Long-Term Excretion of Roxadustat in Urine.

Roxadustat (FG-4592), an orally active hypoxia-inducible factor prolyl hydroxylase stabilizer, has been shown to enhance erythropoiesis by increasing endogenous erythropoietin. It is indicated for the treatment of anemia and chronic kidney disease and is approved for clinical use in several countries, including the European Union, Japan and others. Due to its reasonably anticipated performance-enhancing effect in athletes, roxadustat is prohibited for use in sports at all times. A few cases of adverse analytical findings in routine doping controls have been reported worldwide, some of which were claimed to be the result of contaminated dietary supplements. The present study offers new data demonstrating the long-term excretion pattern of roxadustat. Even after a single-dose administration, roxadustat can remain detectable in urine for 8 months, albeit at very low concentrations (<10 pg/mL). Following three times a week treatment with 70 to 100 mg of roxadustat, the drug was still detectable in the urine of anemic patients for between 9 and 18 months after treatment was discontinued. Lastly, an athlete who admitted use of roxadustat for almost a year (50 mg 3 to 5 times a week) has now tested positive multiple times over the course of 15 months (the first test being 12 months after the drug was discontinued), with an estimated concentration of roxadustat between 3 and 8 pg/mL. Altogether, these findings indicate the unusually prolonged terminal excretion kinetics of roxadustat, a property that testing authorities should consider in their results management process.

Improving the Determination of Carbon Isotope Ratios of Endogenous Steroids Found in Human Serum.

The determination of serum concentrations of testosterone (T) and 4-androstenedione (A4) was implemented into the steroidal module of the Athlete Biological Passport in 2023. Monitoring T, A4, and the ratio of T/A4 in a longitudinal manner enables the detection of the misuse of low-dose T administrations especially in female athletes, whereas urinary markers of the steroid profile may not be influenced significantly. In contrast to the urinary steroid profile, knowledge on confounding factors regarding serum concentrations of T and A4 is yet comparably scarce, and corroborating exogenous sources of the target analytes by isotope ratio mass spectrometry (IRMS) is desirable. In a recent study, it was demonstrated that carbon isotope ratios (CIRs) of serum steroids can be determined if analyte concentrations permit. The therein-employed method utilized 2D-GC/IRMS, and only a limited number of potential endogenous reference compounds were available. The here-presented approach uses complementary analyte purification strategies, addressing previous limitations. A high-performance liquid chromatography cleanup was developed and fully validated for serum steroids in order to enable all doping control laboratories to potentially implement this method alongside existing protocols for urinary steroids. Besides the already-investigated endogenous steroids cholesterol, dehydroepiandrosterone sulfate, androsterone sulfate, and epiandrosterone sulfate, two additional steroids were included in the test menu, namely, pregnenolone sulfate and 5-androstene-3ß,17ß-diol sulfate. Serum steroid concentrations down to 25 ng/mL were found to allow robust CIR determinations, and a reference population encompassing 124 male and female athlete samples was investigated to enable the calculation of population-based thresholds for all relevant steroid combinations.

Identification of human metabolites of fast skeletal troponin activators Tirasemtiv and Reldesemtiv for doping control purposes.

The fast skeletal troponin activators (FSTAs) Reldesemtiv and Tirasemtiv were developed for patients suffering from neuro-degenerative diseases of the motor nervous system, e.g. amyotrophic lateral sclerosis (ALS). The drug candidates can increase the sensitivity of troponin C to calcium by selectively activating the troponin complex resulting in increased skeletal muscle contraction. Although the development of the drug candidates is currently discontinued because of missed end points in phase III clinical studies with patients with ALS, phase I clinical trials showed an increase in muscle contraction force in healthy humans. This effect could be abused by athletes to enhance performance in sports. As the substances are listed on the 2024 edition of the World Anti-Doping Agency's Prohibited List, the aim of this study was to identify and characterize metabolites of Reldesemtiv and Tirasemtiv to ensure their reliable identification in doping control analyses. The biotransformation of the drug candidates was studied in vitro using pooled human liver microsomes and 3D cultivated human hepatic cells of the cell line HepaRG, yielding a total of 11 metabolites of Reldesemtiv and eight of Tirasemtiv. In addition, a human elimination study was conducted to investigate the metabolism and elimination profile of Tirasemtiv and Reldesemtiv in vivo, suggesting the N-glucuronide of Tirasemtiv and hydroxylated 3-fluoro-2-(3-fluoro-1-methylcyclobutyl)pyridine as well as its glucuronide as suitable target analytes for routine doping controls. Applying a validating HPLC-MS/MS method, optimized to detect Reldesemtiv and Tirasemtiv in human urine, microdosing (50 µg) of each substance was traceable for 24-72 h.

