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β-hydroxy β-methyl butyric acid (HMB), either as the free acid or in the form of its calcium salt, is a component of several dietary supplements marketed to enhance sports performance, due to its role in protein synthesis. In this pilot study, we investigated the urinary excretion profile of HMB, an endogenous metabolite of the essential amino acid leucine. The endogenous reference ranges of HMB, in human urine samples, were monitored by collecting samples from 20 volunteers. Data obtained were compared with those from controlled excretion studies, following the intake of a 3-g oral dose of HMB. Urine samples were analyzed through a targeted procedure employing a conventional GC-MS system operating in SIM mode. Our results show that the excreted urinary concentration of HMB significantly exceeds the range of endogenous concentrations for at least 24 h, making possible to detect its exogenous origin., (© 2024 The Author(s). Drug Testing and Analysis published by John Wiley & Sons Ltd.)

Confounding factors including exercise and environments challenge the interpretation of individual Athlete Biological Passports (ABPs). This study aimed to investigate the natural variability of hematological ABP parameters over 1 year in elite athletes compared with healthy control subjects and the validity of a multiparametric model estimating plasma volume (PV) shifts to correct individual ABP thresholds. Blood samples were collected monthly with full blood counts performed by flow cytometry (Sysmex XN analyzers) in 20 elite xc-skiers (ELITE) and 20 moderately trained controls. Individual ABP profiles were generated through Anti-Doping Administration & Management System Training, a standalone version of the ABP's adaptive model developed by the World Anti-Doping Agency. Additionally, eight serum parameters were computed as volume-sensitive biomarkers to run a multiparametric model to estimate PV. Variability in ELITE compared with controls was significantly higher for the Abnormal Blood Profile Scores (P = 0.003). Among 12 Atypical Passport Findings (ATPF) initially reported, six could be removed after correction of PV shifts with the multiparametric modeling. However, several ATPF were additionally generated (n = 19). Our study outlines a larger intraindividual variability in elite athletes, likely explained by more frequent exposure to extrinsic factors altering hematological biomarkers. PV correction for individual ABP thresholds allowed to explain most of the atypical findings while generating multiple new ATPF occurrences in the elite population. Overall, accounting for PV shifts in elite athletes was shown to be paramount in this study outlining the opportunity to consider PV variations with novel approaches when interpreting individual ABP profiles., (© 2024 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

We performed genotyping analysis of human biallelic polymorphisms (single nucleotide polymorphisms) for the detection of homologous blood transfusion in sports doping. DNA was extracted from dried blood spots and quantified real-time fast PCR. The method was proven to allow the detection of transfusions up to a donor percentage of 1%, with a significant improvement in terms of sensitivity with respect to both the reference cytofluorimetric method and a previously proposed strategy based on the DNA STR-based strategy., (© 2024 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

This paper aims to study the metabolism of thyroid hormones (TH) in urine by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was applied to samples collected before and after the administration of sodium triiodothyronine (T3) and sodium levothyroxine (T4) to a euthyroid volunteer and to samples of athletes declaring and not declaring thyroid supplementation. Samples were analyzed by LC-MS/MS after enzymatic hydrolysis, liquid-liquid, and solid-phase extractions. Ratios between T3/thyronine and T4/3,3'-T2 may be used for the detection of the administration of exogenous T3 in urine. Meanwhile, 3-T1 concentrations may be used to detect exogenous T4 administration. Nevertheless, these markers may not work properly in hypothyroid population, as athletes seem to be. The levels of T3 and T4 of athletes were lower than those of a euthyroid state even when they are under administration of TH supplements. The HTP axis high efficiency does not allow observing differences between athletes who do not declare and those who declare having used TH supplementation by direct measurements of T3 and T4 in urine. The detection of TH administration in urine (triiodothyronine and levothyroxine) may work when dealing with euthyroid individuals. Nevertheless, in individuals with hypothyroidism where the tendency is toward the maintenance of homeostasis, and it may be not possible to detect their consumption by applying cut-off values., (© 2024 John Wiley & Sons Ltd.)

Recently, the trend of thyroid hormones (TH) consumption in the sports community has been published. It is known the capacity of the exogenously administered TH to enhance metabolism, being an attractive feature for athletes, who search for weight control and increased caloric expenditure. This paper aimed the validation of a method to measure TH and related compounds in urine by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The method was applied to urine samples collected before and after the administration of a diiodothyronine (3,5-T2) supplement. A method to detect nine TH included an enzymatic hydrolysis, liquid-liquid extraction, and solid-phase extraction. The extracts were analyzed by LC-MS/MS. Validated parameters showed good results for accuracy (85%-104%), precision (3%-16%), LOD (10-40 pg/mL, except for thyronacetic acids that was 200 pg/mL), and the combined uncertainty (2.2%-22%). Maximum concentration of 3,5-T2 in pre-administration samples was 0.71 ng/mL, and after 30 h of the last administration, concentrations returned to pre-administration values. Maximum values of ratios between the analyte and thyronine, T3, and T4 were 0.09, 0.19, and 0.12, respectively, and after 30 h of the last administration, the ratios reached back the basal values. Acidic or basic metabolites were not found in urine at least at the method LOD. A proposed method to assess TH in urine was validated, and as a proof of concept, its efficacy was demonstrated with an excretion study of 3,5-diiodothyronine. The consumption of 3,5-T2 was detected in urine measuring the analyte concentration and ratios between the analyte and thyronine, T3, and T4., (© 2024 John Wiley & Sons Ltd.)

