythropoietic process (linked with the red blood cell functions) may strengthen the hematological module in the future. Therefore, this review aims to provide a global systematic overview of the biomarkers considered to date in the indirect investigation of blood doping., (© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Background: Autologous blood transfusion is one of the illicit strategies, banned by the World Anti-Doping Agency, to increase the levels of hemoglobin, with a consequent improvement in the delivery of oxygen to tissues. At present, this practice is detectable exclusively by the individual, longitudinal monitoring of hematological biomarkers, as in the hematological module of the Athlete Biological Passport; but this indirect approach may suffer from different confounding factors. We are presenting a multi-parametric, analytical strategy to detect autologous blood transfusions by targeting the modification of the red blood cells during storage. We focused on the assessment of "storage lesions", targeting (i) membrane proteins: Glycophorin-A and Band 3 complex, (ii) biomarkers of oxidative stress: Peroxiredoxin-2, (iii) biomarkers of senescence: CD47 and Phosphatidylserine, (iv) erythrocytes microparticles., Results: All of the above markers were monitored, by immunological and flow cytofluorimetric methods, on samples of stored whole blood collected at different time intervals, and on fresh blood samples, collected for official doping control tests, mixed "ex vivo" to simulate an autotransfusion. Although anonymized before the delivery to the laboratory, it was possible to mix samples belonging to the same subject based on the "athlete biological passport" code. Our results showed that the irreversible alteration of RBCs morphology, the loss of membrane integrity, the occurrence of hemolysis phenomena, and, more in general, the "aging" of the erythrocytes during storage are closely related to: (i) the reduced concentration, on the erythrocyte membrane, of Band 3 protein (decrease of 19% and of 39% after 20 and 40 days of storage respectively) and of glycophorin A (- 47% and - 63% respectively); (ii) the externalization of phosphatidyl serine (with a five-fold increase after 20 days and a further 2× increase after 40 days); (iii) the reduced concentration of CD47; and (iv) increased levels of erythrocyte microparticles., Conclusions: The most promising method to detect the presence of transfused blood in whole blood samples can be based on a multi-parametric strategy, considering jointly both protein expression on RBCs membranes and micro-vesiculation phenomena., (© 2023. The Author(s).)

The ratio of testosterone (T) to epitestosterone (E) determined in urine samples is the main biomarker used to prove T and precursors abused by athletes. Analytically, the correction of the ratio is required by the World Anti-Doping Agency. This work describes a series of experiments aimed to study when it is appropriate to correct the T/E ratio value, using different mass spectrometric techniques since only reports on GC-MS exist. Analyses using external calibrators, controls, and routine samples were performed by three different techniques GC-MS, GC-MS n , and LC-MS n . A statistical comparison of the T/E was performed after the application of two corrections previously published: Isotopic contribution peak area correction (corr&#95;1) and use of a verified internal deuterated internal standard (TD3/ED3) correction (corr&#95;2) and the ratio based on T and E concentrations. The use of external calibration samples introduces biases that influence not only the T and E concentrations but also the T/E ratio, even when both methods of correction are applied. The correction after applying corr&#95;1 method barely contributed to the accuracy of the T/E calculation. Nevertheless, the application of the corr&#95;2 method increased the accuracy between 5% and 6% when comparing theoretical and experimental T/E values. Finally, the best results were obtained by the ratio calculated directly from the estimated concentrations of T and E. Attention must be paid when the T/E is measured by LC-MS n since different acquisition modes produced significantly different results, even in MS/MS when the same transition is used for T and E., (© 2023 John Wiley & Sons Ltd.)

Γ-valerolactone (GVL), marketed online as "Tranquilli-G" and "excellent Valium", is used as a legal substitute for γ-hydroxybutyric acid (GHB); however, until now, GVL has only been connected to one Drug-Facilitated Sexual Assault (DFSA) case. Moreover, the pharmaco-toxicological effects of GVL are poorly studied. The aim of this study was to investigate the 1) in vivo effects of gavage administration of GVL (100-3000 mg/kg) on neurological (myoclonia, convulsions), sensorimotor (visual, acoustic, and overall tactile) responses, righting reflex, thermoregulation, motor activity (bar, drag, and accelerod test) and cardiorespiratory changes (heart rate, breath rate, oxygen saturation, and pulse distension) in CD-1 male mice and the 2) in silico ADMET profile of GVL in comparison to GHB and the open active form γ-hydroxyvaleric acid (GHV). The present study demonstrates that GVL inhibits, in a dose-dependent manner, sensorimotor and motor responses and induces cardiorespiratory depression (at a dose of 3000 mg/kg) in mice. The determination of the ED 50 in sensorimotor and motor responses revealed that GVL is about 4-5 times less potent than GHB. In silico prediction of ADMET profiles revealed toxicokinetic similarities between GHB and GHV, and differences with GVL. These results suggest that GVL could be used as a substitute for GHB and should be added to forensic toxicology screenings., Competing Interests: Declaration of Competing Interest Declarations of interest: none., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

This work focused on the possible alterations of the markers of the steroidal module of the athlete biological passport, considering samples of athletes declaring and not-declaring the supplementation of thyroid hormones (TH) in the Doping Control Form (DCF). Concentrations of 5α-androstane-3α,17β-diol (5α-Adiol), 5β-androstane-3α,17β-diol (5β-Adiol), testosterone (T), androsterone (A), etiocholanolone (Etio), epitestosterone (E), pregnanediol (PD), dehydroepiandrosterone (DHEA), and 11β-hydroxy-androsterone (OHA) were calculated using internal standards and external calibration by gas chromatography-tandem mass spectrometry. Also, ratios between the above biomarkers were also estimated. The data set was composed of samples from females and males declaring and not-declaring TH supplementation in the DCF. To corroborate these observations, a controlled urinary excretion study was carried out with multiple doses of sodium liothyronine (T3). Female data showed significant differences for the concentrations of 5α-Adiol, A, DHEA, E, OHA, and T and the ratio A/Etio between FD and FND groups, whereas the male groups only showed significant differences in OHA concentration. In both cases, males and females declaring the consumption of levothyroxine showed narrower data distribution and diminished percentiles from 17% to 67% with respect to the not-declaring corresponding groups (p < 0.05). Concentrations of 5α-metabolites showed a higher depression for the FND, and both FD and MD groups showed a peculiar behavior for the PD concentrations. The controlled study agreed with the observations, mainly for the female group with significant differences for concentrations of E, Etio, 5α-Adiol, and 5β-Adiol after TH administration. The interpretation of the steroid markers of the ABP should consider TH administrations., (© 2023 John Wiley & Sons Ltd.)

