Explore the vast range of titles available.

The added value of dried blood spot (DBS) samples complementing the information obtained from commonly routine doping control matrices is continuously increasing in sports drug testing. In this project, a robotic-assisted non-destructive hematocrit measurement from dried blood spots by near-infrared spectroscopy followed by a fully automated sample preparation including strong cation exchange solid-phase extraction and evaporation enabled the detection of 46 lower molecular mass (< 2 kDa) peptide and non-peptide drugs and drug candidates by means of LC-HRMS. The target analytes included, amongst others, agonists of the gonadotropin-releasing hormone receptor, the ghrelin receptor, the human growth hormone receptor, and the antidiuretic hormone receptor. Furthermore, several glycine derivatives of growth hormone–releasing peptides (GHRPs), arguably designed to undermine current anti-doping testing approaches, were implemented to the presented detection method. The initial testing assay was validated according to the World Anti-Doping Agency guidelines with estimated LODs between 0.5 and 20 ng/mL. As a proof of concept, authentic post-administration specimens containing GHRP-2 and GHRP-6 were successfully analyzed. Furthermore, DBS obtained from a sampling device operating with microneedles for blood collection from the upper arm were analyzed and the matrix was cross-validated for selected parameters. The introduction of the hematocrit measurement method can be of great value for doping analysis as it allows for quantitative DBS applications by managing the well-recognized “hematocrit effect.”

The objective of this pilot study was to compare glucose concentrations in capillary blood (CB) samples analysed in a laboratory by a validated method and glucose concentrations measured in the interstitial fluid (ISF) by continuous glucose monitoring (CGM) under different physical activity levels in a postprandial state in healthy athletes without diabetes. As a physiological shift occurs between glucose concentration from the CB into the ISF, the applicability of CGM in sports, especially during exercise, as well as the comparability of CB and ISF data necessitate an in-depth assessment. Ten subjects (26 ± 4 years, 67 ± 11 kg bodyweight (BW), 11 ± 3 h) were included in the study. Within 14 days, they underwent six tests consisting of (a) two tests resting fasted (HC_Rest/Fast and LC_Rest/Fast), (b) two tests resting with intake of 1 g glucose/kg BW (HC_Rest/Glc and LC_Rest/Glc), (c) running for 60 min at moderate (ModExerc/Glc), and (d) high intensity after intake of 1 g glucose/kg BW (IntExerc/Glc). Data were collected in the morning, following a standardised dinner before test day. Sensor-based glucose concentrations were compared to those determined from capillary blood samples collected at the time of sensor-based analyses and subjected to laboratory glucose measurements. Pearson’s r correlation coefficient was highest for Rest/Glc (0.92, p < 0.001) compared to Rest/Fast (0.45, p < 0.001), ModExerc/Glc (0.60, p < 0.001) and IntExerc/Glc (0.70, p < 0.001). Mean absolute relative deviation (MARD) and standard deviation (SD) was smallest for resting fasted and similar between all other conditions (Rest/Fast: 8 ± 6%, Rest/Glc: 17 ± 12%, ModExerc/Glc: 22 ± 24%, IntExerc/Glc: 18 ± 17%). However, Bland–Altman plot analysis showed a higher range between lower and upper limits of agreement (95% confidence interval) of paired data under exercising compared to resting conditions. Under resting fasted conditions, both methods produce similar outcomes. Under resting postprandial and exercising conditions, respectively, there are differences between both methods. Based on the results of this study, the application of CGM in healthy athletes is not recommended without concomitant nutritional or medical advice.

The substitution of a urine sample that may result in an adverse analytical finding with a previously collected, clean sample is strictly prohibited under the World Anti-Doping Agency (WADA) regulations and is referred to as sample swapping. When an athlete reuses their own clean sample, detection becomes particularly difficult through conventional analytical methods. In this paper, we propose a similarity detection framework that explicitly accounts for pattern complexity in the analysis of urinary steroid profiles. The framework is based on a convolutional network to capture more complex and subtle variations in profile pairs. Using a dataset of 67,651 steroid profiles collected between 2021 and 2023, the framework was evaluated on both synthetic and laboratory-confirmed similar samples, reflecting realistic variability in doping control processes. The results show that the proposed framework outperforms several baseline models, achieving higher accuracy compared to different baselines. These findings demonstrate the potential of machine learning to improve anti-doping workflows by enabling the automated detection of reused or identical urine samples within large-scale sample collection managed by the Athlete Biological Passport.