Supercritical fluid chromatography-mass spectrometry (SFC-MS) has proved to be a beneficial tool for sample analysis for a wide variety of compounds and, as such, has recently gained the attention of the anti-doping community. We have tested the applicability of SFC-MS for routine doping control analysing approximately 3 × 1000 identical anti-doping samples utilising SFC-MS instruments from three different vendors: Agilent Technologies, Waters Corporation and Shimadzu Corporation. A 'dilute and inject' approach either without or after hydrolysis of glucuronide metabolites was applied. Most of the compounds included in our study demonstrated excellent chromatography, whereas some showed co-elution with endogenous interferences requiring MS discrimination. Retention times typically were very stable within batches (%CV ≤ 0.5%), although this appeared to be analyte and column dependent. Chromatographic peak shape was good (symmetrical) and stable over the period of the testing without any change of column. Our results suggest that SFC-MS is a sensitive, reproducible and robust analytical tool ready to be used in anti-doping laboratories alongside the currently applied techniques such as gas and liquid chromatography coupled to mass spectrometry. Even if instruments are designed slightly differently, all three setups demonstrated their fitness for the purpose in anti-doping testing., (© 2024 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

This study investigated the impact of low-volume blood withdrawal on the hematological biomarkers currently considered for anti-doping purposes. After baseline measurement (D - 7), a 140 mL blood withdrawal was completed (D + 0) on 12 healthy volunteers, followed by weekly monitoring for 21 days (D + 7 - 21). Each visit consisted of a full blood count (Sysmex XN-1000) and duplicate blood volume measurements by CO-rebreathing. A significant decrease in total hemoglobin mass (Hbmass) (-2.3%, p = 0.007) and red blood cell volume (RBCV) (-2.8%, p = 0.028) was reported at D + 7. Despite no atypical passport finding (ATPF) when considering the athlete biological passport adaptive longitudinal model, hemoglobin concentration ([Hb]) increased significantly at D + 21 (+3.8%, p = 0.031). Besides, ferritin (FERR) was significantly downregulated at all points following blood withdrawal, with the largest decrease occurring at D + 7 (-26.6%, p < 0.001). Regardless of the presumable effect of blood reinfusion on ABP biomarkers, these results illustrate the challenge of monitoring hematological variables for the detection of low-volume blood withdrawal. Finally, this study outlines the sensitivity of FERR to altered erythropoiesis to support the implementation of iron markers as complementary variables for the longitudinal monitoring of blood doping, despite the potential influence of confounding factors (e.g., iron supplementations)., (© 2023 The Authors. Drug Testing and Analysis published by John Wiley & Sons Ltd.)

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.)

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.)

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.)

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 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.)

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.)

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.)

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.)

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.)

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.)

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.)

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.)

Twenty-two pharmaceutical formulations containing prednisolone or prednisone commercially available in Italy, Belgium, Spain, Brazil, and India were analyzed through a specific gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS) method. All of them showed typical non-endogenous δ 13 C values, except for the Belgian nasal spray, Sofrasolone®, with a less depleted 13 C content (-17.84 ± 0.18‰). Observational studies were performed on two volunteers in therapy with Sofrasolone® to confirm the applicability of the method and to suggest adequate interpretation criteria also in the case of drugs with less negative δ 13 C values. Urine samples were collected before, during, and within the 36 hours after the administration of the spray. Both δ 13 C values and urinary concentrations of prednisolone and prednisone were evaluated. All samples were subjected to an adequate pre-treatment (enzymatic hydrolysis, liquid/liquid extraction, and two sequential HPLC steps) before injection to the GC-C-IRMS instrument, according to the method recently developed and validated in our laboratory. Pregnanediol (PD), tetrahydro-11-deoxycortisol (THS), and pregnanetriol (PT) were selected as endogenous reference compounds (ERC). The excretion profile was estimated through liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method used routinely for the quali-quantitative detection of glucocorticoids. δ 13 C values and urinary levels of prednisolone and prednisone were also determined after the intake of one single vial of Sintredius®, a prednisolone oral formulation with a conventional more negative δ 13 C value (-29.28 ± 0.25‰). Finally, the potential masking effect that combined therapy with Sofrasolone® and Sintredius® could induce on the IRMS findings was investigated., (© 2020 John Wiley & Sons, Ltd.)

The detection of clostebol misuse in sports has been growing recently, especially in Italy, due to the ample availability of pharmaceutical formulations containing clostebol acetate (Trofodermin®) and the use of more sensitive instrumentation by the antidoping laboratories. Most of these cases have been claimed to be related to a nonconscious use of the drug or through contact with relatives or teammates using it. We have investigated, through the application of the well-known and currently used gas chromatographic mass spectrometric procedures, the likelihood of these allegations and have demonstrated that after a single transdermal administration of 5 mg of clostebol acetate and a transient contact with the application area, it is possible to generate adverse analytical findings in antidoping controls. We have reviewed the Phase I and Phase II clostebol metabolism in order to generate evidences that may help the sport authorities reviewing these cases. The main clostebol metabolite (4-chloro-androst-4-en-3α-ol-17-one, M1) generally used at the screening level as well as other three metabolites (M2-M4) are mainly excreted as glucuronides, whereas M5 (4ζ-chloro-5ζ-androstan-3β-ol-17-one) is predominantly excreted as sulfate. Neither the 5α-reductases activity (impaired by the presence of the chlorine in C4) nor specific sulfotransferases present in the skin allowed a clear distinction of the administration route. Studies with a larger number of volunteers and probably investigating another physiological fluid allowed in antidoping such as blood are needed for a deeper investigation. It is not unreasonable to establish a reporting level for M1, maybe creating some false negatives but excluding nonintentional doping scenarios., (© 2020 John Wiley & Sons, Ltd.)