Insulin-like growth factor 1 analogues are prohibited in sport for their ability to enhance athletic performance in several sport disciplines. Their detection presents several analytical challenges, mainly due to the minimum required performance limits fixed by the World Anti-Doping Agency. Here, we are presenting analytical workflows to detect IGF-1 and its analogues in different biological matrices. Several off-line immunocapture techniques and protocols were comparatively evaluated. Separation and detection were performed by using standard flow reverse-phase liquid chromatography coupled to a time-of-flight mass spectrometer. The best recoveries were obtained using magnetic beads or pipette tips functionalized with protein A. The analytical workflows were fully validated for qualitative determinations: all the target analytes were clearly distinguishable from the interference of the matrices, with limits of detection and identification in the range of 0.05-0.30 ng/mL in urine and 0.5-2.0 ng/mL in serum/plasma. The extraction efficiency proved to be repeatable (CV% < 10) with recoveries higher than 50%. Intra- and inter-day precision were found to be smaller than 10 and 15%, respectively. The method was successfully applied to the analysis of authentic matrix samples containing the target peptides at the minimum required performance limits, proving that the method developed can be successfully applied to detect and identify IGF-1 analogues for doping control purposes in all the matrices selected. The analytical workflow developed here to detect the target peptides in different matrices can be readily implemented in anti-doping laboratories and has the potential to be adapted for the simultaneous analysis of different similarly sized peptide hormones of doping relevance., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Background: Most current state-of-the-art strategies to generate individual adaptive reference ranges are designed to monitor one clinical parameter at a time. An innovative methodology is proposed for the simultaneous longitudinal monitoring of multiple biomarkers. The estimation of individual thresholds is performed by applying a Bayesian modeling strategy to a multivariate score integrating several biomarkers (compound concentration and/or ratio). This multimodal monitoring was applied to data from a clinical study involving 14 female volunteers with normal menstrual cycles receiving testosterone via transdermal route, as to test its ability to detect testosterone administration. The study samples consisted of urine and blood collected during 4 weeks of a control phase and 4 weeks with a daily testosterone gel application., Results: Integrating multiple biomarkers improved the detection of testosterone gel administration with substantially higher sensitivity compared with the distinct follow-up of each biomarker, when applied to selected urine and serum steroid biomarkers, as well as the combination of both. Among the 175 known positive samples, 38% were identified by the multimodal approach using urine biomarkers, 79% using serum biomarkers and 83% by combining biomarkers from both biological matrices, whereas 10%, 67% and 64% were respectively detected using standard unimodal monitoring., Significance and Novelty: The detection of abnormal patterns can be improved using multimodal approaches. The combination of urine and serum biomarkers reduced the overall number of false-negatives, thus evidencing promising complementarity between urine and blood sampling for doping control, as highlighted in the case of the use of transdermal testosterone preparations. The generation in a multimodal setting of adaptive and personalized reference ranges opens up new opportunities in clinical and anti-doping profiling. The integration of multiple parameters in a longitudinal monitoring is expected to provide a more complete evaluation of individual profiles generating actionable intelligence to further guide sample collection, analysis protocols and decision-making in clinics and anti-doping., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Rationale: The proposed metabolomic workflow, based on coupling high-resolution mass spectrometry with computational tools, can be an alternative strategy for metabolite detection and identification. This approach allows the extension of the investigation field to chemically different compounds, maximizing the information obtainable from the data and minimizing the time and resources required., Methods: Urine samples were collected from five healthy volunteers before and after oral administration of 3β-hydroxyandrost-5-ene-7,17-dione as a model compound and defining three excretion time intervals. Raw data were acquired in both positive and negative ionization modes using an Agilent Technologies 1290 Infinity II series HPLC coupled to a 6545 Accurate-Mass Quadrupole Time-of-Flight. They were then processed to align peak retention times with the same accurate mass, and the resulting data matrix was subjected to multivariate analysis., Results: Multivariate analysis (PCA and PLS-DA models) demonstrated high similarity between samples belonging to the same collection time interval and clear discrimination between different excretion intervals. The blank and long excretion groups were distinguished, suggesting the presence of long excretion markers, which are of remarkable interest in anti-doping analyses. The correspondence of some significant features with metabolites reported in the literature confirmed the rationale and usefulness of the proposed metabolomic approach., Conclusions: The presented study proposes a metabolomics workflow for the early detection and characterization of drug metabolites by untargeted urinary analysis to reduce the range of substances still excluded from routine screening. Its application has detected minor steroid metabolites, as well as unexpected endogenous alterations, proving to be an alternative strategy that can allow gathering a more complete range of information in the antidoping field., (© 2023 John Wiley & Sons Ltd.)

Scope: The phytosteroid ecdysterone is present in spinach. In this study, the urinary elimination of ecdysterone and its metabolites in humans is investigated following spinach consumption of two different culinary preparations., Methods and Results: Eight participants (four males, four females) ingested 950 (27.1) g sautéed spinach (average [±standard deviation (SD)]) and 912 (70.6) g spinach smoothie as second intervention after washout. Post-administration urines are analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS). After intake of both preparations, ecdysterone and two metabolites, 14-deoxy-ecdysterone, and 14-deoxy-poststerone, are excreted in urine. The maximum concentration of ecdysterone is ranging from 0.09 to 0.41 µg mL -1 after sautéed spinach and 0.08-0.74 µg mL -1 after smoothie ingestion. The total excreted amount (mean% [±SD]) in the urine as a parent drug plus the metabolites is only 1.4 (1.0) for both sautéed spinach and smoothie. The apparent sex related differences in 14-deoxy-poststerone excretion will need further investigations., Conclusion: Only a small proportion of ecdysterone from spinach is excreted into urine. No significant differences are found in concentration and recovered amount (%) of ecdysterone, 14-deoxy-ecdysterone, and 14-deoxy-poststerone in urine between sautéed spinach and smoothie ingestion. A discrimination between ecdysterone from food or preparations will be challenging based on urinary concentrations only, at least for later post-administration samples., (© 2023 The Authors. Molecular Nutrition & Food Research published by Wiley-VCH GmbH.)

Background: Nicotine is a psychostimulant drug with purported use in sports environments, though the use of nicotine among athletes has not been studied extensively., Objective: The aim of this study was to assess the nicotine positivity rate in 60,802 anti-doping urine samples from 2012 to 2020., Methods: Urine samples obtained in-competition at different national and international sports events held in Italy during the period 2012-2020 were analysed. All samples were from anonymous athletes that were collected and analysed at the WADA-accredited antidoping laboratory in Rome, Italy. Samples were analysed by gas chromatography coupled with mass spectrometry, with a cut-off concentration for nicotine of > 50 ng/mL. Results were stratified by year, sport and sex., Results: An overall mean of 22.7% of the samples (n = 13,804; males: n = 11,099; females: n = 2705) showed nicotine intake, with male samples also displaying higher positivity rates than female (24.1% vs 18.5%). Sample positivity was higher during 2012-2014 (25-33%) than 2015-2020 (15-20%). Samples from team sports displayed a higher positivity rate than those from individual sports (31.4 vs 14.1%)., Conclusions: The current data demonstrates that one in five samples from a range of 90 sports test positive for nicotine in-competition. There is a lower positivity rate in endurance versus power/strength athletes and higher positivity rate in team versus individual sports, probably accounted for by differences in physiological and psychological demands and the desire for socialisation. WADA, international and national sports federations should consider these findings with concern, proactively investigate this phenomenon and act in order to protect the health and welfare of its athletes., (© 2023. The Author(s).)

Supercritical fluid chromatography is proving to be a good separation and sample preparation tool for various analytical applications and, as such, has gained the attention of the anti-doping community. Here, the applicability of supercritical fluid chromatography hyphenated to tandem mass spectrometry for routine doping control analysis was tested. A multi-analyte method was developed to cover 197 drugs and metabolites that are prohibited in sport. More than 1000 samples were analyzed by applying a "dilute and inject" approach after hydrolysis of glucuronide metabolites. Additionally, a comparison with routinely used liquid chromatography-mass spectrometry was performed with 250 of the 1000 samples and a number of past positive anti-doping samples. It revealed some features where supercritical fluid chromatography-tandem mass spectrometry was found to be complementary or advantageous to liquid chromatography-mass spectrometry for anti-doping purposes, such as better retention of analytes that are poorly retained in reversed-phase liquid chromatography. Our results suggest that supercritical fluid chromatography-tandem mass spectrometry is sensitive (limit of detection <50% relevant minimum required performance level required by the World Anti-Doping Agency for anti-doping analysis), reproducible, robust, precise (analytes of interest area coefficient of variation <5%; retention time difference coefficient of variation <1%) and complementary to existing techniques currently used for routine analysis in the World Anti-Doping Agency accredited laboratories., (© 2023 Wiley-VCH GmbH.)

We present a novel procedure to monitor the fluctuations of the levels of IGF-1 in capillary blood in the framework of doping control analysis. Being an endogenous hormone, direct methods are not applicable, so the most effective way to detect the intake of the exogenous hormone would be based on the longitudinal monitoring of the athlete. We have therefore followed the individual variability, in four subjects (two males and two females), of the levels of IGF-1 in capillary blood samples collected three times per day for five days, then once a week for at least two months. Analyses were performed by liquid chromatography coupled to tandem mass spectrometry following a bottom-up approach. The whole protocol, from the sample collection to the instrumental analysis, was validated according to the World Anti-Doping Agency's guidelines and ISO17025. The analytical protocol showed to be fit for purpose in terms of sensitivity (LOD 25 ng/mL and LOI 35 ng/mL), selectivity (no interferences were detected at the retention time of IGF-1 and the internal standard), and repeatability (CV<10%). The linearity was confirmed in the range of 50-1000 ng/mL (correlation coefficient R 2 >0.995, with a % relative bias of the experimental concentration of the different calibrators used for the estimation of the linearity lower than 20% for the lowest level and than 15% for the other levels). Stability studies were also performed, also to establish the optimal conditions for transport and storage: samples were stable at 4 °C for up to 72 h and at -20 °C and -80 °C for up to three months. Our preliminary results indicate that, in all subjects, the levels of IGF-1 did not present significant circadian fluctuations and remained stable during the entire period of the study (2-3 months, depending on the subject). The stability over time of IGF-1 levels in capillary blood indicates the possibility of detecting the intake of the non-endogenous hormone based on a longitudinal approach, as it is modeled in the framework of the endocrinological module of the athlete biological passport., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The steroidal module of the athlete biological passport (ABP) targets the use of pseudo-endogenous androgenous anabolic steroids in elite sport by monitoring urinary steroid profiles. Urine and blood samples were collected weekly during two consecutive oral contraceptive pill (OCP) cycles in 15 physically active women to investigate the low urinary steroid concentrations and putative confounding effect of OCP. In urine, testosterone (T) and epitestosterone (E) were below the limit of quantification of 1 ng/ml in 62% of the samples. Biomarkers' variability ranged between 31% and 41%, with a significantly lesser variability for ratios (except for T/E [41%]): 20% for androsterone/etiocholanolone (p < 0.001) and 25% for 5α-androstane-3α,17β-diol/5ß-androstane-3α,17β-diol (p < 0.001). In serum, markers' variability (testosterone: 24%, androstenedione: 23%, dihydrotestosterone: 19%, and T/A4: 16%) was significantly lower than in urine (p < 0.001). Urinary A/Etio increased by >18% after the first 2 weeks (p < 0.05) following withdrawal blood loss. In contrast, serum T (0.98 nmol/l during the first week) and T/A4 (0.34 the first week) decreased significantly by more than 25% and 17% (p < 0.05), respectively, in the following weeks. Our results outline steroidal variations during the OCP cycle, highlighting exogenous hormonal preparations as confounder for steroid concentrations in blood. Low steroid levels in urine samples have a clear negative impact on the subsequent interpretation of steroid profile of the ABP. With a greater analytical sensitivity and lesser variability for steroids in healthy active women, serum represents a complementary matrix to urine in the ABP steroidal module., (© 2022 John Wiley & Sons Ltd.)