A narrative review with an overall aim of indicating the current state of knowledge and the relevance concerning food and supplement contamination and/or adulteration with doping agents and the respective implications for sports drug testing is presented. The identification of a doping agent (or its metabolite) in sports drug testing samples constitutes a violation of the anti-doping rules defined by the World Anti-Doping Agency. Reasons for such Adverse Analytical Findings (AAFs) include the intentional misuse of performance-enhancing/banned drugs; however, also the scenario of inadvertent administrations of doping agents was proven in the past, caused by, amongst others, the ingestion of contaminated dietary supplements, drugs, or food. Even though controversial positions concerning the effectiveness of dietary supplements in healthy subjects exist, they are frequently used by athletes, anticipating positive effects on health, recovery, and performance. However, most supplement users are unaware of the fact that the administration of such products can be associated with unforeseeable health risks and AAFs in sports. In particular anabolic androgenic steroids (AAS) and stimulants have been frequently found as undeclared ingredients of dietary supplements, either as a result of cross-contaminations due to substandard manufacturing practices and missing quality controls or an intentional admixture to increase the effectiveness of the preparations. Cross-contaminations were also found to affect therapeutic drug preparations. While the sensitivity of assays employed to test pharmaceuticals for impurities is in accordance with good manufacturing practice guidelines allowing to exclude any physiological effects, minute trace amounts of contaminating compounds can still result in positive doping tests. In addition, food was found to be a potential source of unintentional doping, the most prominent example being meat tainted with the anabolic agent clenbuterol. The athletes’ compliance with anti-doping rules is frequently tested by routine doping controls. Different measures including offers of topical information and education of the athletes as well as the maintenance of databases summarizing low- or high-risk supplements are important cornerstones in preventing unintentional anti-doping rule violations. Further, the collection of additional analytical data has been shown to allow for supporting result management processes.

Red blood cells (RBC) possess a nitric oxide synthase (RBC-NOS) whose activation depends on the PI3-kinase/Akt kinase pathway. RBC-NOS-produced NO exhibits important biological functions like maintaining RBC deformability. Until now, the cellular target structure for NO, to exert its influence on RBC deformability, remains unknown. In the present study we analyzed the modification of RBC-NOS activity by pharmacological treatments, the resulting influence on RBC deformability and provide first evidence for possible target proteins of RBC-NOS-produced NO in the RBC cytoskeletal scaffold. Blood from fifteen male subjects was incubated with the NOS substrate L-arginine to directly stimulate enzyme activity. Direct inhibition of enzyme activity was induced by L-N5-(1-Iminoethyl)-ornithin (L-NIO). Indirect stimulation and inhibition of RBC-NOS were achieved by applying insulin and wortmannin, respectively, substances known to affect PI3-kinase/Akt kinase pathway. The NO donor sodium nitroprusside (SNP) and the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) were additionally applied as NO positive and negative controls, respectively. Immunohistochemical staining was used to determine phosphorylation and thus activation of RBC-NOS. As a marker for NO synthesis nitrite was measured in plasma and RBCs using chemiluminescence detection. S-nitrosylation of erythrocyte proteins was determined by biotin switch assay and modified proteins were identified using LC-MS. RBC deformability was determined by ektacytometry. The data reveal that activated RBC-NOS leads to increased NO production, S-nitrosylation of RBC proteins and RBC deformability, whereas RBC-NOS inhibition resulted in contrary effects. This study first-time provides strong evidence that RBC-NOS-produced NO modifies RBC deformability through direct S-nitrosylation of cytoskeleton proteins, most likely α- and β-spectrins. Our data, therefore, gain novel insights into biological functions of RBC-NOS by connecting impaired RBC deformability abilities to specific posttranslational modifications of RBC proteins. By identifying likely NO-target proteins in RBC, our results will stimulate new therapeutic approaches for patients with microvascular disorders.

In order to detect the misuse of testosterone (T), urinary steroid concentrations and concentration ratios are quantified and monitored in a longitudinal manner to enable the identification of samples exhibiting atypical test results. These suspicious samples are then forwarded to isotope ratio mass spectrometry (IRMS)–based methods for confirmation. Especially concentration ratios like T over epitestosterone (E) or 5α-androstanediol over E proved to be valuable markers. Unfortunately, depending on the UGT2B17 genotype and/or the gender of the athlete, these markers may fail to provide evidence for T administrations when focusing exclusively on urine samples. In recent years, the potential of plasma steroids has been investigated and were found to be suitable to detect T administrations especially in female volunteers. A current drawback of this approach is the missing possibility to confirm that elevated steroid concentrations are solely derived from an administration of T and cannot be attributed to confounding factors. Therefore, an IRMS method for plasma steroids was developed and validated taking into account the comparably limited sample volume. As endogenous reference compounds, unconjugated cholesterol and dehydroepiandrosterone sulfate were found suitable, while androsterone and epiandrosterone (both sulfo-conjugated) were chosen as target analytes. The developed method is based on multi-dimensional gas chromatography coupled to IRMS in order to optimize the overall assay sensitivity. The approach was validated, and a reference population encompassing n = 65 males and females was investigated to calculate population-based thresholds. As proof-of-concept, samples from volunteers receiving T replacement therapies and excretion study samples were investigated.