The urinary excretion profile of prednisolone and prednisone after both systemic (i.e., oral) and topical (i.e., ocular and intranasal) administration was studied by liquid chromatography coupled to mass spectrometry, also to select the most appropriate marker(s) of intake for doping control purposes. Urines were collected from ten subjects every 3 h before and after the administration of therapeutic doses of pharmaceutical formulations containing either prednisone or prednisolone. Samples were subjected to enzymatic hydrolysis (performed for the investigation on the glucuronide profile) followed by liquid/liquid extraction with tert-butylmethylether in alkaline conditions. The chromatographic separation was carried out on C18 column, employing as mobile phases ultrapurified water and acetonitrile, both containing 0.1% of formic acid. Detection was achieved using as mass spectrometric analyzer a triple quadrupole, with positive ion electrospray ionization and multiple reaction monitoring as acquisition mode. After both systemic and topical use, the compounds excreted in urine in higher concentration were prednisone, prednisolone and 20β-dihydro-prednisolone followed by 20α-dihydro-prednisolone and 20α/β-dihydro-prednisone. All were excreted mainly as unconjugated compounds, with a maximum of excretion in the first 3-9 h after the administration. After systemic use, prednisone and prednisolone were both detectable for at least 24 h in concentrations ranging from 5 to 500 ng/mL and from 5 to 900 ng/mL respectively. Whereas, after topical administration, prednisone and prednisolone were detectable for at least 18 h in concentrations ranging from 5 to 140 ng/mL and from 5 to 50 ng/mL respectively., (© 2019 John Wiley & Sons, Ltd.)

7-keto-DHEA (3β-hydroxy-androst-5-ene-7,17-dione) is included in section S1 of the World Antidoping Agency (WADA) List of Prohibited Substances. The detection of its misuse in sports needs special attention, since it is naturally present in urine samples. The main goal of this study is to investigate the in vivo metabolism of 7-keto-DHEA after a single administration to healthy volunteers and to better describe the relationship between arimistane (androst-5-ene-7,17-dione) and 7-keto-DHEA after the application of the common routine procedures to detect anabolic steroids in WADA accredited antidoping laboratories. Free, glucuro-, and sulpho-conjugated steroids extracted from urine samples obtained before and after the administration of 7-keto-DHEA were analyzed by different gas chromatographic (GC)-mass spectrometric (MS) techniques. Gas chromatography coupled to tandem MS to study the effect on the endogenous steroid profile, coupled to isotope ratio mass spectrometry (IRMS) to investigate the potential formation of androgens derived from DHEA and coupled to high resolution accurate mass spectrometry (HRMS) to investigate new diagnostic metabolites. The analysis by IRMS confirmed that there is no formation of DHEA from 7-keto-DHEA. Ten proposed metabolites, not previously reported, were described. These include reduced and hydroxylated structures that are not considered part of the steroid profile in antidoping analyses. They showed considerable responses in all fractions analyzed. Some deoxidation reactions (including arimistane formation) were found and most probably can be linked to the sample preparation or instrumental analysis. This is important when interpreting the results after the application of procedures to detect steroids in urine currently used in antidoping laboratories. 7-keto-DHEA metabolism in humans for antidoping purposes was studied and unexpected results were found. This could lead to a misinterpretation of the data, depending on the procedure applied and the analytical instrumentation used., (© 2019 John Wiley & Sons, Ltd.)

Prednisone and prednisolone are two anti-inflammatory steroidal drugs listed by the World Anti-Doping Agency (WADA) within the class of glucocorticoids, which are prohibited "in competition" and when administered systemically. Their presence in collected urine samples may be attributed, if no exogenous administration has occurred, to an in situ microbial formation from endogenous steroids. In this work, a gas chromatography coupled to carbon isotope ratio mass spectrometry (GC-C-IRMS) method was developed and validated to distinguish an exogenous origin from an endogenous one. Eight prednisone/prednisolone pharmaceutical preparations commercially available in Italy were analysed to establish an exogenous δ 13 C value reference range (-28.96 ± 0.39‰). No more than 25 mL of urine was processed and no derivatization nor intentional steroids structure modifications were performed before the GC-C-IRMS analysis. A first HPLC purification step was set up to isolate the three endogenous reference compounds (ERCs) selected (tetrahydro-11-deoxycortisol (THS), pregnanediol (PD), and pregnanetriol (PT)), while a second LC purification was necessary to separate prednisone from prednisolone. In the GC-C-IRMS analysis, two different GC run methods were set up to guarantee better sensitivity and selectivity for each compound. Both prednisone and prednisolone showed signals (m/z 44) with amplitudes within the method linearity range to a lower urinary concentration of 20 ng/mL (< WADA reporting level, 30 ng/mL). The method was fully validated according to WADA requirements. As a proof of concept, urine samples collected from two excretion studies in healthy male volunteers, after a prednisone or prednisolone administration, were analysed by the proposed method, demonstrating its applicability for the analysis of real samples., (© 2019 John Wiley & Sons, Ltd.)