The Athlete Biological Passport (ABP) was introduced to complement the direct anti-doping approach by indirectly outlining the possible use of prohibited substances or methods in sports. The ABP proved its effectiveness, at least through a deterrent effect, even though the matrices used for longitudinal monitoring (urine and blood) are subject to many intrinsic (e.g., genetic) and extrinsic (e.g., environmental conditions) confounding factors. In that context, new and more specific biomarkers are currently under development to enhance both the sensitivity and the specificity of the ABP. Multiple strategies are presently being explored to improve this longitudinal monitoring, with the development of the current modules, the investigation of new strategies, or the screening of new types of doping. Nevertheless, due to the variability induced by indirect biomarkers, the consideration of confounding factors should continuously support this research. Beyond tremendous advances in analytical sensitivity, machine learning-based approaches seem inevitable to facilitate an expert interpretation of numerous biological profiles and promote anti-doping efforts. This perspective article highlights the current innovations of the Athlete Biological Passport that seem the most promising. Through different research axes, this short manuscript provides an opportunity to bring together approaches that are more widely exploited (e.g., omics strategies) and others in the early stages of investigation (e.g., artificial intelligence) seeking to develop the ABP., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Krumm, Botrè, Saugy and Faiss.)

The rectal administration of glucocorticoids, as well as any injectable, and oral ones, is currently prohibited by the World Anti-Doping Agency when occurs "in competition." A reporting level of 100 ng/ml for prednisolone and 300 ng/ml for prednisone was established to discriminate the allowed and the prohibited administration. Here, the urinary excretion profiles of prednisone and prednisolone were evaluated in five volunteers in therapy with glucocorticoid-based rectal formulations containing prednisone or prednisolone caproate. The urinary levels of the excreted target compounds were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) following the procedure validated and currently in use in our laboratory to detect and quantitate glucocorticoids in urine. Predictably, the excretion trend of the analytes of interest were generally comparable with those obtained after oral administration, even if the excretion profile showed a broad interindividual variability, with the absorption rate and the systemic bioavailability after rectal administration being strongly influenced by the type of formulations (suppository or rectal cream, in our case) as well as the physiological conditions of the absorption area. Results showed that the target compounds were detectable for at least 30 h after drug administration. After suppository administration, prednisolone levels reached the maximum after 3 h from drug administration and then dropped below the reporting level after 15-21 h; prednisone reached the maximum after 3 h from drug administration, and then dropped below the reporting level after 12-15 h. After cream administration, both prednisone and prednisolone levels remained in a concentration below the reporting level throughout the entire monitored period., (© 2022 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

The detection of testosterone and its precursors' abuse in antidoping sports analysis is based on the longitudinal evaluation of markers of the urinary endogenous steroid profile. A Bayesian statistical approach is applied, allowing the establishment of credible intervals of the selected parameters for every athlete. Samples showing values outside the acceptable boundaries are selected for additional confirmation by isotope ratio mass spectrometric (IRMS) analysis. The alterations of the IRMS values last longer than the alterations of the steroid profile. Then the application of IRMS to a larger number of samples, at a screening level, would presumably allow detection of additional positive cases. The steroid profile and IRMS data can be treated using the same Bayesian inference procedure. In nonsports population, we have demonstrated the stability of IRMS data. In this work, we studied the variability of these data in real conditions, in samples collected on athletes subjected to antidoping analyses over the years. The data obtained confirmed previous observations and the applicability of the proposed approach. The results of cases where confounding factors of the steroid profile were reported are discussed, showing that in most of the cases no significant changes are observed over the absolute delta values. Changes in diet may significantly change the absolute delta values but not the ones relative to endogenous reference compounds. Finally, a case that could have been evaluated as normal with the current approach without a thorough review of the data was detected as positive by the proposed approach, demonstrating the benefit of its application., (© 2022 John Wiley & Sons Ltd.)

We present a quick and simple multi-targeted analytical workflow based on ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry for the screening in dried blood spots and dried plasma spots of a wide variety of drugs with different chemical properties. Seven different microsampling devices were evaluated in view of their application for the detection of the selected target analytes in the framework of doping control analysis. The extraction of the analytes was optimized by assessing the efficacy of protocols based on ultrasonication with aqueous buffers and/or organic solvents of different polarities. Optimal recoveries were obtained by using pure methanol or mixtures of methanol/acetonitrile and methanol/isopropanol, depending on both the device and the target analytes. The method was fully validated according to both ISO17025 and the requirements of the World Anti-Doping Agency: all the analytes were clearly distinguishable from the matrix, with limits of detection in the range of 0.1-3.0 ng mL -1 . Stability studies simulating the storage of samples before the analysis and in view of a possible re-analysis showed that most of the analytes were stable for at least 24 h at 50 °C and for at least 3 weeks at 25 and at 4 °C. The real applicability of the method was assessed by analyzing the samples collected after the administration of two model drugs, acetazolamide and deflazacort. The performance of the method was confirmed to be fit for purpose, and data obtained in blood can also be used to complement those available in urine, allowing to refine the knowledge concerning the pharmacokinetic profiles., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

This paper aimed to assess a method to measure eight thyroid-related compounds in serum by liquid chromatography-mass spectrometry (LC-MS/MS), to verify the correlation with radioimmunoassay (RIA), to evaluate the possible cross-reactivity, and to observe differences between athletes declaring the consumption of sodium levothyroxine and nonathletes serum samples. Validation was carried out to assess carryover, working range and linearity, limit of detection and limit of quantification, precision, matrix influence, recovery, accuracy, and uncertainty. Comparison between RIA and LC-MS/MS results was done. The assay was applied to serum samples, and comparison with RIA was done for T3 and T4 levels supported by RIA Thyroid-stimulating hormone (TSH) measurements. Validation parameters showed satisfactory results. Correlation between RIA and LC-MS/MS for T3 and T4 showed good results, but a cross-reactivity between T3 and T3AA was observed. Although no significant differences were proved, preliminary comparison between athletes and nonathletes serum samples showed a shift towards high values of TSH and lower for T4 values in the athletes' group. Differences between thyronine and T4AA concentrations and ratios were observed. The trend of T4 values supported by TSH measures might indicate subclinical hypothyroidism in athletes. This represents one of the most controversial thyroid statuses as different criteria about its treatment are described, especially since one of the exogenous causes is inadequate levothyroxine therapy. Because the variation of thyroid hormones and TSH has been extensively studied in high-performance sports, it is worth considering the need to set an adequate reference interval to accurately assess the thyroid status in athletes., (© 2022 John Wiley & Sons, Ltd.)

3-(1-Naphthalenylmethyl)-1-pentyl-1H-indole (JWH-175) is a synthetic cannabinoid illegally marketed for its psychoactive cannabis-like effects. This study aimed to investigate and compare in vitro and in vivo pharmacodynamic activity of JWH-175 with that of 1-naphthalenyl (1-pentyl-1H-indol-3-yl)-methanone (JWH-018), as well as evaluate the in vitro (human liver microsomes) and in vivo (urine and plasma of CD-1 male mice) metabolic profile of JWH-175. In vitro binding studies showed that JWH-175 is a cannabinoid receptor agonist less potent than JWH-018 on mouse and human CB1 and CB2 receptors. In agreement with in vitro data, JWH-175 reduced the fESPS in brain hippocampal slices of mice less effectively than JWH-018. Similarly, in vivo behavioral studies showed that JWH-175 impaired sensorimotor responses, reduced breath rate and motor activity, and increased pain threshold to mechanical stimuli less potently than JWH-018. Metabolic studies demonstrated that JWH-175 is rapidly bioactivated to JWH-018 in mice blood, suggesting that in vivo effects of JWH-175 are also due to JWH-018 formation. The pharmaco-toxicological profile of JWH-175 was characterized for the first time, proving its in vivo bio-activation to the more potent agonist JWH-018. Thus, it highlighted the great importance of investigating the in vivo metabolism of synthetic cannabinoids for both clinical toxicology and forensic purposes.

