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BACKGROUND: Atherosclerotic plaques form unevenly due to disturbed blood flow, causing localized endothelial cell (EC) dysfunction. Obesity exacerbates this process, but the underlying molecular mechanisms are unclear. The transcription factor EPAS1 (HIF2A) has regulatory roles in endothelium, but its involvement in atherosclerosis remains unexplored. This study investigates the potential interplay between EPAS1, obesity, and atherosclerosis. METHODS: Responses to shear stress were analyzed using cultured porcine aortic EC exposed to flow in vitro coupled with metabolic and molecular analyses and by en face immunostaining of murine aortic EC exposed to disturbed flow in vivo. Obesity and dyslipidemia were induced in mice via exposure to a high-fat diet or through Leptin gene deletion. The role of Epas1 in atherosclerosis was evaluated by inducible endothelial Epas1 deletion, followed by hypercholesterolemia induction (adeno-associated virus-PCSK9 [proprotein convertase subtilisin/kexin type 9]; high-fat diet). RESULTS: En face staining revealed EPAS1 enrichment at sites of disturbed blood flow that are prone to atherosclerosis initiation. Obese mice exhibited substantial reduction in endothelial EPAS1 expression. Sulforaphane, a compound with known atheroprotective effects, restored EPAS1 expression and concurrently reduced plasma triglyceride levels in obese mice. Consistently, triglyceride derivatives (free fatty acids) suppressed EPAS1 in cultured EC by upregulating the negative regulator PHD2. Clinical observations revealed that reduced serum EPAS1 correlated with increased endothelial PHD2 and PHD3 in obese individuals. Functionally, endothelial EPAS1 deletion increased lesion formation in hypercholesterolemic mice, indicating an atheroprotective function. Mechanistic insights revealed that EPAS1 protects arteries by maintaining endothelial proliferation by positively regulating the expression of the fatty acid-handling molecules CD36 and LIPG to increase fatty acid beta-oxidation. CONCLUSIONS: Endothelial EPAS1 attenuates atherosclerosis at sites of disturbed flow by maintaining EC proliferation via fatty acid uptake and metabolism. This endothelial repair pathway is inhibited in obesity, suggesting a novel triglyceride-PHD2 modulation pathway suppressing EPAS1 expression. These findings have implications for therapeutic strategies addressing vascular dysfunction in obesity.

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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.

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Current guidelines for prolonged altitude exposure suggest altitude levels ranging from 2000 to 2500 m to optimize an increase in total hemoglobin mass (Hbmass). However, natural low altitude locations (<2000 m) remain popular, highlighting the interest to investigate any possible benefit of low altitude camps for endurance athletes. Ten elite racewalkers (4 women and 6 men) underwent a 4-week "live high-train high" (LHTH) camp at an altitude of 1720 m (PIO2 = 121 mmHg; 20.1°C; 67% relative humidity [RH]), followed by a 3-week tapering phase (20 m; PIO2 = 150 mmHg; 28.3°C; 53% RH) in preparation for the World Athletics Championships (WC). Venous blood samples were withdrawn weekly during the entire observation period. In addition, blood volumes were determined weekly by carbon monoxide rebreathing during altitude exposure and 2 weeks after return to sea level. High-level performances were achieved at the WC (five placings among the Top 10 WC races and three all-time career personal bests). A slight but significant increase in absolute (+1.7%, p = 0.03) and relative Hbmass (+2.3%, p = 0.02) was observed after 4-week LHTH. In addition, as usually observed during LHTH protocols, weekly training distance (+28%, p = 0.02) and duration (+30%, p = 0.04) significantly increased during altitude compared to the pre-LHTH period. Therefore, although direct causation cannot be inferred, these results suggest that the combination of increased training load at low altitudes with a subsequent tapering period in a warm environment is a suitable competition-preparation strategy for elite endurance athletes.

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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.