An analytical procedure based on ultra-performance liquid chromatography-mass spectrometry was developed to screen and to confirm dutasteride and its metabolites in human urine. Sample preparation included an enzymatic hydrolysis followed by solid-phase extraction using the strong cation exchange cartridges OASIS ® MCX. The chromatographic separation was carried out on C18 column, employing as mobile phases ultra purified water and acetonitrile, both containing 0.1% formic acid. Detection was achieved using a triple quadrupole as a mass spectrometric analyzer, with positive ion electrospray ionization and multiple reaction monitoring as acquisition mode. The analytical procedure developed was validated according to ISO 17025 and World Anti-Doping Agency guidelines. The extraction efficiency was estimated to be greater than 75% for both dutasteride and its hydroxylated metabolites. Detection capability was determined in the range of 0.1-0.4 ng/mL. Specificity and repeatability of the relative retention times (CV% < 0.5) and of the relative abundances of the characteristic ion transitions selected (CV% < 10) were confirmed to be fit for purpose to ensure the unambiguous identification of dutasteride and its metabolites in human urine. The developed method was used to characterize the urinary excretion profile of dutasteride after both chronic and acute administration of therapeutic doses. After chronic administration, dutasteride and its hydroxylated metabolites were easily detected and confirmed. After acute administration, instead, only the two hydroxylated metabolites were detected for 3-4 days., (© 2019 John Wiley & Sons, Ltd.)

Anectodical information suggests that flavonoids may be widely used among athletes for their multiple biochemical and pharmacological effects. We have evaluated in vitro the effects of two synthetic isoflavones, methoxyisoflavone and ipriflavone, on the catalytic activity of human aromatase (CYP19), the enzyme catalyzing the conversion of androgens (ie, testosterone or androstenedione) to estrogens (ie, estradiol and estrone). The potential inhibitory effect was evaluated by measuring the rate of aromatization of testosterone, monitored by gas chromatography-mass spectrometry (GC-MS), both in the presence and in the absence of methoxyisoflavone or ipriflavone, comparing their effects with those of synthetic aromatase inhibitors (formestane, anastrozole, and aminoglutethimide) presently included in the list of prohibited substances and methods, and of natural flavonoids (chrysin, quercetin, and daidzein), that are known inhibitors of CYP19. The preliminary results of our in vitro study show that methoxyisoflavone and ipriflavone act as competitive inhibitors of aromatase, the degree of inhibition measured in vitro being of the same order of magnitude of that of the aromatase inhibitors commonly used in anti-estrogenic therapies. Our preliminary in vitro results indicate that, in principle, a sufficiently large intake of isoflavones could alter the kinetics of the dynamic equilibria between androgens and estrogens, suggesting their monitoring in doping control routine analysis., (© 2018 John Wiley & Sons, Ltd.)

The reactions involved in the metabolic pathways of SR9009 were characterized by liquid chromatography-mass spectrometry (LC-MS) to identify the most appropriate marker(s) of use. The effects of gender, genetic polymorphism, and drug-drug interaction on the metabolic profile of SR9009 were also evaluated. In vitro approaches were based on the use of human liver microsomes and cytochrome P450 isoforms. Sample preparation included an enzymatic hydrolysis (performed only for the phase II investigation) followed by liquid-liquid extraction. The chromatographic separation was carried out using a reverse-phase column; detection was performed by either a triple-quadrupole or a time-of-flight system in positive electrospray ionization and different acquisition modes. In the presence of human liver microsomes, SR9009 was biotransformed to 13 metabolites by CYP3A4, CYP3A5, CYP2C19, and CYP2D6 isoenzymes. The reactions included hydroxylation, de-alkylation, oxidation, and combinations thereof, the de-alkylated metabolites being the most abundant. Once formed the mentioned metabolites underwent glucuronidation. Concerning the effects of gender, genetic polymorphism, and drug-drug interaction on the metabolic profile of SR9009, our observation have shown the following: (a) No significant alterations were measured between female and male, (b) significant differences were registered using either the CYP2D6 or CYP2C19 allelic variants, and finally (c) significant alterations were registered in the presence of ketoconazole, miconazole, fluoxetine, nefazodone and paroxetine; moderate variation were instead registered with fluconazole, itraconazole, gestodene, and levonorgestrel. This observation put in evidence the importance to take into account both genetic polymorphism and drug-drug interaction to select the most appropriate marker(s) of use in doping analysis., (© 2018 John Wiley & Sons, Ltd.)

