Simultaneous quantification of endogenous biomarkers and detection of testosterone esters in human serum using gas chromatography-high-resolution mass spectrometry.

RATIONALE: High-resolution mass spectrometry (HRMS) has been demonstrated to be an alternative platform for quantitative analyses, identifying unknown compounds and gathering information for the elucidation of chemical structures. This work describes a method to detect 13 esters of testosterone (T) and 5 biomarkers in 0.1 mL of human serum using gas chromatography (GC) coupled to HRMS. METHODS: Analytes were extracted from serum after deproteinization and liquid-liquid extraction. The trimethylsilyl derivatives were analyzed using a gas chromatograph coupled to HRMS at low electron energy to minimize molecule fragmentation. The acquisition in profiling full-scan mode was applied with a resolving power of 30 000 at m/z 400. Linearity, lower limit of quantitation, and measurement uncertainty were assessed. Precision and accuracy were assessed at 0.5 and 2 ng/mL, respectively. Mass accuracy (MA) and mass extraction window (MEW) were also evaluated. RESULTS: T esters showed a linear response between 0.25 and 10 ng/mL (except for undecanoate, enanthate, and propionate that showed lineal responses between 0.5 and 10 ng/mL and isocaproate between 2 and 10 ng/mL); detection limits remained between 0.1 and 0.5 ng/mL and accuracy between 81% and 119%. The MA (MEW = 10 ppm) was maintained between -2.4 and 4.8 ppm. The biomarkers (T, androstenedione, dehydroepiandrosterone [DHEA], estradiol, and 17-OH-progesterone) showed a linear response within the evaluated range; quantification limits remained between 0.1 and 0.5 ng/mL (except for DHEA), the accuracy between 88% and 99%, and precision between 3.5% and 10.8%. Measurement uncertainties were found between 5.6% and 17.2%. MA (MEW = 3 ppm) was maintained between -0.47 and 0.12 ppm. CONCLUSIONS: The method to detect T esters and five endogenous biomarkers in serum using GC coupled to HRMS showed linear responses up to 10 ng/mL with adequate precision, accuracy, and uncertainties. It was possible to distinguish cholesterol from T-isocaproate based on the MEW of 10 ppm, preventing false positives. In addition, this method allows searching for other biomarkers and/or unknown metabolites and other ester forms not included here but at a later stage if necessary.

Quinoa as phytopharmaceutical? Urinary elimination of ecdysterone after consumption of quinoa alone and in combination with spinach.

The phytosteroid ecdysterone is classified as an anabolic agent and has been included on the monitoring list of the World Anti-Doping Agency since 2020. Therefore, the consumption of food rich in ecdysterone, such as quinoa and spinach, is the focus of a lively debate. Thus, the urinary excretion of ecdysterone and its metabolites in humans was investigated following quinoa consumption alone and in combination with spinach. Eight participants (four male and four female) were included, and they ingested 368 ± 61 g cooked quinoa alone and in combination with 809 ± 115 g spinach after a washout. Post-administration urines were analyzed by LC-MS/MS. After intake of both preparations, ecdysterone and two metabolites were excreted in the urine. The maximum concentration of ecdysterone ranged from 0.44 to 5.5 µg/mL after quinoa and from 0.34 to 4.1 µg/mL after quinoa with spinach. The total urinary excreted amount as parent drug plus metabolites was 2.61 ± 1.1% following quinoa intake and 1.7 ± 0.9% in combination with spinach. Significant differences were found in the total urinary excreted amount of ecdysterone, 14-deoxy-ecdysterone, and 14-deoxy-poststerone. Only small portions of ecdysterone from quinoa and the combination with spinach were excreted in the urine, suggesting that both quinoa and spinach are poor sources of ecdysterone in terms of bioavailability.

Serum myokines as potential biomarkers of myostatin inhibition in sport doping: a preliminary study on their baseline levels in elite athletes.

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

LC-HRMS-based metabolomics workflow: An alternative strategy for metabolite identification in the antidoping field.

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 3ß-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.

Metabolomics workflow as a driven tool for rapid detection of metabolites in doping analysis. Development and validation.

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

Detection of urinary arimistane metabolites in humans using liquid chromatography-mass spectrometry: Complementary results to gas chromatography mass spectrometric data and its application to antidoping analyses.

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

Low-energy electron ionization optimization for steroidomics analysis using high-resolution mass spectrometry.