Objectives: This paper aimed to consider those features that may suggest a link between thyroid hormones pharmacology and athletes' health based on current consumption trends in a population of athletes., Methods: Methods used were observation, description, and synthesis, mainly. Among the documents reviewed were books, scientific articles, and review articles peer-reviewed. The review covered sources published in the period 1961 to 2021. Only references with a traceable origin were accepted (DOI numbering, ISSN, and ISBN, as well as peer-reviewed journals). The data on the consumption of thyroid hormones derivatives were extracted from the Doping Control Forms of athlete samples received at Laboratorio Antidoping FMSI of Rome from 2017 to 2021., Results: An overview of the biosynthesis, pharmacology, and metabolism of thyroid hormones, including thyronamines and thyronacetic acids, was presented. Likewise, a summary is presented on the relationship between thyroid hormones and ethnic and gender differences, their physiology in sport, and the reasons why their use could be considered attractive for athletes., Conclusion: Today, thyroid hormones are not listed as a prohibited substance by the World Anti-Doping Agency. However, several requests to include levothyroxine on the prohibited list are documented. The observation that the number of athletes taking thyroid hormones is growing, particularly in sports such as cycling, triathlons, and skating, should prompt an update on this topic., (© 2022 John Wiley & Sons, Ltd.)

The hematological module of the Athlete's Biological Passport (ABP) identifies doping methods and/or substances used to increase the blood's capacity to transport or deliver oxygen to the tissues. Recombinant human erythropoietin (rhEPOs) are doping substances known to boost the production of red blood cells and might have an effect on the blood biomarkers of the ABP. However, hypoxic exposure influences these biomarkers similarly to rhEPOs. This analogous impact complicates the ABP profiles' interpretation by antidoping experts. The present study aimed to collect and identify, through a literature search, the physiological effects on ABP blood biomarkers induced by these external factors. A total of 43 studies were selected for this review. A positive correlation (R 2 = 0.605, r = 0.778, p < 0.001) was identified between the hypoxic dose and the increase in hemoglobin concentration (HGB) percentage. In addition, the change in the reticulocyte percentage (RET%) has been identified as one of the most sensitive parameters to rhEPO use. The mean effects of rhEPO on blood parameters were greater than those induced by hypoxic exposure (1.7 times higher for HGB and RET% and 4 times higher for hemoglobin mass). However, rhEPO micro-doses have shown effects that are hardly distinguishable from those identified after hypoxic exposure. The results of the literature search allowed to identify temporal and quantitative evolution of blood parameters in connection with different hypoxic exposure doses, as well as different rhEPOs doses. This might be considered to provide justified and well-documented interpretations of physiological changes in blood parameters of the Athlete Biological Passport., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Saugy, Schmoutz and Botrè.)

Background: Humanin and the mitochondrial open reading frame of the 12S rRNA-c (MOTS-c) are mitochondrial encoded peptides involved in energy metabolism, cytoprotection, longevity, insulin sensitivity and their expression decrease with age. Levels of these molecules have been shown to respond to acute exercise, however little is known about their modulation under different chronic exercise conditions. In this study, we aim to compare levels of Humanin and MOTS-c in non-athletes vs professional (moderate and high endurance) athletes., Methods: Serum samples were collected from 30 non-athlete controls and 75 professional athletes (47 low/moderate endurance and 28 high endurance athletes). Levels of Humanin and MOTS-c were measured by the enzyme linked immunosorbent aaasy (ELISA) and linear models were generated to compare the effect of different levels of endurance exercise on these factors in different age groups. Spearman correlation was used to assess the correlation between these factors in athletes and non-athletes., Results: We showed that professional athletes had lower levels of MOTS-c and higher levels of Humanin than sedentary controls. Within the athletic groups, high endurance athletes had lower levels of Humanin than low/moderate endurance athletes of the same gender/age groups, whereas MOTS-c levels did not change between the subgroups. Humanin and MOTS-c levels were highly correlated in athletes, but not in sedentary controls., Conclusions: This pilot data suggests that serum levels of the mitochondrial proteins MOTS-c and Humanin change in response to chronic exercise with implications on energy metabolism and performance., Competing Interests: The authors declare no conflict of interest., (Copyright: © 2022 The Author(s). Published by IMR Press.)

Thyroid hormones and their derivatives are structurally related to the non-essential amino acid tyrosine (4-hydroxyphenylalanine). However, there are physicochemical differences that make it difficult to apply an analytical method for their simultaneous detection. This work focused on the optimization of a method using liquid chromatography-electrospray ionization mass spectrometry to measure eight compounds related to levothyroxine (T4). In addition, the influence of the additives to the mobile phase, the solvents for liquid-liquid extraction and the influence of the hydrolysis of the conjugated analytes were studied. Optimization of MRM transitions and collision energy for analytes and capillary voltage, nebulizer gas pressure, nozzle voltage, sheath gas flow, sheath gas temperature, drying gas flow and drying gas temperature for ionization source was done. The recovery of analytes was studied using five solvents and six solvent systems to introduce them into the liquid-liquid extraction and matrix cleanup steps. Different additives to the mobile phase were evaluated as well as the effectiveness of enzymatic and chemical hydrolysis. The best MRM transitions and source parameters were settled in order to generate an optimized instrumental method. The addition of ammonium formate, ammonium fluoride, and acetic acid to the mobile phase showed no improvement in responses compared to classic 0.1% formic acid. The use of tert-butyl methyl ether: isopropanol (75: 25, V: V) showed a suitable recovery of analytes to perform a liquid-liquid extraction, and n-hexane might be an appropriate solvent if a cleanup step is needed. The stability of T3, T4 and thyronine, checked after the hydrolysis with extracts of E. coli and H. pomatia showed good results, contrary to chemical hydrolysis that showed a total degradation of T3 and T4., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Endurance training is associated with physiological changes in elite athletes, but little is known about female-specific effects of endurance training. Despite the significant rise in female sports participation, findings from studies performed on male athletes are largely extrapolated to females without taking into consideration sex-specific differences in metabolism. Subsequently, this study aimed to investigate the steroid hormone profiles of elite female endurance athletes in comparison with their non-athletic counterparts. Untargeted metabolomics-based mass spectroscopy combined with ultra-high-performance liquid chromatography was performed on serum samples from 51 elite female endurance athletes and 197 non-athletic females. The results showed that, compared to non-athletic females, certain androgen, pregnenolone, and progestin steroid hormones were reduced in elite female endurance athletes, while corticosteroids were elevated. The most significantly altered steroid hormones were 5alpha-androstan-3alpha,17alpha-diol monosulfate (FDR = 1.90 × 10 -05 ), androstenediol (3alpha, 17alpha) monosulfate (FDR = 2.93 × 10 -04 ), and cortisol (FDR = 2.93 × 10 -04 ). Conclusively, the present study suggests that elite female endurance athletes have a unique steroid hormone profile with implications on their general health and performance., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

We present a liquid chromatography tandem mass spectrometry method for the simultaneous analysis of 16 endogenous steroids (androgens, estrogens, glucocorticoids and progestogens) in human serum. Samples (250 μl of matrix) were extracted with t-butylmethyl ether prior to LC-MS/MS analysis. The chromatographic separation was achieved on a reversed-phase column using a methanol-water gradient. The HPLC was coupled to a triple quadrupole mass spectrometer equipped with an electrospray ionization source with acquisition in multiple reaction monitoring mode. The method was validated using surrogate matrices and human serum samples. The specificity of the method was confirmed for all of the considered steroids; linearity was also assessed (R 2  > 0.99, lack-of-fit test) in the ranges of concentrations investigated. The lower limits of quantification were in the range 10-400 pg/ml depending on the target steroid. Accuracy was in the range 85-115% for all target steroids except for the lower limit of quantitation levels where it was 80-120%. The extraction recovery was always >65%. No significant matrix effects were observed. To test the reliability of the method, the analysis of serum samples collected from 10 healthy subjects (5 M/5F) was performed. The present method can be used to identify the trajectories of deviation from the concentration normality ranges applied to disorders of the gonadal and adrenal axes., (© 2022 John Wiley & Sons, Ltd.)