The potential consequences of drug-drug interactions on the excretion profile of the anabolic androgenic steroid methandienone (17β-hydroxy-17α-methylandrosta-1,4-dien-3-one) are discussed. More specifically, we have evaluated by in vitro and in vivo experiments the effects of 7 non-prohibited drugs (fluconazole, ketoconazole, itraconazole, miconazole, fluoxetine, paroxetine, and nefazodone) on the main metabolic pathways of methandienone. These are selected among those most commonly used by the athletes. The in vitro assays were based on the use of human liver microsomes, specific recombinant enzyme isoforms of cytochrome P450 and uridine 5'-diphospho-glucuronosyl-transferase. The in vivo study was performed by analyzing urines collected after the oral administration of methandienone with and without the co-administration of ketoconazole. Methandienone and its metabolites were determined by liquid chromatography-mass spectrometry-based techniques after sample pretreatment including an enzymatic hydrolysis step (performed only for the investigation on phase II metabolism) and liquid/liquid extraction with t-butyl methyl-ether. The results from the in vitro experiments showed that the formation of the hydroxylated and dehydrogenated metabolites was significantly reduced in the presence of itraconazole, ketoconazole, miconazole and nefazodone, whereas the production of the 18-nor-hydroxylated metabolites and glucuronidation reactions was reduced significantly only in the presence of ketoconazole and miconazole. The analysis of the post-administration samples confirmed the in vitro observations, validating the hypothesis that drug-drug interaction may cause significant alterations in the metabolic profile of banned drugs, making their detection during doping control tests more challenging., (© 2018 John Wiley & Sons, Ltd.)

The potential consequences of drug-drug interaction on the strategies adopted by anti-doping laboratories to report an adverse analytical finding for morphine were investigated. We evaluated in vitro the effects of 14 drugs on the principal metabolic pathways of morphine. The selected drugs are among those most commonly used by the athletes, none of them presently included in the World Anti-Doping Agency (WADA) Prohibited List. The non-prohibited drugs included 4 antifungals (fluconazole, itraconazole, ketoconazole, and miconazole), 6 benzodiazepines (alprazolam, bromazepam, clonazepam, lorazepam, lormetazepam, and triazolam), and 4 non-steroidal anti-inflammatory drugs (diclofenac, ibuprofen, ketoprofen, and nimesulide). The in vitro assays were based on the use of either human liver microsomes or uridine 5'-diphospho-glucuronosyl-transferases. Morphine and its glucuronides were determined by developed liquid chromatography-mass spectrometry procedure after dilution with an aqueous solution containing their deuterated isotopologues as internal standards. Morphine is mainly excreted as phase II metabolites: about 70% of the parent compound is found to be biotransformed by UGT2B7 to morphine-3-glucuronide (6065%) and morphine-6-glucuronide (5-10%). A reduction of the enzymatic activity of the UGT2B7 was recorded in the presence of 9 of the 14 drugs under investigation (ketoconazole, miconazole, itraconazole, diclofenac, ibuprofen, clonazepam, lorazepam, lormetazepam, and triazolam), with a consequent significant reduction of the levels of the glucuronide metabolites. This phenomenon in vivo may affect the rate of the urinary excretion of morphine with the risk of reporting "false negative" results, especially in case of results close to the decision limit value set by WADA., (Copyright © 2017 John Wiley & Sons, Ltd.)

The detection of the abuse of pseudoendogenous steroids (testosterone and/or its precursors) is currently based, when possible, on the application of the steroid module of the World Anti-Doping Agency (WADA), athlete biological passport (ABP), implemented through the global database, ADAMS. When a suspicious sample is detected, the confirmation by isotope ratio mass spectrometry (IRMS) is required. It is well known that this confirmation procedure is time consuming and expensive and can be only applied on a reduced number of samples. In previous studies we have demonstrated that the longitudinal evaluation of the IRMS data is able to detect positive samples that otherwise will be evaluated as negative, improving the efficacy of the fight against doping in sport. This would require the analysis of a much larger volume of samples by IRMS. The aim of the present work is to describe an IRMS screening method allowing to increase the throughput of samples that can be analyzed by IRMS. The detection efficacy of the method is compared with the confirmation method in use, and to assess its robustness and applicability, all the samples of a major cycling stage competition were analyzed, with the agreement of the testing authority, under routine conditions and response times. The results obtained permit to conclude that the IRMS screening method here proposed has adequate selectivity and produces results that overlap with the already validated method currently in use permitting to analyze a much higher volume of samples even during a major event without compromising the detection capacity. Copyright © 2017 John Wiley & Sons, Ltd., (Copyright © 2017 John Wiley & Sons, Ltd.)

Urine collection containers used in the doping control collection procedure do not provide a protective environment for urine, against degradation by microorganisms and proteolytic enzymes. An in-house chemical stabilization mixture was developed to tackle urine degradation problems encountered in human sport samples, in cases of microbial contamination or proteolytic activity. The mixture consists of antimicrobial substances and protease inhibitors for the simultaneous inactivation of a wide range of proteolytic enzymes. It has already been tested in lab-scale, as part of World Anti-Doping Agency's (WADA) funded research project, in terms of efficiency against microbial and proteolytic activity. The present work, funded also by WADA, is a follow-up study on the improvement of chemical stabilization mixture composition, application mode and limitation of interferences, using pilot urine collection containers, spray-coated in their internal surface with the chemical stabilization mixture. Urine in plastic stabilized collection containers have been gone through various incubation cycles to test for stabilization efficiency and analytical matrix interferences by three WADA accredited Laboratories (Athens, Ghent, and Rome). The spray-coated chemical stabilization mixture was tested against microorganism elimination and steroid glucuronide degradation, as well as enzymatic breakdown of proteins, such as intact hCG, recombinant erythropoietin and small peptides (GHRPs, ipamorelin), induced by proteolytic enzymes. Potential analytical interferences, observed in the presence of spray-coated chemical stabilization mixture, were recorded using routine screening procedures. The results of the current study support the application of the spray-coated plastic urine container, in the doping control collection procedure. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

