Search

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.)

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.)

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.)

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.)

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

Cytochrome P450 enzymes (CYPs) are capable of catalyzing regio- and stereo-specific oxy functionalization reactions, which otherwise are major challenges in organic chemistry. In order to make the best possible use of these biocatalysts it is imperative to understand their specificities. Human CYP21A2 (steroid 21-hydroxylase) acts on the side-chain attached to C-17 in ring D of a steroid substrate, but the configuration of ring A also plays a prominent role in substrate cognition. Here, we comprehensively investigated this relationship using sixteen 17,17-dimethyl-18-nor-13-ene steroids with different arrangements of hydroxy-, oxo-, fluoro- and chloro-groups and in the presence or absence of double bonds (Δ1 and/or Δ4) and heteroatoms in ring A. The results show that presence of a 3-oxo group is a strict requirement for a CYP21A2 substrate, while the other configurations tested were all tolerated. This was also confirmed by control experiments using endogenous steroids. While progesterone and 17-hydroxyprogesterone were hydroxylated at C-21, (17-hydroxy-) pregnenolone did not react. Molecular docking experiments indicate that the interaction of the carbonyl group at C-3 to the side-chain Arg234 of the enzyme is indispensable., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

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.)

The urinary steroidal fraction has been extensively explored as non-invasive alternative to monitor pathological conditions as well as to unveil the illicit intake of pseudo-endogenous anabolic steroids in sport. However, the majority of previous approaches involved the a priori selection of potentially relevant target analytes. Here we describe the non-targeted analysis of the urinary steroidal profiles. The workflow includes minimal sample pretreatment and normalization according to the specific gravity of urine, a 20 min reverse phase ultra-performance liquid chromatographic separation hyphenated to electrospray time-of-flight mass spectrometry. As initial validation, we analyzed a set of quality control urines spiked with glucurono- and sulfo-conjugated steroids at physiological ranges. We then applied the method for the analysis of samples collected after single transdermal administration of testosterone in hypogonadal men. The method allowed profiling of approximately three thousand metabolic features, including steroids of clinical and forensic relevance. It successfully identified metabolic pathways mostly responsible for groups clustering even in the context of high inter-individual variability and allowed the detection of currently unknown metabolic features correlating with testosterone administration. These outcomes set the stage for future studies aimed at implementing currently monitored urinary steroidal markers both in clinical and forensic analysis., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A rapid sample treatment procedure for the gas chromatography/mass spectrometry (GC/MS) determination of anabolic steroids in human urine has been developed. The new procedure makes use of ultrasonic energy to reduce reaction times and increase the overall sensitivity. The following variables affecting the performance of the ultrasonic treatment were optimised: (i) time, (ii) device, (iii) frequency, and (iv) temperature. It was found that, under an ultrasonic field, the hydrolysis of conjugated steroids with beta-glucuronidase from Escherichia coli K12 was possible with a treatment time of 10 min. The accuracy and precision of the ultrasonic method were found to be in agreement with those achieved with the conventional thermal conductivity procedure (Student's t-test; p = 0.05, n = 10). After the enzymatic hydrolysis, the derivatisation of the target compounds with trimethylsilyl (TMS) reagent, methyl-N-trimethylsilyltrifluoroacetamide (MSTFA)/NH(4)I/dithioerythritol (DTE) (1000:2:4, v/w/w), was also accelerated using ultrasonic energy. In order to test the applicability of the use of ultrasonic energy in the acceleration of the derivatisation reaction with TMS, the classic method of thermal conductivity was applied for comparative purposes to a pool of 35 androgenic anabolic steroids (AAS) and/or their metabolites. The results demonstrated that after 3 min of sonication in a Sonoreactor device (50% amplitude), 19 of the 35 compounds studied showed similar reaction yield to those obtained with the classic procedure requiring 30 min (Student's t-test; p = 0.05, n = 5); 13 increased to higher silylation yields; and for the steroids 1-testosterone, danazol and etiocholanolone-D5, the same results were obtained using a sonication time of 5 min.The overall applicability of the ultrasonic-based sample treatment method is shown by the analysis of five urine samples. The results are similar to those achieved by the routine procedure. The new method is fast, robust, and allows high sample throughput., (2010 John Wiley & Sons, Ltd.)

