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.

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.

Routine application of SFC-MS in doping control: Analysis of 3 × 1000 urine samples using three different SFC-MS instruments.

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.

Liquid vs dried blood matrices: Application to longitudinal monitoring of androstenedione, testosterone, and IGF-1 by LC-MS-based techniques.

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.

Prevalence of Drug Use in Ultraendurance Athletes.

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.

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.

Yearly intrasubject variability of hematological biomarkers in elite athletes for the Athlete Biological Passport.

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.

Indirect biomarkers of blood doping: A systematic review.

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.

Biotransformation of anabolic androgenic steroids in human skin cells.

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.

Age-Markers on the Red Blood Cell Surface and Erythrocyte Microparticles may Constitute a Multi-parametric Strategy for Detection of Autologous Blood Transfusion.

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.

Comprehensive evaluation of the pharmacological and toxicological effects of γ-valerolactone as compared to γ-hydroxybutyric acid: Insights from in vivo and in silico models.

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.

Detection of synthetic analogues of insulin-like growth factor 1 in different biological fluids by liquid chromatography quadrupole time-of-flight mass spectrometry: comparison of different immunoaffinity protocols.

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.

Effect of thyroid hormones administration on urinary endogenous steroid profile of the athlete biological passport.

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.

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.

Urinary Excretion of Ecdysterone and Its Metabolites Following Spinach Consumption.

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.

A new multimodal paradigm for biomarkers longitudinal monitoring: a clinical application to women steroid profiles in urine and blood.

BACKGROUND: Most current state-of-the-art strategies to generate individual adaptive reference ranges are designed to monitor one clinical parameter at a time. An innovative methodology is proposed for the simultaneous longitudinal monitoring of multiple biomarkers. The estimation of individual thresholds is performed by applying a Bayesian modeling strategy to a multivariate score integrating several biomarkers (compound concentration and/or ratio). This multimodal monitoring was applied to data from a clinical study involving 14 female volunteers with normal menstrual cycles receiving testosterone via transdermal route, as to test its ability to detect testosterone administration. The study samples consisted of urine and blood collected during 4 weeks of a control phase and 4 weeks with a daily testosterone gel application. RESULTS: Integrating multiple biomarkers improved the detection of testosterone gel administration with substantially higher sensitivity compared with the distinct follow-up of each biomarker, when applied to selected urine and serum steroid biomarkers, as well as the combination of both. Among the 175 known positive samples, 38% were identified by the multimodal approach using urine biomarkers, 79% using serum biomarkers and 83% by combining biomarkers from both biological matrices, whereas 10%, 67% and 64% were respectively detected using standard unimodal monitoring. SIGNIFICANCE AND NOVELTY: The detection of abnormal patterns can be improved using multimodal approaches. The combination of urine and serum biomarkers reduced the overall number of false-negatives, thus evidencing promising complementarity between urine and blood sampling for doping control, as highlighted in the case of the use of transdermal testosterone preparations. The generation in a multimodal setting of adaptive and personalized reference ranges opens up new opportunities in clinical and anti-doping profiling. The integration of multiple parameters in a longitudinal monitoring is expected to provide a more complete evaluation of individual profiles generating actionable intelligence to further guide sample collection, analysis protocols and decision-making in clinics and anti-doping.

Capillary blood as a complementary matrix for doping control purposes. Application to the definition of the individual longitudinal profile of IGF-1.

We present a novel procedure to monitor the fluctuations of the levels of IGF-1 in capillary blood in the framework of doping control analysis. Being an endogenous hormone, direct methods are not applicable, so the most effective way to detect the intake of the exogenous hormone would be based on the longitudinal monitoring of the athlete. We have therefore followed the individual variability, in four subjects (two males and two females), of the levels of IGF-1 in capillary blood samples collected three times per day for five days, then once a week for at least two months. Analyses were performed by liquid chromatography coupled to tandem mass spectrometry following a bottom-up approach. The whole protocol, from the sample collection to the instrumental analysis, was validated according to the World Anti-Doping Agency's guidelines and ISO17025. The analytical protocol showed to be fit for purpose in terms of sensitivity (LOD 25 ng/mL and LOI 35 ng/mL), selectivity (no interferences were detected at the retention time of IGF-1 and the internal standard), and repeatability (CV<10%). The linearity was confirmed in the range of 50-1000 ng/mL (correlation coefficient R2 >0.995, with a % relative bias of the experimental concentration of the different calibrators used for the estimation of the linearity lower than 20% for the lowest level and than 15% for the other levels). Stability studies were also performed, also to establish the optimal conditions for transport and storage: samples were stable at 4 °C for up to 72 h and at -20 °C and -80 °C for up to three months. Our preliminary results indicate that, in all subjects, the levels of IGF-1 did not present significant circadian fluctuations and remained stable during the entire period of the study (2-3 months, depending on the subject). The stability over time of IGF-1 levels in capillary blood indicates the possibility of detecting the intake of the non-endogenous hormone based on a longitudinal approach, as it is modeled in the framework of the endocrinological module of the athlete biological passport.

Analysis of doping control samples using supercritical fluid chromatography-tandem mass spectrometry: Ready for routine use.

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

Should We be Concerned with Nicotine in Sport? Analysis from 60,802 Doping Control Tests in Italy.

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