A comprehensive gas chromatography electron ionization high resolution mass spectrometry study of a new steroidal selective androgen receptor modulator (SARM) compound S42.

The synthetic 20-keto-steroid S42 (1) demonstrated selective androgen receptor modulator (SARM) properties in preclinical studies and, consequently, received growing attention also in the context of sports drug testing programs. Fundamental understanding of the behavior of S42 (1) and of relevant derivatives in gas chromatography-electron ionization MS experiments at high resolution (GC-EI-HRMS) is indispensable to develop a reliable qualitative and quantitative doping control method for S42 (1) and its metabolites in body fluid matrices. We present important fundamental mechanistic data on the EI fragmentation behavior of S42 (1) and of silyl ether derivatives as well as of stable isotope-labelled reference material.

Investigations into the concentrations and metabolite profiles of doping agents and antidepressants in human seminal fluid using liquid chromatography-mass spectrometry.

Exogenous substances, including drugs and chemicals, can transfer into human seminal fluid and influence male fertility and reproduction. In addition, substances relevant in the context of sports drug testing programs, can be transferred into the urine of a female athlete (after unprotected sexual intercourse) and trigger a so-called Adverse Analytical Finding. Here, the question arises as to whether it is possible to distinguish analytically between intentional doping offences and unintentional contamination of urine by seminal fluid. To this end, 480 seminal fluids from non-athletes were analysed to identify concentration ranges and metabolite profiles of therapeutic drugs that are also classified as doping agents. Therefore, a screening procedure was developed using liquid chromatography connected to a triple quadrupole mass spectrometer, and suspect samples (i.e. samples indicating the presence of relevant compounds) were further subjected to liquid chromatography-high-resolution accurate mass (tandem) mass spectrometry. The screening method yielded 90 findings (including aromatase inhibitors, selective estrogen receptor modulators, diuretics, stimulants, glucocorticoids, beta-blockers, antidepressants, and the non-approved PPARδ agonist GW1516) in a total of 81 samples, with 91 % of these suspected cases being verified by the confirmation method. Besides the intact drug, phase-I and -II metabolites were also occasionally observed in the seminal fluid. This study demonstrated that various drugs including those categorized as doping agents partition into seminal fluid. Monitoring substances and metabolites may contribute to a better understanding of the distribution and metabolism of exogenous substances in seminal fluid that may be responsible for the impairment of male fertility. Significance Statement This study demonstrates that doping agents as well as clinically relevant substances are transferred/eliminated into seminal fluid to a substantial extent and that knowledge about drug levels (and potential consequences for the male fertility and female exposure) is limited. The herein generated new dataset provides new insights into an important and yet little explored area of drug deposition and elimination, and hereby a basis for the assessment of contamination cases by seminal fluid in sports drug testing.

Longitudinal evaluation of laboratory results and method precision in worldwide erythropoietin external quality assessments.

Introduction: This study presents a longitudinal analysis of external quality assessment (EQA) results for erythropoietin (EPO) determinations conducted between 2017 and 2022 with a continuously increasing number of participating laboratories. The aim of this work was to evaluate participant performance and methodological aspects. Methods: In each of the eleven EQA surveys, a blinded sample set of lyophilized human serum containing one sample with lower EPO concentrations (L) and one with higher EPO concentrations (H) was sent to the participating laboratories. Results: A total of 1,256 measurements were included. The median (interquartile range) fraction of participants not meeting the criteria of acceptance set at 20% around the robust mean of the respective survey was 9.5% (6.1%-10.7%) (sample L) and 9.1% (5.8%-11.8%) (sample H) but lacked a clear trend in the observed period. Some surveys exhibited unusually high interlaboratory variation, suggesting interfering components in the EQA samples. Different immunological methods and reagent manufacturers also showed variability in measurement outcomes to some extent. Conclusion: These findings highlight the need for continuous quality assessment in EPO measurements to ensure patient safety and identify areas for further research and investigation.