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Methyltestosterone (MT) is one of the most frequently misused anabolic androgenic steroids detected in doping control analysis. The metabolism of MT in humans leads to several phase І metabolites and their corresponding phase Ⅱ conjugates. Previous studies have postulated the 3α-sulfoconjugate of 17α-methyl-5ß-androstane-3α,17ß-diol (S2) as principal sulfate metabolite of MT, with a detection window exceeding 10 days. However, a final direct and unambiguous confirmation of the structure of this metabolite is missing until now. In this study, we established an approach to detect and identify S2, using intact analysis by liquid chromatography hyphenated with tandem mass spectrometry (LC-MS/MS) without complex sample pretreatment. An in vitro study yielded the LC-MS/MS reference retention times of all 3-sulfated 17-methylandrostane-3,17-diol diastereomers, allowing for accurate structure assignment of potentially detected metabolites. In an in vivo excretion study with a single healthy male volunteer, the presence of the metabolite S2 was confirmed after a single oral dose of 10 mg MT. The reference standard was chemically synthesized, characterized by accurate mass mass spectrometry (MS) and nuclear magnetic resonance (NMR), and quantified by quantitative NMR (qNMR). Thus, this study finally provides accurate structure information on the S2 metabolite and a direct analytical method for detection of MT misuse. The availability of the reference material is expected to facilitate further evaluation and subsequent analytical method validation in anti-doping research.

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

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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.

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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.

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BACKGROUND: Dried blood spots have recently been approved by the World Anti-Doping Agency as an alternative biological matrix for testing of doping substances. However, their use is limited to the detection of non-threshold compounds without a Minimum Reporting Level due to the numerous issues related to quantitative analyses and the limitation on testing capabilities of a haemolysed matrix. AIM: In this study androstenedione, testosterone and IGF-1 were longitudinally monitored in four different blood matrices to evaluate the potential of liquid capillary blood as an alternative matrix for quantitative determination in doping control analysis. METHODOLOGY: The analytical protocols developed to pretreat 20 µL of the blood matrices selected were based: i) for testosterone and androstenedione, on supported liquid extraction for liquid blood matrices, and on ultrasonication in the presence of methanol for dried blood matrices; ii) for IGF-1, proteins precipitation followed by evaporation of the supernatant was used to pretreat both liquid and dried blood matrices. The detection for all the target analytes was performed using liquid chromatography coupled to mass spectrometry. The analytical workflows, once optimized, were fully validated according to the requirements of World Anti-Doping Agency and ISO 17025 standard and used for the analysis of venous (serum) and capillary (liquid plasma and dried whole blood collected using either volumetric or non-volumetric devices) blood samples collected from 7 healthy subjects. RESULTS: The validation results showed satisfactory performance as related to specificity, sensitivity, matrix effects, linearity, accuracy, and precision in all the blood matrices evaluated despite the limited volume of sample used. The analysis of the different blood matrices collected from the subjects showed non-significant differences between the levels of testosterone and androstenedione measured in dried (fixed volume collected) and liquid matrices. An acceptable underestimation (lower than 15 %) was observed in capillary plasma compared to venous serum. The testosterone/androstenedione ratio was similar in all the blood matrices considered (bias lower than 5 %), indicating this parameter was not affected by either the blood matrix or collection device selected. For IGF-1, the levels measured in liquid blood matrices differed significantly (bias higher than 20 %) from those measured in dried whole blood matrices, suggesting haemolyzed blood might represent a challenge for the determination of macromolecules, mainly due to the complexity of the whole blood matrix in comparison to plasma/serum. NOVELTY: The outcomes of our study suggest that liquid capillary blood might open new avenues to blood microsampling in doping control field. It represents an efficient alternative to overcome the issues related to venous blood and dried blood spot sampling. Furthermore, it also allows greater frequency of blood sampling, with minor discomfort and without needing a phlebotomist, for analyses that can only be performed in blood samples, with an increased probability to detect and report Adverse Analytical Finding.

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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.

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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.

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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.