In the last decade, the market for new psychoactive substances has been enriched by numerous psychedelic phenethylamines, which mimic the psychoactive effect of lysergic acid diethylamide (LSD). In particular, the -NBOMe series, which are more potent than their 2C compounds analogs, are considered worthy substitutes for LSD by users. The purpose of this study was to assess the effects of 25 H -NBOMe and its halogenated derivatives (25 I -NBOMe and 25 B -NBOMe) in comparison to their 2C compounds analogs and LSD on the sensorimotor (visual, acoustic, and overall tactile), reaction time, spontaneous (total distance traveled) and stimulated (drag, accelerod test) motor activity, grip strength test, and prepulse inhibition (PPI) responses in mice. Systemic administration of -NBOMe, 2C compounds analogs, and LSD (0.001-10 mg/kg) differently impaired the sensorimotor, reaction time, motor, and PPI responses in mice. In particular, halogenated (25I and 25B)-NBOMe derivatives appear to be more effective than the entire class of 2C compounds analogs in altering visual and acoustic responses, affecting reaction time, and motor and sensory gating in PPI test. In fact, the specific rank order of compounds potency for nearly all of the experiments showed that (25I and 25B)-NBOMe were more potent than 2C compounds analogs and LSD. -NBOMe and 2C compounds analogs impaired not only the reception of incoming sensory stimuli (visual and acoustic), but their correct brain processing (PPI) in an equal and sometimes stronger way than LSD. This sensory impairment directly affected the spontaneous motor response and reaction time of mice, with no change in performance in stimulated motor activity tests. These aspects should be carefully considered to better understand the potential danger that psychedelic phenethylamines, in particular -NBOMe, may pose to public health, with particular reference to decreased performance in driving and hazardous works that require special sensorimotor skills., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tirri, Bilel, Arfè, Corli, Marchetti, Bernardi, Boccuto, Serpelloni, Botrè, De-Giorgio, Golembiowska and Marti.)

This article presents the results of a study aimed to give new suggestions and strategies for improving the implementation of the flow cytofluorimetry-based method for the detection of homologous blood transfusions in doping control. The method is based on the recognition of the phenotypic mismatch between minority blood group antigens possessed by the donor and the recipient. Two strategies have been followed to reduce the risk of false-negative results: (i) the monitoring of a broader range of erythrocytes surface antigens; and (ii) the application of different surface erythrocyte staining protocols, tailored on the different antigens and the type of antigenic mismatch that had to be detected (whether it is the donor or the recipient who expresses or not the antigen to be detected). Special attention has also been focused on the time factor, to avoid prolonged sample storage, since hemolysis may have a significant impact on the reliability and quality of the results. Our experimental evidence suggests that the risk of false-negative results can be minimized by (i) the expansion of the antigen panel, with the inclusion of four additional targets; (ii) a more accurate selection of the gating area of the red blood cells; (iii) the choice of a better fluorochrome (alexa fluor 488) to be conjugated to the secondary antibody; and (iv) the implementation of different staining protocols depending on the nature of the double population to be detected (donor expressing vs. recipient non-expressing and vice versa). The combination of the above approaches allowed a significant reduction of false-negative results, assessed on samples simulating a homologous blood transfusion between two compatible subjects., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Donati, de la Torre, Pagliarosi, Pirri, Prevete and Botrè.)

Mexedrone is a synthetic cathinone structurally related to mephedrone, which belongs to the class of N-alkyl cathinone derivatives, whose metabolic profile has not been fully clarified yet. This study considers the in vitro phase I metabolism of mexedrone, to pre-select the most appropriate marker(s) of intake. Mexedrone was incubated in the presence of either human liver microsomes or single recombinant CYP450 isoforms. The metabolic profile was outlined by ultra-high-performance liquid chromatography coupled to both high- and low-resolution mass spectrometry. In detail, the phase I metabolic profile of mexedrone was initially defined by a time-of-flight analyzer, while the chemical structures of the detected metabolites and the potential presence of minor metabolites were subsequently studied by tandem mass spectrometry, using a triple quadrupole analyzer. The main phase I metabolic reactions were hydroxylation and N- and O-dealkylation. The CYP450 isoforms most involved were CYP2C19, responsible for the formation of both hydroxylated and dealkylated metabolites, followed by CYP2D6 and CYP1A2, involved in the hydroxylation reactions only. Finally, a significant fraction of mexedrone unchanged was also detected. Based on this evidence, the most appropriate markers of intake are mexedrone unchanged and the hydroxylated metabolites., (© 2021 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Rationale: This work demonstrates the high potential of combining high-resolution mass spectrometry with chemometric tools, using metabolomics as a guided tool for anti-doping analysis. The administration of 7-keto-DHEA was studied as a proof-of-concept of the effectiveness of the combination of knowledge-based and machine-learning approaches to differentiate the changes due to the athletic activities from those due to the recourse to doping substances and methods., Methods: Urine samples were collected from five healthy volunteers before and after an oral administration by identifying three time intervals. Raw data were acquired by injecting less than 1 μL of derivatized samples into a model 8890 gas chromatograph coupled to a model 7250 accurate-mass quadrupole time-of-flight analyzer (both from Agilent Technologies), by using a low-energy electron ionization source; the samples were then preprocessed to align peak retention times with the same accurate mass. The resulting data table was subjected to multivariate analysis., Results: Multivariate analysis showed a high similarity between the samples belonging to the same collection interval and a clear separation between the different excretion intervals. The discrimination between blank and long excretion groups may suggest the presence of long excretion markers, which are particularly significant in anti-doping analysis. Furthermore, matching the most significant features with some of the metabolites reported in the literature data demonstrated the rationality of the proposed metabolomics-based approach., Conclusions: The application of metabolomics tools as an investigation strategy could reduce the time and resources required to identify and characterize intake markers maximizing the information that can be extracted from the data and extending the research field by avoiding a priori bias. Therefore, metabolic fingerprinting of prohibited substance intakes could be an appropriate analytical approach to reduce the risk of false-positive/negative results, aiding in the interpretation of "abnormal" profiles and discrimination of pseudo-endogenous steroid intake in the anti-doping field., (© 2021 John Wiley & Sons Ltd.)

We have investigated the metabolic profile of N-ethyl heptedrone, a new designer synthetic stimulant drug, by using data independent acquisition mass spectrometry. Phase I and phase II metabolism was studied by in vitro models, followed by liquid-chromatography coupled to mass spectrometry, to characterize and pre-select the most diagnostic markers of intake. N-ethyl heptedrone was incubated in the presence of pooled human liver microsomes. The contribution of individual enzymatic isoforms in the formation of the phase I and phase II metabolites was further investigated by using human recombinant cDNA-expressed cytochrome P450 enzymesand uridine 5'-diphospho glucuronosyltransferases. The analytical workflow consisted of liquid-liquid extraction with tert-butyl-methyl-ether at alkaline pH, performed before (to investigate the phase I metabolic profile) and after (to investigate the glucuronidation profile) enzymatic hydrolysis. The separation, identification, and determination of the compounds formed in the in vitro experiments were carried out by using liquid chromatography coupled to either high- or low-resolution mass spectrometry. Data independent acquisition method, namely sequential window acquisition of all theoretical fragment-ion spectra (SWATH®) and product ion scan were selected for high-resolution mass spectrometry, whereas multiple reaction monitoring was used for low-resolution mass spectrometry. Thirteen phase-I metabolites were isolated, formed from reactions being catalyzed mainly by CYP1A2, CYP2C9, CYP2C19 and CYP2D6 and, to a lesser degree, by CYP3A4 and CYP3A5. The phase I biotransformation pathways included hydroxylation in different positions, reduction of the ketone group, carbonylation, N-dealkylation, and combinations of the above. Most of the hydroxylated metabolites underwent conjugation reactions to form the corresponding glucurono-conjugated metabolites. Based on our in vitro observation, the metabolic products resulting from reduction of the keto group, N-dealkylation and hydroxylation of the aliphatic chain appear to be the most diagnostic target analytes to be selected as markers of exposure to N-ethyl heptedrone., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

5α-reductase inhibitors (5-ARIs) are considered by the World Anti-doping Agency as potential confounding factors in evaluating the athlete steroid profile, since they may interfere with the urinary excretion of several diagnostic compounds. We herein investigated 5α-reductase inhibitors from a different perspective, by verifying their influence on the carbon isotopic composition of 5α- and 5β-reduced testosterone and nandrolone metabolites. The GC-C-IRMS analysis was performed on a set of urine samples collected from three male Caucasian volunteers after the acute and chronic administration of finasteride in combination with the intake of 19-norandrostenedione, a nandrolone precursor. The excretion and the isotopic profile of androsterone (A), etiocholanolone (Etio) 5α-androstane-3α,17β-diol (5αAdiol), and 5β-androstane-3α,17β-diol (5βAdiol) were determined as well as those of 19-norandrosterone (19-NA) and 19-noretiocholanolone (19-NE). Pregnanediol (PD) and pregnanetriol (PT) were also measured as endogenous reference compounds to define the individual endogenous isotopic profile. Our results confirmed the impact of finasteride, especially if chronically administered, on the enzymatic pathway of testosterone and nandrolone, and pointed out the influence of 5-ARIs on δ 13 C values of the selected target compounds determined in the IRMS confirmation analysis., (© 2021 John Wiley & Sons, Ltd.)