Detection of the GH analog somatrogon in sports drug testing: Immunological approaches and LC-HRMS/MS.

Due to the presumed lipolytic and anabolic properties, the misuse of human growth hormone (hGH) and its synthetic analogs in sports is prohibited both in- and out-of-competition. Within this research project, the detectability of somatrogon, a recombinant fusion glycoprotein of 22 kDa hGH and the C-terminal peptide (CTP) of the human chorionic gonadotropin (hCG) ß-subunit, with current WADA-approved doping control assays for hGH and hCG was investigated. For that purpose, cross-reactivity tests and a somatrogon administration study were conducted, and only "Kit 2" of the GH isoform differential immunoassays proved applicable to the detection of somatrogon administration in serum. In urine, the immunoassay specific for total hCG yielded presumptively positive findings for several post-administration samples, which can probably be attributed to the presence of an immunoreactive fragment of the hCG ß-subunit. As the detectability of somatrogon with these approaches was found to be limited, a highly specific detection assay (LOD: 10 ng/mL) for the drug in serum samples was developed by using affinity purification with GH receptor (GHR)-conjugated magnetic beads, proteolytic digestion, and liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS). Following optimization, the approach was comprehensively characterized, and authentic post-administration serum samples were successfully analyzed as proof-of-concept, indicating a detection window of at least 96 h. Consequently, the presented method can be employed to confirm the presence of somatrogon in serum samples, where only "Kit 2" of the currently used immunoassay kits yielded an abnormally high Rec/Pit ratio.

Acute exercise activates the AHR in peripheral blood mononuclear cells in an intensity-dependent manner.

The kynurenine pathway (KP) of tryptophan degradation generates several metabolites such as kynurenine (KYN) or kynurenic acid (KA) that serve as endogenous ligands of the aryl hydrocarbon receptor (AHR). Due to its distinct biological roles particularly modulating the immune system, the AHR is a current therapeutic target across different inflammation-related diseases. Here, we show an acute exercise-induced increase in AHR ligand availability on a systemic level and a kynurenine pathway activation in peripheral blood mononuclear cells (PBMCs). Concurrently, the AHR is activated in PBMCs following acute exercise. Exercise effects on both, kynurenic acid and AHR activation in PBMCs were greater in response to high-intensity interval exercise (HIIE) (50 min, six 3-min intervals at 90% V̇o2peak, and 3-min intervals at 50% V̇o2peak in between) compared with workload-matched moderate-intensity continuous exercise (MICE) (50 min). In conclusion, these data indicate a novel mechanistic link in how exercise modulates the immune system through the kynurenine pathway-AHR axis, potentially underlying exercise-induced benefits in various chronic diseases.NEW & NOTEWORTHY The findings of this study show that acute endurance exercise activates a receptor that has been described to integrate metabolic signals into the immune system. We uncover a potential mechanistic link on how exercise modulates the immune system through the kynurenine pathway-AHR axis, potentially underlying exercise-induced benefits in various chronic diseases and of relevance for other cell types.

Development of an LC-HRMS/MS Method for Quantifying Steroids and Thyroid Hormones in Capillary Blood: A Potential Tool for Assessing Relative Energy Deficiency in Sport (RED-S).

Relative energy deficiency in sport (RED-S) is a condition that arises from persistent low energy availability (LEA), which affects the hypothalamic-pituitary axis and results in alterations of several hormones in both male and female athletes. As frequent blood hormone status determinations using venipuncture are rare in sports practice, microsampling offers promising possibilities for preventing and assessing RED-S. Therefore, this study aimed at developing a liquid chromatography-high-resolution tandem mass spectrometry (LC-HRMS/MS) method for quantifying relevant steroids and thyroid hormones in 30 µL of capillary blood obtained using Mitra® devices with volumetric absorptive microsampling technology (VAMS®). The results of the study showed that all validation criteria were met, including a storage stability of more than 28 days in a frozen state (-18 °C) and 14 days at room temperature (20 °C). The validated assay provided precise (<12%) and accurate (<13%) results for all the target analytes. Furthermore, as a proof of concept, autonomously collected VAMS® samples from 50 female and male, healthy, active adults were analyzed. The sensitivity of all analytes was adequate to quantify the decreased hormone concentrations in the RED-S state, as all authentic samples could be measured accordingly. These findings suggest that self-collected VAMS® samples offer a practical opportunity for regular hormone measurements in athletes and can be used for early RED-S assessment and progress monitoring during RED-S recovery.