This work presents an analytical procedure for the identification and characterization of liposome-entrapped haemoglobins, based on flow cytofluorimetry. Flow cytofluorimetric detection is carried out following labelling by two distinct fluorescent reagents, an anti-haemoglobin antibody, fluorescein isothiocyanate conjugated, and an anti-poly(ethylene glycol) antibody, streptavidin-phycoerythrin conjugated. This experimental strategy allows the detection of liposome-entrapped haemoglobins in aqueous media, including plasma; the efficacy of the proposed approach has been verified on whole blood samples added with the liposomal formulation (ex-vivo). Additionally, the proposed technique allows the characterization of several key parameters in the study of liposomal haemoglobins, including, for instance (1) the determination of the degree of haemoglobin entrapment by liposomes; (2) the poly(ethylene glycol) insertion efficiency; and (3) the evaluation of liposome-entrapped haemoglobins stability following storage at 4 °C, allowing to follow both the process of haemoglobin loss from liposomes and the liposome degradation. The procedure is proposed for the detection and characterization of liposome-entrapped haemoglobin formulations to control their misuse in sport, but is also suggested for further applications in biological and clinical laboratory investigations. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

In the present work, aqueous normal-phase liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), in different acquisition modes, was employed for the direct analysis and profiling of nine phospholipid classes (phosphatidic acids, phosphatidylserines, phosphatidylethanolamines, lysophosphatidylethanolamines, phosphatidylglycerols, phosphatidylinositols, phosphatidylcholines, lysophosphatidylcholines, and sphingomyelins) in biological and pharmaceutical matrices. After chromatographic separation by a diol column, detection and elucidation of phospholipid and sphingomyelin classes and molecular species were performed by different scan acquisition modes. For screening analysis, molecular ions [M + H] + were detected in positive precursor ion scan of m/z 184 for the classes of phosphatidylcholines, lyso-phosphatidylcholines and sphingomyelins; while phosphatidylethanolamines and lyso-phosphatidylethanolamines were detected monitoring neutral loss scan of 141 Da; and phosphatidylserines detected using neutral loss scan of 184 Da. Molecular ions [M-H] - were instead acquired in negative precursor ion scan of m/z 153 for the classes of phosphatidic acids and phosphatidylglycerols; and of m/z 241 for the phosphatidylinositols. For the identification of the single molecular species, product ion scan mass spectra of the [M + HCOO] - ions for phosphatidylcholines and [M + H] + ions for the other phospholipids considered were determined for each class and compared with the fragmentation pattern of model phospholipid reference standard. By this approach, nearly 100 phospholipids and sphingomyelins were detected and identified. The optimized method was then used to characterize the phospholipid and sphingomyelin profiles in human plasma and urine samples and in two phospholipid-based pharmaceutical formulations, proving that it also allows to discriminate compounds of endogenous origin from those resulting from the intake of pharmaceutical products containing phospholipidic liposomes. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

The detection of the abuse of pseudo-endogenous steroids (testosterone and/or its precursors) is currently based on the application of the steroid module of the World Anti-Doping Agency (WADA) Athletes' Biological Passport (ABP), implemented through ADAMS. Diagnostic metabolites are monitored for every athlete and statistically evaluated with a predictive Bayesian approach. In the case of suspicious samples, the data of the ABP are confirmed and the isotope ratio mass spectrometry (IRMS) test is activated. We have previously demonstrated that IRMS enables confirmation of the non-endogenous origin of pseudo-endogenous steroids in otherwise non-suspicious samples, after a longitudinal evaluation of the ABP, even after the inclusion of additional long-term diagnostic hydroxylated metabolites, and that the delta values of the parameters obtained during the IRMS confirmation process presented much less variability compared to the parameters of the ABP. The aim of the present work is to evaluate the application of the same methodology used for the evaluation of the ABP, on the delta values of the pseudo-endogenous steroids monitored. The effectiveness of the proposed model has been assessed on samples obtained after controlled administrations of oral androstenedione and transdermal testosterone. The results support the conclusion that, if applied, the longitudinal evaluation of the IRMS data allows the detection of positive samples that otherwise will be reported as atypical findings (ATF), improving the efficacy of the fight against doping in sport. This approach, by narrowing the individual acceptable range, could possibly improve the detection of the intake of preparations of synthetic origin with delta values close to or overlapping those of endogenously produced steroids. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

We have studied whether the phase II metabolism of 19-norandrosterone, the most representative metabolite of 19-nortestosterone (nandrolone), can be altered in the presence of other drugs that are not presently included on the Prohibited List of the World Anti-Doping Agency. In detail, we have evaluated the effect of non-prohibited drugs belonging to the classes of anti-fungals, benzodiazepines, and non-steroidal anti-inflammatory drugs on the glucuronidation of 19-norandrosterone. In vitro assays based on the use of either pooled human liver microsomes or specific recombinant isoforms of uridine diphosphoglucuronosyl-transferase were designed and performed to monitor the formation of 19-norandrosterone glucuronide from 19-norandrosterone. Determination of 19-norandrosterone (free and conjugated fraction) was performed by gas chromatography - mass spectrometry after sample pretreatment consisting of an enzymatic hydrolysis (performed only for the conjugated fraction), liquid/liquid extraction with tert-butylmethyl ether, and derivatization to form the trimethylsilyl derivative. In parallel, a method based on reversed-phase liquid chromatography coupled to tandem mass spectrometry in positive electrospray ionization with acquisition in selected reaction monitoring mode was also developed to identify the non-prohibited drugs considered in this study. Incubation experiments have preliminarily shown that the glucuronidation of 19-norandrosterone is principally carried out by UGT2B7 (39%) and UGT2B17 (31%). Inhibition studies have shown that the yield of the glucuronidation reaction is reduced in the presence of the anti-fungals itraconazole, ketoconazole, and miconazole, of the benzodiazepine triazolam and of the non-steroidal anti-inflammatory drugs diclofenac and ibuprofen, while no alteration was recorded in the presence of all other compounds considered in this study. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