RATIONALE: Systematic electron ionization fragmentation studies of steroids have been performed to elucidate and trace their characteristic fragmentation patterns. However, the electron ionization source setting at 70 eV electron energy is much higher than the ionization potential (7-15 eV) of most organic compounds, leading to extensive fragmentation. We present a multifactorial study on optimizing a low-energy electron ionization source to maximize molecular ion formation while minimizing the extent of fragmentation to improve the analytical sensitivity of steroids, especially the more thermolabile ones, while preserving the information that can be extracted from the data. METHODS: Twenty-seven steroid reference materials, chosen to cover four main classes of urinary steroids, were considered; gas chromatography/quadrupole time-of-flight (GC/qTOF) analyses were carried out using an Agilent Technologies model 8890 gas chromatograph coupled to an Agilent Technologies model 7250 accurate-mass quadrupole time-of-flight (GC/qTOF) instrument. The effects of electron energy, emission current, and source temperature, as well as their potential interactions on steroid fragmentation pathways, have been assessed in full factorial experimental designs. RESULTS: Three parameters were specifically evaluated to improve the chromatographic/spectrometric response of the selected steroids: (i) degree of fragmentation; (ii) relative abundance of the molecular ion; and (iii) peak width. The first two were evaluated by screening designs that highlighted collision energy and source temperature as the most influential factors on the analytical responses of the considered steroids, while emission current always showed a non-significant influence. Then, an optimization design was performed to select the final source setting by searching for the combination of factors that minimize peak tailing. CONCLUSIONS: The proposed analytical approach permits a faster selection of optimal experimental conditions for steroidomics analysis using low-energy electron ionization and high-resolution mass spectrometry. The development of these designs of experiments (DoE) in full factorial design (FFD) allowed multiple inputs to be monitored at the same time, highlighting the possible interactions and estimating the effects of a factor in the different levels of the other factors considered.

New Insights into the Metabolism of Methyltestosterone and Metandienone: Detection of Novel A-Ring Reduced Metabolites.

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

Mass spectrometric analysis of 7-oxygenated androst-5-ene structures. Influence in trimethylsilyl derivative formation.

Several authors have described the generation of androsta-3,5-diene-7-one structures from androst-5-ene-3,7-dione or androst-5-ene-3ß-ol-7-one under acidic conditions and/or at high temperatures. The goal of this study was to observe and to describe the results obtained after the chromatographic analysis of the trimethylsilyl derivatives of reference materials of 7-oxo-DHEA, 7α-hydroxy-DHEA, 7ß-hydroxy-DHEA, and androsta-3,5-diene-7,17-dione known as arimistane. METHODS: The purity of the analyte reference materials was verified by liquid chromatography/quadrupole mass spectrometry. The trimethylsilyl derivatives obtained using several mixtures with MSTFA (N-methyl-N-trimethylsilyl trifluoroacetamide) in comparison with solely MSTFA were analyzed by gas chromatography coupled to a time-of-flight detector equipped with a multimode inlet or to a simple quadrupole detector with a split/splitless inlet. RESULTS: The study showed that the formation of arimistane from 7-oxo-DHEA occurs using common derivatization reagents used for the analyses by gas chromatography (GC). In addition, the formation of the enolized TMS derivative of 7-oxo-DHEA was observed in considerable amount when it was reacted with MSTFA. The analysis of 7α-hydroxy-DHEA resulted in the detection of ~1% of arimistane. The formation of unexpected artifacts from derivatization is influenced by the reagent itself, the reaction temperature, the inlet used and its configuration. CONCLUSIONS: The derivatization reagent, instrumental conditions (inlet), as well as the chemical structures of the analytes present in the matrix, can influence the results. So, before describing a new feature as a potential "new" metabolite, special caution must be taken since we could actually be dealing with an artifact.

In-depth gas chromatography/tandem mass spectrometry fragmentation analysis of formestane and evaluation of mass spectral discrimination of isomeric 3-keto-4-ene hydroxy steroids.

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 H9 ]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 H2 18 O to obtain derivatives doubly labelled with [2 H9 ]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 (CH3 )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 MS3 may be suitable in future bioanalytical applications requiring discrimination of the 2- and 4-hydroxy compounds.

A further insight into methyltestosterone metabolism: New evidences from in vitro and in vivo experiments.