Dehydrochloromethyltestosterone (DHCMT) is an anabolic-androgenic steroid that was developed by Jenapharm in the 1960s and was marketed as Oral Turinabol®. It is prohibited in sports at all times; nevertheless, there are several findings by anti-doping laboratories every year. New long-term metabolites have been proposed in 2011/12, which resulted in adverse analytical findings in retests of the Olympic games of 2008 and 2012. However, no controlled administration trial monitoring these long-term metabolites was reported until now. In this study, DHCMT (5 mg, p.o.) was administered to five healthy male volunteers and their urine samples were collected for a total of 60 days. The unconjugated and the glucuronidated fraction were analyzed separately by gas chromatography coupled to tandem mass spectrometry. The formation of the described long-term metabolites was verified, and their excretion monitored in detail. Due to interindividual differences there were several varieties in the excretion profiles among the volunteers. The metabolite M3, which has a fully reduced A-ring and modified D-ring structure, was identified by comparison with reference material as 4α-chloro-17β-hydroxymethyl-17α-methyl-18-nor-5α-androstan-13-en-3α-ol. It was found to be suitable as long-term marker for the intake of DHCMT in four of the volunteers. In one of the volunteers, it was detectable for 45 days after single oral dose administration. However, in two of the volunteers M5 (already published as long-term metabolite in the 1990s) showed longer detection windows. In one volunteer M3 was undetectable but another metabolite, M2, was found as the longest detectable metabolite. The last sample clearly identified as positive was collected between 9.9 and 44.9 days. Furthermore, the metabolite epiM4 (partially reduced A-ring and a modified D-ring structure which is epimerized in position 17 compared to M3) was identified in the urine of all volunteers with the help of chemically synthesized reference as 4-chloro-17α-hydroxymethyl-17β-methyl-18-nor-androsta-4,13-dien-3β-ol. It may serve as additional confirmatory metabolite. It is highly recommended to screen for all known metabolites in both fractions, glucuronidated and unconjugated, to improve identification of cheating athletes. This study also offers some deeper insights into the metabolism of DHCMT and of 17α-methyl steroids in general., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

In this work we have investigated the influence of the intake of two synthetic isoflavones, methoxyisoflavone and ipriflavone, on the urinary concentration of endogenous steroids, and on their relative ratios, of doping relevance. Specifically, the concentrations of testosterone (T), epitestosterone (E), androsterone (A), etiocholanolone (Etio), 5α-androstan-3α,17α-diol (5αAdiol), 5β-androstan-3α,17α-diol (5βAdiol), and the ratios T/E, A/T, A/Etio, 5αAdiol/5βAdiol, 5αAdiol/E, were considered, in the framework of the Steroidal Module of the Athlete Biological Passport (ABP). The above set of parameters were complemented by the urinary levels of luteinizing hormone (total LH) and the ratio between T and LH (T/total LH), to assess the possible effects on the biosynthesis of the mentioned steroids. Five healthy Caucasian male volunteers were selected for the study. Urine samples were collected before and during the administration of (i) methoxyisoflavone (Methoxyisoflavone, MyProtein) and (ii) ipriflavone (Osteofix ®, Chiesi Farmaceutici). For the analysis of the urinary steroid profile, after enzymatic hydrolysis with β-glucuronidase from Escherichia Coli (E. Coli) and liquid-liquid extraction with tert-buthylmethyl ether, all samples were analyzed by gas chromatography coupled to tandem mass spectrometry (GC-MS/MS), while for the determination of total LH all urine samples were directly analyzed by a chemiluminescent immunometric assay technique (Siemens Immulite 2000 LH). Our results show that the administration of either methoxyisoflavone or ipriflavone causes an alteration of the urinary concentrations and concentration ratios of the investigated steroids, in the range 55-80% from the baseline values. Furthermore, an oversecretion of LH after the daily intake of methoxyisoflavone or ipriflavone was also recorded in all volunteers, corresponding to an increase in the biosynthesis and excretion of T and some of its metabolites. These changes trigger a disregulation in the pattern of urinary excretion of the steroids included in the Steroidal Module of the ABP, which makes more difficult the interpretation of the longitudinal steroid profile based on the definition of individual normality ranges for each athlete. Our data are also consistent with previous evidence regarding the in vitro effects of natural and synthetic isoflavones, suggesting that their monitoring in doping control routine analysis would be very beneficial for the result management activities., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

This article reports the results obtained from the investigation of the influence of miconazole administration on the physiological fluctuation of the markers of the steroid profile included in the "steroidal module" of the Athlete Biological Passport. Urines collected from male Caucasian subjects before, during, and after either systemic (i.e., oral and buccal) or topical (i.e., dermal) treatment with miconazole were analyzed according to validated procedures based on gas chromatography coupled to tandem mass spectrometry (GC-MS/MS) (to determine the markers of the steroid profile) or liquid chromatography coupled to MS/MS (LC-MS/MS) (to determine miconazole urinary levels). The results indicate that only after systemic administration, the markers of the steroid profile were significantly altered. After oral and buccal administration, we have registered (i) a significant increase of the 5α-androstane-3α,17β-diol/5β-androstane-3α,17β-diol ratio and (ii) a significant decrease of the concentration of androsterone, etiocholanolone, 5β-androstane-3α,17β-diol, and 5α-androstane-3α,17β-diol and of the androsterone/etiocholanolone, androsterone/testosterone, and 5α-androstane-3α,17β-diol/epitestosterone ratios. Limited effects were instead measured after dermal intake. Indeed, the levels of miconazole after systemic administration were in the range of 0.1-12.5 μg/ml, whereas after dermal administration were below the limit of quantification (50 ng/ml). Significant alteration started to be registered at concentrations of miconazole higher than 0.5 μg/ml. These findings were primarily explained by the ability of miconazole in altering the kinetic/efficacy of deglucuronidation of the endogenous steroids by the enzyme β-glucuronidase during the sample preparation process. The increase of both incubation time and amount of β-glucuronidase was demonstrated to be effective countermeasures in the presence of miconazole to reduce the risk of uncorrected interpretation of the results., (© 2021 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

4,4'-Dimethylaminorex (4,4'-DMAR) is a new synthetic stimulant, and only a little information has been made available so far regarding its pharmaco-toxicological effects. The aim of this study was to investigate the effects of the systemic administration of both the single (±) cis (0.1-60 mg/kg) and (±) trans (30 and 60 mg/kg) stereoisomers and their co-administration (e.g., (±) cis at 1, 10 or 60 mg/kg + (±) trans at 30 mg/kg) in mice. Moreover, we investigated the effect of 4,4'-DMAR on the expression of markers of oxidative/nitrosative stress (8-OHdG, iNOS, NT and NOX2), apoptosis (Smac/DIABLO and NF-κB), and heat shock proteins (HSP27, HSP70, HSP90) in the cerebral cortex. Our study demonstrated that the (±) cis stereoisomer dose-dependently induced psychomotor agitation, sweating, salivation, hyperthermia, stimulated aggression, convulsions and death. Conversely, the (±) trans stereoisomer was ineffective whilst the stereoisomers' co-administration resulted in a worsening of the toxic (±) cis stereoisomer effects. This trend of responses was confirmed by immunohistochemical analysis on the cortex. Finally, we investigated the potentially toxic effects of stereoisomer co-administration by studying urinary excretion. The excretion study showed that the (±) trans stereoisomer reduced the metabolism of the (±) cis form and increased its amount in the urine, possibly reflecting its increased plasma levels and, therefore, the worsening of its toxicity.

Rationale: The metabolism of arimistane (Arim) was first described in 2015, and androst-3,5-diene-7β-ol-17-one was proposed as the main metabolite excreted in urine. Recently, a more detailed study describing the findings in urine after the administration of Arim has been published. This study corroborated the previously described metabolite but also described several phase I and II metabolites, analyzing trimethylsilylated urinary extracts using accurate mass spectrometry coupled to gas chromatography (GC/qTOF). The present communication is an extension of this late investigation aiming to implement the results of Arim metabolism using either accurate mass spectrometry and/or triple quadrupole tandem mass spectrometry, both coupled to liquid chromatography (LC/qTOF and LC/QqQ)., Methods: The samples used in this study were the same as previously studied using GC/qTOF. One single oral dose of Arim was administered to three volunteers, and samples collected before and up to 10 h after the Arim administration were analyzed. The unconjugated fraction of urine was removed, and the hydrolysis was performed with β-glucuronidase from Escherichia coli. The extracts were reconstituted in water:acetonitrile before the LC/qTOF and LC/QqQ analysis., Results: The presence of the proposed metabolites studied using GC was verified by accurate mass measurements. Twelve metabolites not found in the blank urine samples were identified by the accurate mass spectra with acceptable errors between -7.5 and 8.1 ppm: 4 reduced metabolites, 4 monohydroxylated metabolites, and 4 with an additional hydroxylation (bis-hydroxylated metabolites). Unlike in the study carried out using GC/qTOF, Arim itself was found in the samples of the three volunteers., Conclusions: Twelve metabolites were identified, and specific transitions were proposed. Despite the good results, some limitations remain. As for GC/qTOF, the α- or β configuration of hydroxy groups, as well as the exact position for some unsaturation, cannot be assigned with certainty. Because certified reference materials of these metabolites are not yet available, the molecular structures were hypothesized considering the previous study using GC., (© 2021 John Wiley & Sons Ltd.)