This paper describes the effects of oral administration of non-steroidal anti-inflammatory drugs on the endogenous and synthetic anabolic androgenic steroids urinary excretion as assessed by gas-chromatography mass-spectrometry. Experiments were carried out on 5 male subjects, with pathologies and/or diseases, treated with non-steroidal anti-inflammatory drugs. To set up the individual baseline variability of testosterone and its main metabolites, urine samples were collected for 3 days, every 2 h prior to the administration of the drug(s); whereas the study of the effects of a single dose of each drug, here considered, on the endogenous androgen steroid urinary concentrations, was assessed by collecting urine samples for 2 days, every 2 h. Data obtained after drugs administration were then evaluated taking into account the individual baseline variability. The results showed that, only in the case of propyphenazone administration, the relative urinary concentrations of some testosterone metabolites were significantly altered. More specifically, the urinary levels of dehydroepiandrosterone, 11keto-etiocholanolone, 11beta-hydroxyandrosterone, 11beta-hydroxyetiocholanolone, androsterone, etiocholanolone and some metabolite ratios decrease significantly, generally between 2 and 10 h after administration of the drug, whereas no effects were observed on urinary calculated concentrations of testosterone, epitestosterone, 5alpha-androstane-3alpha,17beta-diol, 5beta-androstane-3alpha,17beta-diol and testosterone/epitestosterone ratio. The observed effects do not depend on alterations on pharmacokinetics (excretion/metabolism), but on steroid sample preparation steps (hydrolysis and derivatization) inhibition. More specifically the significant decrease of dehydroepiandrosterone and testosterone metabolites urinary levels was due to a reduced yield of the steroid derivatization step for the presence in urine of the main metabolites of propyphenazone, namely hydroxyl-propyphenazone metabolites.

This paper presents a general screening method, based on liquid chromatography/mass spectrometry (LC/MS), for the simultaneous detection in human urine of 72 xenobiotics (21 diuretics, 16 synthetic glucocorticoids, 17 beta-adrenergic drugs, 10 stimulants, 5 anti-oestrogens and 3 anabolic steroids), excreted free or as glucuro-conjugates in urine. Although the method has been specifically designed and evaluated in view of its potential application to anti-doping analyses, it can also be effective in other areas of analytical toxicology. Sample preparation was based on two liquid/liquid separation steps (performed at alkaline and at acid pH, respectively) of hydrolyzed human urine, and then an assay by LC/MS-MS in positive and negative ionization mode using an electrospray ionization source (ESI) and multiple reaction monitoring (MRM) as the acquisition mode. The overall time needed for an LC run was less than 15 minutes. All compounds showed good reproducibility in terms of both the retention times (CV%<1) and the relative abundances of the diagnostic transitions (CV%<10). The limits of detection (LOD) were in the range of 1-50 ng/mL for glucocorticoids, anti-oestrogens and steroids, and 50-500 ng/mL for diuretics, beta-adrenergic drugs and stimulants, thus satisfying the minimum required performance limits (MRPL) set by the World Anti-Doping Agency (WADA) for the accredited anti-doping laboratories.

This paper describes a liquid chromatographic/tandem mass spectrometric (LC/MS-MS) method specifically designed for the screening of synthetic glucocorticosteroids in human urine. The method is designed to recognize a common mass spectral fragment formed from the particular portion of the molecular structure that is common to all synthetic glucocorticosteroids and that is fundamental to their pharmacological activity. As such, the method is also suitable for detecting unknown substances, provided they contain the portion of the molecular structure selected as the analytical target. The effectiveness of this approach was evaluated on seventeen synthetic glucocorticosteroids. Urine samples, including blank urines spiked with one or more synthetic glucocorticosteroids, were treated according to a standard procedure (enzymatic hydrolysis, liquid/liquid extraction and evaporation to dryness) and analyzed using LC/MS-MS with electrospray ionization (ESI). MS-MS acquisition was carried out in a precursor ion scan, and the results were compared with those obtained by a previously developed reference technique based on acquisition in the multiple reaction monitoring (MRM) mode. All of the glucocorticosteroids considered in this study are clearly detectable in urine, with a limit of detection in the concentration range 5-20 ng/mL, depending on the glucocorticosteroid structure. The proposed method is therefore suitable for the detection of glucocorticosteroids in urine samples taken for "in competition" sport anti-doping control tests, matching the requirements of the World Anti-Doping Agency (WADA) for accredited anti-doping laboratories.

The aim of this study is to investigate the effects in vitro induced by androgenic anabolic steroids (AAS) (testosterone, nandrolone, androstenedione, norandrostenedione, and norandrostenediol) used illicitly in sport competitions, on the proliferation ability, apoptosis and the intracellular calcium concentration ([Ca2+]i) in human umbilical vein endothelial cells (HUVECs), selected as a prototype of a biological target system whose structure and function can be affected by steroids. For this purpose, we evaluated the proliferation inhibition by cytotoxic assay expressed as the concentration of drug inducing a 50% decrease in growth (IC50). The IC50 was reached for testosterone at 100 microM, androstenedione at 375 microM, nandrolone at 9 microM, norandrostenedione at 500 microM. The IC50 value for norandrostenediol was not reached until a concentration of 6000 microM. The apoptotic effect was evaluated by flow cytometry at IC50 for each drug. We observed that testosterone induced 31% of apoptotic cells, norandrostenedione 25%, androstenedione 15% and nandrolone 18%. We have analyzed the effects of these drugs on [Ca2+]i both in the immediate and long-term continuous presence of each compound. Our data show a statistically significant increase of [Ca2+]i in the acute condition and in long-term treated cultures, suggesting that androgen steroids modulate intracellular levels of calcium independent of incubation time or compound identity. As a whole, this study demonstrates that AAS might alter endothelial homeostasis, predisposing to the early endothelial cell activation that is responsible for vascular complications observed frequently in AAS users.