RATIONALE: Although the metabolism of methyltestosterone (MT) has been extensively studied since the 1950s using different techniques, the aim of this study was to investigate the hydroxylation in positions C2, C4 and C6 after in vitro experiments and in vivo excretion studies using gas chromatography time-of-flight (GC/TOF) and gas chromatography/tandem mass spectrometry (GC/MS/MS). The results could be influenced by the mass spectrometric analyser used. METHODS: Incubations were carried out with human liver microsomes and six enzymes belonging to the cytochrome P450 family using MT as a substrate. The trimethylsilyl derivatives of the samples were analysed using GC/TOF and GC/MS/MS once the correct MS/MS transitions had been selected, mainly for 6-hydroxymethyltestosterone (6-OH-MT) to avoid artefact interferences. A urinary excretion study was then performed after the administration of a 10 mg single oral dose of MT to a volunteer. RESULTS: The formation of hydroxylated metabolites of MT in the C6, C4 and C2 positions after both in vitro and in vivo experiments was observed. Sample evaluation using GC/TOF showed an interference for 6-OH-MT that could only be resolved in GC/MS/MS by monitoring specific transitions. The transitory detection of these hydroxylated metabolites in urine agrees with previous investigations that had described this metabolic route as being of little significance. CONCLUSIONS: In doping analysis, the formation of 4-hydroxymethyltestosterone (oxymesterone) from MT cannot be underestimated. Although it is only detected as a minor and short-term excretion metabolite, it cannot be overlooked as it was found in both in vitro and in vivo experiments. The use of a combination of different mass spectrometric instruments allowed reliable conclusions to be reached, and it was shown that special attention must be given to artefact formation.

Detection of urinary metabolites of arimistane in humans by gas chromatography coupled to high-accuracy mass spectrometry for antidoping analyses.

RATIONALE: The selection of the most appropriate metabolites of the substances included in the Prohibited List of the World Anti-Doping Agency (WADA) is fundamental for setting up methods allowing the detection of their intake by mass spectrometric methods. The aim of this work is to investigate the metabolism of arimistane (an aromatase inhibitor included in the WADA list) in order to improve its detection capacity among the antidoping community. METHODS: Urinary samples collected after controlled single administration of arimistane in three healthy volunteers were analysed using the common routine sample preparation in antidoping laboratories to determine the steroid profile parameters considered in the steroid module of the Athlete Biological Passport by gas chromatography coupled to tandem mass spectrometry (GC/MS/MS). For the elucidation of the proposed metabolites, GC coupled to high-accuracy MS (GC/qTOFMS) was used. Both mass spectrometers were operated in electron ionization mode. Non-conjugated (free), glucuronated and sulfated fractions were analysed separately. RESULTS: No relevant effects on the steroid profile could be detected after a single oral dose (25 mg). Up to 15 metabolites, present only in the post-administration samples, were detected and some structures were postulated. These metabolites are mainly excreted as glucuro-conjugated into urine and only minor amounts of two metabolites are also excreted unconjugated or as sulfates. CONCLUSIONS: Arimistane itself was not observed in the free or glucuronated fractions, but only in the sulfate fraction. The peaks showing mass spectra in agreement with hydroxylated metabolites did not match with those for 7-keto-DHEA, 7α- or 7ß-hydroxy-DHEA. This suggests that the first hydroxylation did not occur on C3, but on C2. These newly described metabolites allow the specific detection of arimistane misuse in sports.

Isotope ratio mass spectrometry in antidoping analysis: The use of endogenous reference compounds.

RATIONALE: Isotope ratio mass spectrometry (IRMS) is an analytical technique required by the World Antidoping Agency (WADA) before releasing of an adverse finding for the abuse of pseudoendogenous steroids (i.e. testosterone). For every single individual, the delta 13 C values (‰) of the selected target compounds (TCs, i.e. testosterone and/or its precursors/metabolites) are compared with those of endogenous reference compounds (ERCs). The aim of this work is to investigate the individual variation in the delta values of four different commonly used ERCs to establish the maximum acceptable variation, in order to detect potential outliers. METHODS: Routine urine samples collected for antidoping purposes were submitted to IRMS confirmation. After a specific liquid chromatographic purification of the analytes of interest, the final extracts were analyzed by gas chromatography/combustion (GC/C)-IRMS. The selected ERCs monitored were pregnanediol, pregnanetriol, 11-keto-etiocholanolone and 11ß-hydroxyandrosterone. The obtained 13 C delta values were statistically analyzed to evaluate their inter- and intra-individual distribution. RESULTS: The delta values of the ERCs studied showed a normal distribution and no major differences among genders were observed. As expected, there are differences depending on the geographical origin of the samples, reflecting different dietary habits and food sources. The intra-individual dispersion, expressed as the standard deviation (SD) of the values of the studied ERCs, did not greatly exceed the instrumental error (0.5‰), demonstrating the good preservation of the delta values along the metabolic pathway. CONCLUSIONS: For the selected ERCs of non-sporting volunteers and the urinary specimens from more than 1000 sportsmen, we can propose a maximum SD of 0.54‰ and range of 1.2‰ for delta 13 C values as acceptance criteria to detect potential outliers. These cases can be caused by the external masking effect of the administration of a substance modifying the delta values or outliers due to unforeseen procedural artifacts.