Ecdysterone is a phytosteroid widely discussed for its various pharmacological, growth-promoting, and anabolic effects, mediated by the activation of estrogen receptor beta (ERbeta). Performance-enhancement in sports was demonstrated recently, and ecdysterone was consequently included in the Monitoring Program, to detect potential patterns of misuse in sport. Only few studies on the pharmacokinetics of ecdysterone in humans have been reported so far. In this study, post-administration urine samples in twelve volunteers (single dose of 50 mg of ecdysterone) were analyzed using dilute-and-inject liquid-chromatography-tandem mass spectrometry. Identification and quantitation of ecdysterone and of two metabolites, 14-deoxy-ecdysterone and 14-deoxy-poststerone, was achieved. Ecdysterone was the most abundant analyte present in post-administration urine samples, detected for more than 2 days, with a maximum concentration (C max ) in the 2.8-8.5 h urine (C max = 4.4-30.0 µg/mL). The metabolites 14-deoxy-ecdysterone and 14-deoxy-poststerone were detected later, reaching the maximum concentrations at 8.5-39.5 h (C max = 0.1-6.0 µg/mL) and 23.3-41.3 h (C max = 0.1-1.5 µg/mL), respectively. Sex-specific differences were not observed. Cumulative urinary excretion yielded average values of 18%, 2.3%, and 1.5% for ecdysterone, 14-deoxy-ecdysterone, and 14-deoxy-poststerone, respectively. Ecdysterone and 14-deoxy-ecdysterone were excreted following first-order kinetics with half-lives calculated with three hours, while pharmacokinetics of 14-deoxy-poststerone needs further evaluation.

Cytochrome P450s (CYPs) are an essential family of enzymes in the human body. They play a crucial role in metabolism, especially in human steroid biosynthesis. Reactions catalyzed by these enzymes are highly stereo- and regio-specific. Lack or severe malfunctions of CYPs can cause severe diseases and even shorten life. Hence, investigations on metabolic reactions and structural requirements of substrates are crucial to gain further knowledge on the relevance of different enzymes in the human body functions and the origin of diseases. One key enzyme in the biosynthesis of gluco- and mineralocorticoids is CYP21A2, also known as steroid 21-hydroxylase. To investigate the steric and regional requirements of substrates for this enzyme, we performed whole-cell biotransformation assays using a strain of fission yeast Schizosaccharomyces pombe recombinantly expressing CYP21A2. The progestogens progesterone, pregnenolone, and their 17α-hydroxy-derivatives were used as substrates. After incubation, samples were analyzed using gas chromatography coupled to mass spectrometry. For progesterone and 17α-hydroxyprogesterone, their corresponding 21-hydroxylated metabolites 11-deoxycorticosterone and 11-deoxycortisol were detected, while after incubation of pregnenolone and 17α-hydroxypregnenolone, no hydroxylated product was observed. Findings were confirmed with authentic reference material. Molecular docking experiments agree with these results and suggest that interaction between the 3-oxo group and arginine-234 of the enzyme is a strict requirement. The presented results demonstrate once more that the presence of an oxo-group in position 3 of the steroid is indispensable, while a 3-hydroxy group prevents hydroxylation in position C-21 by CYP21A2. This knowledge may be transferred to other CYP21A2 substrates and hence help to gain essential insights into steroid metabolism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Loke, Stoll, Machalz, Botrè, Wolber, Bureik and Parr.)

Predictive models have been developed for the early identification of novel anabolic androgenic steroids and to obtain information on their molecular structure. To this purpose, gas-chromatographic and mass spectrometric characteristic parameters of 136 anabolic androgenic steroids have been specifically considered. Starting from Principal Component Analysis, different chemometric methods were applied, such as classification and clustering techniques, outlining a spectral and structural characterization for each steroid subclass, and considering the contribution of more than 30 variables. Mass spectrometric data on the TMS-derivatives of the target steroids were obtained by gas chromatography coupled to quadrupole-time of flight mass spectrometry using electron ionization. Steroids included in the training set were grouped in 5 subclasses according to their structural similarity, and the experimental data, processed by the chemometric models, allowed the identification of class-specific common fragments and spectral trends. The results of this study, validated on a test set of 21 steroids, have confirmed that the proposed approach allows tracing novel "designer anabolic steroids", including those previously unknown new structures that may have been designed and illicitly synthesized to be invisible to the current anti-doping tests., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Transcranial Direct Current Stimulation (tDCS) is a non-invasive brain stimulation that may enhance mental and physical performance in sports, representing a potential new form of doping ("brain doping" or "electromagnetic doping"). This study aims to identify diagnostic biomarkers for detecting the possible abuse of tDCS in sport. Brain-Derived Neurotrophic Factor (BDNF) and other neurotrophins (NT, such as beta nerve growth factor, NGF) were pre-selected as potential candidates since their serum values have been observed to change following tDCS. Neurotrophins were measured using ELISA assays in 92 serum samples collected from elite athletes, classified by sex (males = 74; females = 18), age (range 17-25 n = 27, 26-35 n = 36, and over 35 n = 14; age not known n = 15), type of sports practiced (endurance n = 74; power n = 18), and type of sample collection ("in competition" n = 24; "out of competition" n = 68). Single nucleotide polymorphisms (rs6265, rs11030099, and rs11030100) were genotyped on 88 samples to determine their influence on the analytes' basal levels. Athletes older than 35 presented higher BDNF values than younger individuals ( p < 0.05). Samples collected "in competition" showed higher BDNF concentrations than those collected "out of competition" ( p < 0.05). The studied polymorphisms appeared to affect only on proBDNF, not altering BDNF serum concentrations. NT-3 and NT-4 were poorly detectable in serum. Our results suggest that BDNF can be considered as a first biomarker to detect the abuse of tDCS in sport doping. Further studies are necessary to assess whether proBDNF and beta NGF can also be considered suitable biomarkers to detect the recourse to electromagnetic brain stimulation in sports, especially in the case their serum levels can be monitored longitudinally. To the best of our knowledge, this is the first study aimed to pre-select serum biomarkers to identify the use of tDCS, and represents the first step toward the development of an indirect strategy, preferably based on the longitudinal monitoring of individual data, for the future detection of "brain doping" in sports., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Donati, Sian, Biasini, de la Torre, Folchitto and Botrè.)

The detection of 19-norsteroids abuse in doping controls currently relies on the determination of 19-norandrosterone (19-NA) by gas chromatography-tandem mass spectrometry (GC-MS/MS). An additional confirmatory analysis by gas chromatography coupled to isotope ratio mass spectrometry (GC-C-IRMS) is performed on samples showing 19-NA concentrations between 2.5 and 15 ng/ml and not originated from pregnant female athletes or female treated with 19-norethisterone. 19-Noretiocholanolone (19-NE) is typically produced to a lesser extent as a secondary metabolite. The aim of this work was to improve the GC-C-IRMS confirmation procedure for the detection of 19-norsteroids misuse. Both 19-NA and 19-NE were analyzed as target compounds (TCs), whereas androsterone (A), pregnanediol (PD), and pregnanetriol (PT) were selected as endogenous reference compounds (ERCs). The method was validated and applied to urine samples collected by three male volunteers after the administration of nandrolone-based formulations. Before the instrumental analysis, urine samples (<25 ml) were hydrolyzed with β-glucuronidase from Escherichia coli and extracted with n-pentane. Compounds of interest were purified through a single (for PT) or double (for 19-NE, 19-NA, A, and PD) liquid chromatographic step, to reduce the background noise and eliminate interferences that could have affect the accuracy of δ 13 C values. The limit of quantification (LOQ) of 2 ng/ml was ensured for both 19-NA and 19-NE. The 19-NE determination could be helpful in case of "unstable" urine samples, in late excretion phases or when coadministration with 5α-reductase inhibitors occur., (© 2020 John Wiley & Sons, Ltd.)