Members of the transforming growth factor beta superfamily such as myostatin, activin A, and GDF-11, are dimeric cytokines signaling through activin receptors. They play important regulative roles in different biological processes as the formation of muscle and red blood cells. Therefore, inhibitors of the activin receptor signaling pathways (IASPs) are potential performance-enhancing agents in sports, which are included in sections S2 (“Peptide hormones, growth factors, related substances and mimetics”) and S4 (“Hormone and metabolic modulators”) of the WADA Prohibited List. Within this research project, a multiplexed detection assay for nine IASPs in doping control serum/plasma samples by means of (immuno-)affinity purification, tryptic digestion and LC-HRMS/MS was developed. The method was validated and proved to be specific and sensitive (LOD: 10–50 ng/mL). Additionally, it was modified to allow for using urine. As proof-of-concept, authentic Luspatercept serum and urine and Sotatercept serum post-administration samples were successfully analyzed. Luspatercept could be detected in both matrices up to 70 days after the initial and 7 weeks after the second dose. Sotatercept was successfully detected in a serum sample collected 43 h following injection. The presented method can be employed in doping control routine analysis as a qualitative initial testing procedure.

LGD-4033 (ligandrol) is a selective androgen receptor modulator (SARM), which is prohibited in sports by the World Anti-Doping Agency (WADA) and led to 62 adverse analytical findings (AAFs) in 2019. But not only deliberate doping with LGD-4033 constitutes a problem. In the past years, some AAFs that concerned SARMs can be attributed to contaminated dietary supplements (DS). Thus, the urgency to develop methods to differentiate between inadvertent doping and abuse of SARMs to benefit from the performance-enhancing effect of the compound in sports is growing. To gain a better understanding of the metabolism and excretion patterns of LGD-4033, human micro-dose excretion studies at 1, 10, and 50 µg LGD-4033 were conducted. Collected urine samples were prepared for analysis using enzymatic hydrolysis followed by solid-phase extraction and analyzed via LC-HRMS/MS. Including isomers, a total of 15 phase I metabolites were detected in the urine samples. The LC-HRMS/MS method was validated for qualitative detection of LGD-4033, allowing for a limit of detection (LOD) of 8 pg/mL. The metabolite M1, representing the epimer of LGD-4033, was synthesized and the structure elucidated by NMR spectroscopy. As the M1/LGD-4033 ratio changes over time, the ratio and the approximate LGD-4033 concentration can contribute to estimating the time point of drug intake and dose of LGD-4033 in doping control urine samples, which is particularly relevant in anti-doping result management.

Background: In order to address complex scenarios in anti-doping science, especially in cases where an unintentional exposure of athletes to prohibited substances and a corresponding contamination of doping control samples at the collection event are argued, an understanding of tissue-specific drug metabolism is essential. Hence, in this study, the metabolic capacity of the seminal vesicle using in vitro assays was investigated. Methods: The aim was to assess whether selected doping-relevant substances—stanozolol, LGD-4033, GW1516, trimetazidine, and anastrozole—are metabolised in seminal vesicle cellular fractions (SV-S9) and how that metabolism compares to biotransformations induced by human liver S9 fractions (HL-S9). Liquid chromatography coupled to high-resolution/accurate mass spectrometry (LC HRAM MS) enabled the sensitive detection and identification of metabolites, revealing a limited metabolic activity of SV-S9. Results: For LGD-4033, GW1516, and trimetazidine, minor metabolic transformations were observed, whereas no metabolites of stanozolol or anastrozole were detected. Gene expression analysis using digital polymerase chain reaction (dPCR) confirmed transcripts of CYP2D6, CYP2E1, and CYP2C9 in SV-S9, though no enzymatic activity was detected. Gene expression and enzymatic activity in CYP3A4 and CYP1A2—major hepatic enzymes—were absent in SV-S9. Conclusions: Overall, these pilot study results suggest that the seminal vesicle has only a low capacity for xenobiotic metabolism, which translates into a limited role in the biotransformation of drugs and, hence, the metabolic pattern.

Among anabolic agents, selective androgen receptor modulators (SARMs) represent a new class of potential drugs that can exhibit anabolic effects on muscle and bone with reduced side effects due to a tissue-selective mode of action. Besides possible medical applications, SARMs are used as performance-enhancing agents in sports. Therefore, they are prohibited by the World Anti-Doping Agency (WADA) in and out of competition. Since their inclusion into the WADA Prohibited List in 2008, there has been an increase in not only the number of adverse analytical findings, but also the total number of SARMs, making continuous research into SARMs an ongoing topic in the field of doping controls. 4-((2R,3R)-2-Ethyl-3-hydroxy-5-oxopyrrolidin-1-yl)-2-(trifluoromethyl)benzonitrile (SARM 2f) is a novel SARM candidate and is therefore of particular interest for sports drug testing. This study describes the synthesis of SARM 2f using a multi-step approach, followed by full characterization using liquid chromatography–high-resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance spectroscopy (NMR). To provide the first insights into its biotransformation in humans, SARM 2f was metabolized using human liver microsomes and the microsomal S9 fraction. A total of seven metabolites, including phase I and phase II metabolites, were found, of which three metabolites were chemically synthesized in order to confirm their structure. Those can be employed in testing procedures for routine doping controls, further improving anti-doping efforts.