An investigation on the metabolic pathways of synthetic isoflavones by gas chromatography coupled to high accuracy mass spectrometry.

RATIONALE: Isoflavones are a group of flavonoids that may be of interest in sport doping because they can be used by athletes in the recovery periods after the administration of anabolic steroids, with the aim of increasing the natural production of luteinizing hormone (LH) and, consequently, the biosynthesis of endogenous androgens. METHODS: The in vivo metabolism of methoxyisoflavone (5-methyl-7-methoxyisoflavone) and ipriflavone (7-isopropoxyisoflavone), respectively present in a dietary supplement and in a pharmaceutical preparation, was investigated. The study was carried out by the analysis of urinary samples collected from male Caucasian subjects before, during and after the oral administration of methoxyisoflavone or ipriflavone. After enzymatic hydrolysis and liquid-liquid extraction, all urinary samples were analyzed by gas chromatography/quadrupole time-of-flight (qTOF MS system/qTOF) electron ionization mass spectrometry (EI-MS). RESULTS: Eight metabolites of methoxyisoflavone and six metabolites of ipriflavone were isolated. The corresponding accurate mass spectra are specific for isoflavone structures and revealed also a retro-Diels-Alder fragmentation. CONCLUSIONS: When excreted in large amounts, the urinary metabolites of methoxyisoflavone and ipriflavone can be traced to potential confounding factors in doping analysis. As methoxyisoflavone and ipriflavone have been shown to inhibit the enzyme aromatase, thus interfering with the normal metabolic pathways of testosterone, the detection of their intake, by screening for the presence of their main metabolites in urine, might be helpful in routine doping control analysis.

Detection of recombinant insulins in human urine by liquid chromatography-electrospray ionization tandem mass spectrometry after immunoaffinity purification based on monolithic microcolumns.

This work describes an analytical procedure based on automated affinity purification followed by liquid chromatography-electrospray tandem mass spectrometry with a conventional triple quadrupole analyzer, in order to detect synthetic insulins (Apidra®, Humalog®, Levemir®, NovoRapid®, and Tresiba®) in human urine. Sample preparation included ultrafiltration followed by immunoaffinity purification on monolithic microcolumns. Chromatographic separation was performed by a C18 microbore column, while mass spectrometric identification of the analytes was achieved by a triple quadrupole mass spectrometer under positive ion electrospray ionization and acquisition mode in selected reaction monitoring. Identification of the synthetic insulins was performed by selecting at least two characteristic ion transitions for each analyte. The newly developed method was validated in terms of specificity, recovery, matrix effect, sensitivity, robustness, and repeatability of retention times and relative ion transition abundance. The specificity and the reproducibility of the relative retention times and the relative abundance of the characteristic ion transitions selected was confirmed to be fit for purposes of ensuring the unambiguous identification of all target analytes, also in the forensic field. The extraction yield was estimated at greater than 60% and the matrix effect smaller than 35%. The lower limits of detection were in the range of 0.02-0.05 ng/mL, proving the method to be sufficiently sensitive to detect the abuse of insulins in cases where they are used as performance-enhancing agents in sport. The applicability of the developed method was assessed by the analysis of urine samples obtained from diabetic subjects treated with Tresiba® and/or Humalog®, whose presence was confirmed in urine samples collected after the administration of therapeutic doses. Graphical abstract A hybrid assay comprising MSIA-based immunoextraction combined with liquid chromatography-electrospray tandem mass spectrometry was developed and validated for the detection of recombinant insulins in human urine.