Urinary metabolites indicative of the administration of hypoxen monitored by liquid chromatography-high resolution/accurate mass tandem mass spectrometry.

Hypoxen, a poly(dihydroxyphenylene) thiosulfonate-based drug, has been investigated concerning its effect on mitochondrial respiration and the utilization of lactate, especially in the context of strenuous exercise. Since 2023, patterns of use regarding hypoxen amongst the athletic population are monitored by the World Anti-Doping Agency (WADA) and its accredited anti-doping laboratories, necessitating information on suitable urinary markers indicative of the administration of hypoxen. In this exploratory study, urine samples collected post-administration of 1.5 and 2.0 g of hypoxen were analyzed by means of liquid chromatography-high resolution/high mass accuracy (tandem) mass spectrometry, which allowed for the identification of eight analytes that were plausibly attributable to metabolites of hypoxen. The identified species were assigned to the unconjugated species of S-(2,2',5,5'-tetrahydroxy-[1,1'-biphenyl]-3-yl) sulfurothioate and its glucuronide and additional tentatively identified analytes comprising a mercaptobenzene core structure. Including the identified markers into routine doping control analytical procedures enabled the detection of hypoxen use in athletes' doping control samples, thus contributing relevant information to WADA's monitoring program.

Detection of sgRNA via SHERLOCK as Potential CRISPR Related Gene Doping Control Strategy.

Apprehensions about gene doping have grown consistently due to advancements in gene engineering techniques, particularly with the emergence of clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas)-based tools. These tools not only provide unprecedented possibilities for illicit performance enhancement by athletes but also offer new avenues for the detection of gene doping through biosensing of nucleic acids. Hence, pursuing on a previous study, an analytical method based on reverse transcriptase-recombinase polymerase amplification (RT-RPA) and subsequent qualitative nucleic acid detection by means of Specific High Sensitive Enzymatic Reporter UnLOCKing (SHERLOCK) was optimized for the direct detection of sgRNA associated with Streptococcus pyogenes in serum. Detection device, assay parameters, and sample handling were adjusted, to overcome previously determined assay limitations. The conducted method characterization confirmed the methods' specificity and increased detection sensitivity from 100 pM to 1 fM sgRNA in 100 µL of serum. Furthermore, reanalysis of in vivo mouse administration samples collected in a previous proof-of-concept study was conducted with successful identification of sgRNA in all anticipated postadministration samples within the 24-h collection period. Those findings support the applicability of the refined analytical procedure for the detection of illegal doping attempts via ribonucleoprotein-based CRISPR/Cas application through sgRNA identification, offering a new potential doping control strategy for CRISPR related gene doping.

Establishing Personalized Blood Protein Reference Ranges Using Noninvasive Microsampling and Targeted Proteomics: Implications for Antidoping Strategies.

To prevent doping practices in sports, the World Anti-Doping Agency implemented the Athlete Biological Passport (ABP) program, monitoring biological variables over time to indirectly reveal the effects of doping rather than detect the doping substance or the method itself. In the context of this program, a highly multiplexed mass spectrometry-based proteomics assay for 319 peptides corresponding to 250 proteins was developed, including proteins associated with blood-doping practices. "Baseline" expression profiles of these potential biomarkers in capillary blood (dried blood spots (DBS)) were established using multiple reaction monitoring (MRM). Combining DBS microsampling with highly multiplexed MRM assays is the best-suited technology to enhance the effectiveness of the ABP program, as it represents a cost-effective and robust alternative analytical method with high specificity and selectivity of targets in the attomole range. DBS data were collected from 10 healthy athlete volunteers over a period of 140 days (28 time points per participant). These comprehensive findings provide a personalized targeted blood proteome "fingerprint" showcasing that the targeted proteome is unique to an individual and likely comparable to a DNA fingerprint. The results can serve as a baseline for future studies investigating doping-related perturbations.