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PURPOSE: In competitive sport, classic methods of measuring drug prevalence, such as doping controls or questionnaires, are challenging. Here we describe a novel urine sampling method to measure drug use in athletes. We hypothesize that the prevalence of drug use in ultramarathon runners is measured more accurately with our sampling method than randomized-response questionnaires. METHODS: Urine samples and associated demographic data were collected from male participants using blind, automated urinals at the start of ultramarathon races. Various nonprohibited and prohibited substances were subsequently screened. Concomitantly, 2931 male and female runners participating in the same ultramarathons completed an anonymized, randomized-response questionnaire regarding drug use. RESULTS: Among 412 individual urine samples, 205 (49.8%) contained at least one substance, and 16.3% of the samples contained one or more prohibited substances. Substances detected in urine included nonsteroid anti-inflammatory drugs (NSAID) (22.1%), acetaminophen (15.5%), opioids (6.6%), diuretics (4.9%), hypnotics (4.4%), glucocorticoids (2.7%), beta-2 agonists (2.2%), cannabinoids (1.9%), and stimulants (1.2%). None of the samples contained erythropoietin-receptor agonists or suspicious testosterone. Drug use was not associated with the participants' characteristics or ranking. Respondents to the questionnaire reported using acetaminophen (13.6%) and NSAID (12.9%); however, no prohibited substances were declared. CONCLUSIONS: There was a high prevalence of drug use among male ultramarathon runners, in particular, NSAID and painkillers; however, performance-enhancing drugs were marginally used. Blind urine sampling highlighted prohibited drug use not declared in questionnaires, and it is useful to assess the prevalence of drug use and/or doping in competitive athletes.

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In comparison to well-known drug-metabolizing organs such as the liver, the metabolic capacity of human skin is still not well elucidated despite the widespread use of topical drug application. To gain a comprehensive insight into anabolic steroid metabolism in the skin, six structurally related anabolic androgenic steroids, testosterone, metandienone, methyltestosterone, clostebol, dehydrochloromethyltestosterone, and methylclostebol, were applied to human keratinocytes and fibroblasts derived from the juvenile foreskin. Phase I metabolites obtained from incubation media were analyzed by gas chromatography-mass spectrometry. The 5α-reductase activity was predominant in the metabolic pathways as supported by the detection of 5α-reduced metabolites after incubation of testosterone, methyltestosterone, clostebol, and methylclostebol. Additionally, the stereochemistry structures of fully reduced metabolites (4α,5α-isomers) of clostebol and methylclostebol were newly confirmed in this study by the help of inhouse synthesized reference materials. The results provide insights into the steroid metabolism in human skin cells with respect to the characteristics of the chemical structures.

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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.

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

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Γ-valerolactone (GVL), marketed online as "Tranquilli-G" and "excellent Valium", is used as a legal substitute for γ-hydroxybutyric acid (GHB); however, until now, GVL has only been connected to one Drug-Facilitated Sexual Assault (DFSA) case. Moreover, the pharmaco-toxicological effects of GVL are poorly studied. The aim of this study was to investigate the 1) in vivo effects of gavage administration of GVL (100-3000 mg/kg) on neurological (myoclonia, convulsions), sensorimotor (visual, acoustic, and overall tactile) responses, righting reflex, thermoregulation, motor activity (bar, drag, and accelerod test) and cardiorespiratory changes (heart rate, breath rate, oxygen saturation, and pulse distension) in CD-1 male mice and the 2) in silico ADMET profile of GVL in comparison to GHB and the open active form γ-hydroxyvaleric acid (GHV). The present study demonstrates that GVL inhibits, in a dose-dependent manner, sensorimotor and motor responses and induces cardiorespiratory depression (at a dose of 3000 mg/kg) in mice. The determination of the ED50 in sensorimotor and motor responses revealed that GVL is about 4-5 times less potent than GHB. In silico prediction of ADMET profiles revealed toxicokinetic similarities between GHB and GHV, and differences with GVL. These results suggest that GVL could be used as a substitute for GHB and should be added to forensic toxicology screenings.

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

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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,17ß-diol (5α-Adiol), 5ß-androstane-3α,17ß-diol (5ß-Adiol), testosterone (T), androsterone (A), etiocholanolone (Etio), epitestosterone (E), pregnanediol (PD), dehydroepiandrosterone (DHEA), and 11ß-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 5ß-Adiol after TH administration. The interpretation of the steroid markers of the ABP should consider TH administrations.

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

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