Ecdysteroids as non-conventional anabolic agent: performance enhancement by ecdysterone supplementation in humans.

Recent studies suggest that the anabolic effect of ecdysterone, a naturally occurring steroid hormone claimed to enhance physical performance, is mediated by estrogen receptor (ER) binding. In comparison with the prohibited anabolic agents (e.g., metandienone and others), ecdysterone revealed to be even more effective in a recent study performed in rats. However, scientific studies in humans are very rarely accessible. Thus, our project aimed at investigating the effects of ecdysterone-containing products on human sport exercise. A 10-week intervention study of strength training of young men (n = 46) was carried out. Different doses of ecdysterone-containing supplements have been administered during the study to evaluate the performance-enhancing effect. Analysis of blood and urine samples for ecdysterone and potential biomarkers of performance enhancement has been conducted. To ensure the specificity of the effects measured, a comprehensive screening for prohibited performance-enhancing substances was also carried out. Furthermore, the administered supplement has been tested for the absence of anabolic steroid contaminations prior to administration. Significantly higher increases in muscle mass were observed in those participants that were dosed with ecdysterone. The same hypertrophic effects were also detected in vitro in C2C12 myotubes. Even more relevant with respect to sports performance, significantly more pronounced increases in one-repetition bench press performance were observed. No increase in biomarkers for liver or kidney toxicity was noticed. These data underline the effectivity of an ecdysterone supplementation with respect to sports performance. Our results strongly suggest the inclusion of ecdysterone in the list of prohibited substances and methods in sports in class S1.2 "other anabolic agents".

SFC-MS/MS as an orthogonal technique for improved screening of polar analytes in anti-doping control.

HPLC is considered the method of choice for the separation of various classes of drugs. However, some analytes are still challenging as HPLC shows limited resolution capabilities for highly polar analytes as they interact insufficiently on conventional reversed-phase (RP) columns. Especially in combination with mass spectrometric detection, limitations apply for alterations of stationary phases. Some highly polar sympathomimetic drugs and their metabolites showed almost no retention on different RP columns. Their retention remains poor even on phenylhexyl phases that show different selectivity due to π-π interactions. Supercritical fluid chromatography (SFC) as an orthogonal separation technique to HPLC may help to overcome these issues. Selected polar drugs and metabolites were analyzed utilizing SFC separation. All compounds showed sharp peaks and good retention even for the very polar analytes, such as sulfoconjugates. Retention times and elution orders in SFC are different to both RP and HILIC separations as a result of the orthogonality. Short cycle times could be realized. As temperature and pressure strongly influence the polarity of supercritical fluids, precise regulation of temperature and backpressure is required for the stability of the retention times. As CO2 is the main constituent of the mobile phase in SFC, solvent consumption and solvent waste are considerably reduced. Graphical Abstract SFC-MS/MS vs. LC-MS/MS.

Narrowing the gap between the number of athletes who dope and the number of athletes who are caught: scientific advances that increase the efficacy of antidoping tests.

The analytical methods developed and applied by the antidoping laboratories have been continuously evolving over the past 50 years, with the aim of keeping pace with the constant evolution of doping strategies. Despite this, the number of adverse analytical tests reported worldwide by the network of the WADA-accredited laboratories still seems to underestimate the actual number of doped athletes. We investigate the most likely causes for this gap between the likely doping rate and the detection of athletes with positive doping tests. We consider laboratory and non-laboratory reasons that contribute to this gap. More specifically, laboratory issues are focused not only on those doping practices that may still be 'invisible' at the time of a doping test, but also on the possible role of non-conventional masking strategies. These include (1) the intake of banned drugs by specific novel drug delivery systems and (2) the coadministration of prohibited and non-prohibited drugs, taking advantage of the capacity of the latter to affect the metabolism, and consequently the detection, of the former. Non-laboratory issues include the lack of a sufficient level of 'intelligent testing', with the result that, even in the cases of doped athletes, the biological samples delivered to the antidoping laboratories for analysis may not contain those target analytes whose detection (and if necessary quantification above a decision limit) constitutes an adverse analytical finding. We present proposals to improve the efficacy of the doping control policies based on the analysis of biological samples and suggest how to constantly keep up with the continuous developments of new forms of doping.

A SNP-based genotyping strategy to detect the abuse of homologous blood transfusion from dried blood spots.

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.

Detection of thyroid hormones in urine by liquid chromatography coupled to tandem mass spectrometry.

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.