Metandienone and methyltestosterone are orally active anabolic-androgenic steroids with a 17α-methyl structure that are prohibited in sports but are frequently detected in anti-doping analysis. Following the previously reported detection of long-term metabolites with a 17ξ-hydroxymethyl-17ξ-methyl-18-nor-5ξ-androst-13-en-3ξ-ol structure in the chlorinated metandienone analog dehydrochloromethyltestosterone ("oral turinabol"), in this study we investigated the formation of similar metabolites of metandienone and 17α-methyltestosterone with a rearranged D-ring and a fully reduced A-ring. Using a semi-targeted approach including the synthesis of reference compounds, two diastereomeric substances, viz. 17α-hydroxymethyl-17β-methyl-18-nor-5β-androst-13-en-3α-ol and its 5α-analog, were identified following an administration of methyltestosterone. In post-administration urines of metandienone, only the 5β-metabolite was detected. Additionally, 3α,5β-tetrahydro-epi-methyltestosterone was identified in the urines of both administrations besides the classical metabolites included in the screening procedures. Besides their applicability for anti-doping analysis, the results provide new insights into the metabolism of 17α-methyl steroids with respect to the order of reductions in the A-ring, the participation of different enzymes, and alterations to the D-ring.

Analytical procedures to detect the misuse of selective androgen receptor modulators in human urine, targeting either the parent drugs and/or their main metabolites, were developed and validated. In detail, 19 target compounds belonging to 9 different chemical classes were considered: arylpropionamide (i.e., andarine (S4), ostarine (S22), S1, S6, S9 and S23), diarylhydantoin (i.e., GLPG0492), indole (i.e., LY2452473, GSK2881078), isoquinoline-carbonyle (i.e., PF-02620414), phenyl-oxadiazole (i.e., RAD140), pyrrolidinyl-benzonitrile (i.e., LGD4033), quinolinone (i.e., LGD2226, LGD3303), steroidal (i.e., Cl-4AS-1, MK0773 and TFM-4AS-1), and tropanol (i.e., AC-262536 and ACP105) derivatives. The metabolites of the target compounds considered were enzymatically synthesized by using human liver microsomes. Sample pre-treatment included enzymatic hydrolysis followed by liquid-liquid extraction at neutral pH. The instrumental analysis was performed by ultra-high-performance liquid chromatography coupled to either high- or low-resolution mass spectrometry. Validation was performed according to the ISO 17025 and the World Anti-Doping Agency guidelines. The analyses carried out on negative samples confirmed the method's selectivity, not showing any significant interferences at the retention times of the analytes of interest. Detection capability was determined in the range of 0.1-1.0 ng/mL for the screening procedure and 0.2-1.0 ng/mL for the confirmation procedure (except for GLPG0492 and GSK2881078). The recovery was greater than 80 % for all analytes, and the matrix effect was smaller than 35 %. The method also matched the criteria of the World Anti-Doping Agency in terms of repeatability of the relative retention times (CV% < 1.0) and of the relative abundances of the selected ion transitions (performed only in the case of triple quadrupole, CV% < 15), ensuring the correct identification of all the analytes considered. Urine samples containing andarine, ostarine, or LGD4033 were used to confirm the actual applicability of the selected analytical strategies. All target compounds (parent drugs and their main metabolites) were detected and correctly identified., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2020. Published by Elsevier B.V.)

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.)

The detection of the abuse of pseudo-endogenous steroids in sport is articulated in two different levels: an initial testing procedure, based on the longitudinal evaluation of the urinary androgenic steroid profile by gas-chromatography mass spectrometry (GC-MS n ), and a confirmation analysis, based on the differentiation between the endogenous and exogenous origin of the pseudo-endogenous steroids by gas-chromatography coupled to isotopic ratio mass spectrometry (GC/C/IRMS). The abuse of pharmaceutical preparations displaying a carbon isotopic composition values within a range similar to those reported for endogenous urinary steroids makes more difficult the application of GC/C/IRMS technique. To overcome this limitation, the direct detection of an intact synthetic anabolic steroid ester in blood matrices (plasma and/or serum) could supply the unequivocal proof of exogenous administration of pseudo-endogenous steroids. Here we are presenting a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the analysis of 14 testosterone (T) esters and 2 nandrolone (Nand) esters in human serum. Sample pre-treatment consisted of protein precipitation, liquid-liquid extraction and derivatization. The formation of three different derivatives (oxime derivatives, Girard P and Girard T hydrazones) is considered, in order to guarantee an improvement in the detection capability of the assay with respect to underivatized compounds. Once the most suitable derivative was selected, the method was validated, according to the World Anti-Doping Agency (WADA) criteria, in terms of specificity, linearity, limit of detection (LOD), extraction recovery, matrix effect (ion suppression/enhancement), carry over and autosampler stability. The formation of Girard P hydrazones of T and Nand esters provides the best results compared to the underivatized compounds, oxime and Girard T derivatives, respectively. The presented analytical method is specific for all considered compounds and linear in the range of concentrations investigated (0.25-10 ng/mL). The LODs are between 0.03 and 0.30 ng/mL, the extraction recovery higher than 70 % for all esters and no remarkable matrix effect, expressed in terms of ion enhancement and ion suppression, was observed. Finally, the developed and validate method was applied in the analysis of serum samples collected after the administration of a single dose (40 mg, 1 capsule) of testosterone undecanoate (Andriol ®) demonstrating its applicability., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We have considered the urinary excretion profile of methiopropamine (MPA), a thiophene ring-based structural analog of methamphetamine with similar stimulant effects, with the aim of selecting the most appropriate marker(s) of intake that may be useful in forensic analysis. For this purpose, in vitro studies were preliminarily performed on human liver microsomes for tracing the phase I metabolic pathways of MPA, preselecting the best candidates as potential target analytes, and designing the optimal experimental strategy. In vivo studies were then conducted on mice, after the intraperitoneal administration of a 10-mg/kg dose. Urine samples were collected every 3 h in the first 9 h and, subsequently, from 24 to 36 h, and stored at -80°C until further analysis. The measurements were performed using a targeted procedure based on liquid/liquid extraction followed by liquid chromatography-tandem mass spectrometry analysis. Our results show that in the time interval 0-9 h after administration, MPA was extensively oxidized mainly to nor-MPA, oxo-MPA, and two hydroxylated metabolites (ie, hydroxy-aryl-methiopropamine and hydroxy-alkyl-methiopropamine). All phase I metabolites underwent phase II metabolism, with the formation of nor-hydroxy-methiopropamine only in phase II, confirmed by the results obtained after enzymatic hydrolysis with β-glucuronidase and arylsulfatase. In the time interval 24-36 h after administration, only unchanged MPA and nor-MPA were detected, suggesting that these two markers are those endowed with the highest diagnostic value. The method was validated for these two principal markers, proving to be fit for anti-doping, toxicological, and forensic analyses., (© 2020 John Wiley & Sons, Ltd.)

Rationale: The aromatase inhibitor formestane (4-hydroxyandrost-4-ene-3,17-dione) is included in the World Anti-Doping Agency's List of Prohibited Substances in Sport. However, it also occurs endogenously as do its 2-, 6- and 11-hydroxy isomers. The aim of this study is to distinguish the different isomers using gas chromatography/electron ionization mass spectrometry (GC/EI-MS) for enhanced confidence in detection and selectivity for determination., Methods: Established derivatization protocols to introduce [ 2 H 9 ]TMS were followed to generate perdeuterotrimethylsilylated and mixed deuterated derivatives for nine different hydroxy steroids, all with 3-keto-4-ene structure. Formestane was additionally labelled with H 2 18 O to obtain derivatives doubly labelled with [ 2 H 9 ]TMS and 18 O. GC/EI-MS spectra of labelled and unlabelled TMS derivatives were compared. Proposals for the generation of fragment ions were substantiated by high-resolution MS (GC/QTOFMS) and tandem mass spectrometry (MS/MS) experiments., Results: Subclass-specific fragment ions include m/z 319 for the 6-hydroxy and m/z 219 for the 11-hydroxy compounds. Ions at m/z 415, 356, 341, 313, 269 and 267 were indicative for the 2- and 4-hydroxy compounds. For their discrimination the transition m/z 503 → 269 was selective for formestane. In 2-, 4- and 6-hydroxy steroids loss of a TMSO radical takes place as cleavage of a TMS-derived methyl radical and a neutral loss of (CH 3 ) 2 SiO. Further common fragments were also elucidated., Conclusions: With the help of stable isotope labelling, the structures of postulated diagnostic fragment ions for the different steroidal subclasses were elucidated. 18 O-labelling of the other compounds will be addressed in future studies to substantiate the obtained findings. To increase method sensitivity MS 3 may be suitable in future bioanalytical applications requiring discrimination of the 2- and 4-hydroxy compounds., (© 2020 The Authors. Rapid Communications in Mass Spectrometry published by John Wiley & Sons Ltd.)