Glucocorticoids are included in the S9 section of the World Anti-doping Agency (WADA) prohibited list international standard. Some among them are pseudo-endogenous steroids, like cortisol and cortisone, which present the same chemical structure as endogenously produced steroids. We are proposing an analytical method based on gas chromatography coupled to isotope ratio mass spectrometry (GC-C-IRMS) which allows discrimination between endogenous and synthetic origin of the urinary metabolites of the pseudo-endogenous glucocorticoids. A preliminary purification treatment by high-performance liquid chromatography (HPLC) of the target compounds (TC) (i.e., cortisol, tetrahydrocortisone (THE) 5α-tetrahydrocortisone (aTHE), tetrahydrocortisol (THF), and 5α-tetrahydrocortisol (aTHF)) allows collection of extracts with adequate purity for the subsequent analysis by IRMS. A population of 40 urine samples was analyzed for the TC and for the endogenous reference compounds (ERC: i.e., 11-desoxy-tetrahydrocortisol (THS) or pregnanediol). For each sample, the difference between the delta values of the ERCs and TCs (Δδ values) were calculated and based on that, some decision limits for atypical findings are proposed. The limits are below 3% units except for cortisol. The fit to purpose of the method has been confirmed by the analysis of urine samples collected in two patients under treatment with 25 mg of cortisone acetate (p.o). The samples showed Δδ values higher than 3 for at least 24 h following administration depending on the TC considered. The method can easily be integrated into existing procedures already used for the HPLC purification and IRMS analysis of pseudo-endogenous steroids with androgenic/anabolic activity., (Copyright © 2015 John Wiley & Sons, Ltd.)

Formestane (4-hydroxy-androstenedione) is an aromatase inhibitor prohibited in sports and included, since 2004, in the list of prohibited substances updated yearly by the World Anti-Doping Agency (WADA). Since the endogenous production of formestane has been described, it is mandatory for the anti-doping laboratories to use isotope ratio mass spectrometry (IRMS) to establish the exogenous origin before issuing an adverse analytical finding. The described IRMS methods for formestane detection are time-consuming, requiring usually two consecutive liquid chromatographic sample purifications in order to have final extracts of adequate purity before the mass spectrometric analysis. After establishing a procedure for the determination of the origin of formestane by IRMS without the need of derivatization, and integrated in the overall analytical strategy of the laboratory for pseudo-endogenous steroids, a mass spectrometric analysis by gas chromatography-mass spectrometry (GC-MS) and gas chromatography-tandem mass spectrometry (GC-MS/MS) of formestane metabolites was carried out in order to investigate whether other biomarkers of formestane abuse could be integrated in order to avoid time-consuming and expensive IRMS confirmations for formestane. From the metabolic studies performed, the inclusion of 3β,4α-dihydroxy-5α-androstan-17-one (4α-hydroxy-epiandosterone) in the routine GC-MS procedures has demonstrated to be diagnostic in order to reduce the number of unnecessary confirmations of the endogenous origin of formestane., (Copyright © 2014 John Wiley & Sons, Ltd.)

The present study was designed to provide preliminary information on the potential impact of metabolic drug-drug interaction on the effectiveness of doping control strategies currently followed by the anti-doping laboratories to detect the intake of prohibited agents. In vitro assays based on the use of human liver microsomes and recombinant cytochrome P450 isoforms were developed and applied to characterize the phase I metabolic profile of the prohibited agent stanozolol, both in the absence and in the presence of substances (ketoconazole, itraconazole, miconazole, cimetidine, ranitidine, and nefazodone) not included in the World Anti-Doping Agency (WADA) list of prohibited substances and methods and frequently administered to athletes. The results show that the in vitro model utilized in this study is adequate to simulate the in vivo metabolism of stanozolol. Furthermore, our data showed that ketoconazole, itraconazole, miconazole, and nefazodone caused a marked modification in the production of the metabolic products (3'-hydroxy-stanozolol, 4β-hydroxy-stanozolol and 16β-hydroxy-stanozolol) normally selected by the anti-doping laboratories as target analytes to detect stanozolol intake. On the contrary, moderate variations were registered in the presence of cimetidine and no significant modifications were measured in the presence of ranitidine. This evidence confirms that the potential effect of drug-drug interactions is duly taken into account also in anti-doping analysis., (Copyright © 2014 John Wiley & Sons, Ltd.)

The present study was designed to provide preliminary information on the potential impact of metabolic drug-drug interaction on the effectiveness of doping control strategies currently followed by the anti-doping laboratories to detect the intake of banned agents. In vitro assays based on the use of human liver microsomes and recombinant CYP isoforms were designed and performed to characterize the phase I metabolic profile of the prohibited agent toremifene, selected as a prototype drug of the class of selective oestrogen receptor modulators, both in the absence and in the presence of medicaments (fluconazole, ketoconazole, itraconazole, miconazole, cimetidine, ranitidine, fluoxetine, paroxetine, nefazodone) not included in the World Anti-Doping Agency list of prohibited substances and methods and frequently administered to athletes. The results show that the in vitro model developed in this study was adequate to simulate the in vivo metabolism of toremifene, confirming the results obtained in previous studies. Furthermore, our data also show that ketoconazole, itraconazole, miconazole and nefazodone cause a marked modification in the production of the metabolic products (i.e. hydroxylated and carboxylated metabolites) normally selected by the anti-doping laboratories as target analytes to detect toremifene intake; moderate variations were registered in the presence of fluconazole, paroxetine and fluoxetine; while no significant modifications were measured in the presence of ranitidine and cimetidine. This evidence imposes that the potential effect of drug-drug interactions is duly taken into account in anti-doping analysis, also for a broader significance of the analytical results., (Copyright © 2014 John Wiley & Sons, Ltd.)

Boldione is an anabolic androgenic steroid (AAS) related to boldenone, androstenedione, and testosterone bearing two double bonds in C1 and C4 positions. Boldione is rapidly transformed to the well-known AAS boldenone, being both compounds included in the list of prohibited substances and methods published yearly by the World Anti-Doping Agency (WADA). After the administration of boldione to a male volunteer, the already described urinary metabolites of boldenone produced after reduction in C4, oxydoreduction in C3 and C17, and hydroxylation have been detected. In addition, minor new metabolites have been detected and their structure postulated after mass spectrometric analyses. Finally, the reduction of the double bound in C1 produces metabolites identical to the endogenously produced ones. A method based on gas chromatography coupled to isotope ratio mass spectrometry (GC/C/IRMS) after a urine sample purification by high performance liquid chromatography (HPLC) permitted to confirm the main synthetic like boldione/boldenone metabolite (17β-hydroxy-5β-androst-1-en-3-one) and boldenone at trace levels (< 5 ng/mL) and then to establish its synthetic or endogenous origin, and to determine the exogenous origin of metabolites with the same chemical structure of the endogenous ones. The detection of pseudoendogenous androgens of synthetic origin partially overlapped boldenone and its main metabolite detection, being an additional proof of synthetic steroids misuse. By the use of IRMS, the correct evaluation of the modifications of the steroid profile after the administration of synthetic AAS that could be converted into endogenous like ones is possible., (Copyright © 2013 John Wiley & Sons, Ltd.)

A screening procedure specifically developed for the detection of saccharides and polyalcohols in human urine in the framework of doping control analysis is presented. The proposed method, set-up, and validated to detect the abuse of dextran, hydroxyethyl starch and mannitol as a doping practice in sport, involves only one enzymatic hydrolysis step and the direct injection into a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. The chromatographic conditions were optimized to allow the efficient separation of compounds with the same molecular weight. Good linearity (R(2) 0.990-0.995) and reproducibility of relative retention times (CV% lower than 1) and of relative abundances of characteristic ion transitions (CV% lower than 10) were obtained. The lower limits of detection and quantification were in the range of 30-100 µg/ml. Since the analytes studied are present also in non-doping products (e.g. in fruit as well as in food products and drugs additives), the developed method was also used to establish a range of reference urinary concentrations: 600 doping control samples and 30 samples from volunteers not using any medication were considered. While the hydrolysis products (isomaltose and maltose hydroxyl-ethylated), used as specific markers for the detection of dextran and hydroxyethyl starch abuse, were not detected in urine; mannitol was present in all urines in a concentration range of 30-1200 µg/ml. Since no criteria of positivity for mannitol has been established yet, the results obtained in this study could be considered, in combination with those of previous researches, as a starting point to fix a threshold value for doping control purpose., (Copyright © 2011 John Wiley & Sons, Ltd.)

This study presents a fast multi-analyte screening method specifically developed for the detection of xenobiotics in urine. The proposed method allows the screening of several classes of substance in a single chromatographic method with a run-time of 11 min, inclusive of post-run and reconditioning times. Chromatographic separation is achieved in 7.2 min using a reversed-phase 2.7 µm fused-core particle column, generating a back-pressure not exceeding 400 bar and therefore enabling the use of traditional high performance liquid chromatography (HPLC) instruments. The effectiveness of this approach was evaluated, by liquid-chromatography tandem mass spectrometry (LC-MS/MS) in positive electrospray ionization, using 20 blank urine samples spiked with 45 compounds prohibited in sport: 11 diuretics, 16 glucocorticoids, 9 stimulants, 5 anti-oestrogens, as well as formoterol, carboxy-finasteride (previously prohibited by the World Anti-Doping Agency (WADA) in 2008), gestrinone and tetrahydrogestrinone. Qualitative validation shows the proposed method to be specific with no significant interference. All of the analytes considered in this study were clearly distinguishable in urine, with limits of detection ranging from 5 ng/mL to 350 ng/mL, significantly below the Minimum Required Performance Levels (MRPL) set by WADA for the accredited sports anti-doping laboratories. All compounds of interest were separated, including synthetic and endogenous glucocorticoids with similar retention times and fragmentation patterns., (Copyright 2010 John Wiley & Sons, Ltd.)