Employing 11-Ketotestosterone as a Target Analyte for Adrenosterone (11OXO) Administration in Doping Controls.

Adrenosterone (Androst-4-ene-3,11,17-trione, 11OXO) is forbidden in sports according to the Prohibited List of the World Anti-Doping Agency. The administration of 11OXO may be detected by monitoring the urinary concentrations of its main human metabolites 11ß-hydroxy-androsterone and 11ß-hydroxy-etiocholanolone. Preliminary urinary concentration and concentration ratio thresholds have been established for sports drug testing purposes, but adaptations are desirable as the suggested limits would result in numerous suspicious findings due to naturally elevated concentrations and ratios. Recently, the metabolism of 11-oxo-testosterone (KT) was investigated in the context of anti-doping research, resulting in a preliminary urinary concentration threshold and a confirmation procedure based on the determination of carbon isotope ratios (CIRs). Gas chromatography coupled to isotope ratio mass spectrometry was employed to investigate the CIRs of selected steroids. As KT is also a metabolite of 11OXO, the developed protocols for KT have been tested to elucidate their potential to detect the administration of 11OXO after a single oral dose of 100 mg. In order to further improve the analytical approach, the threshold for urinary concentrations of KT was re-investigated by employing a reference population of n = 5232 routine doping control samples. Quantification of urinary steroids was conducted by employing gas chromatography coupled to triple quadrupole mass spectrometry. Derived from these, a subset of n = 106 samples showing elevated concentrations of KT was investigated regarding their CIRs. By means of this, potentially positive samples due to the illicit administration of 11OXO or KT could be excluded, and the calculation of reference population-derived thresholds for the concentrations and CIR of KT was possible. Based on the results, the urinary concentration threshold for KT is suggested to be established at 130 ng/mL.

Longitudinal analysis of external quality assessment of immunoassay-based steroid hormone measurement indicates potential for improvement in standardization.

As hormonal disorders are linked to several diseases, the accurate quantitation of steroid hormone levels in serum is crucial in order to provide patients with a reliable diagnosis. Mass spectrometry-based methods are regarded as having the highest level of specificity and sensitivity. However, immunoassays are more commonly used in routine diagnostics to measure steroid levels as they are more cost effective and straightforward to conduct. This study analyzes the external quality assessment results for the measurement of testosterone, progesterone and 17ß-estradiol in serum using immunoassays between early 2020 and May 2022. As reference measurement procedures are available for the three steroid hormones, the manufacturer-specific biases were normalized to the reference measurement values. The manufacturer-specific coefficients of variation were predominantly inconspicuous, below 20% for the three hormones when outliers are disregarded, however there were large differences between the various manufacturer collectives. For some collectives, the median bias to the respective reference measurement value was repeatedly greater than ±35%, which is the acceptance limit defined by the German Medical Association. In the case of testosterone and progesterone determination, some collectives tended to consistently over- or underestimate analyte concentrations compared to the reference measurement value, however, for 17ß-estradiol determination, both positive and negative biases were observed. This insufficient level of accuracy suggests that cross-reactivity continues to be a fundamental challenge when antibody detection is used to quantify steroids with a high structural similarity. Distinct improvements in standardization are required to provide accurate analysis and thus, reliable clinical interpretations. The increased accuracy of the AX immunoassay for testosterone measurement, as observed in the INSTAND EQAs between 2020 and 2022, could be the result of a recalibration of the assay and raises hope for further improvement of standardization of immunoassay-based steroid hormone analyses in the coming years.

Recent advances in mass spectrometry for the detection of doping.

INTRODUCTION: The analysis of doping control samples is preferably performed by mass spectrometry, because obtained results meet the highest analytical standards and ensure an impressive degree of reliability. The advancement in mass spectrometry and all its associated technologies thus allow for continuous improvements in doping control analysis. AREAS COVERED: Modern mass spectrometric systems have reached a status of increased sensitivity, robustness, and specificity within the last decade. The improved sensitivity in particular has, on the other hand, also led to the detection of drug residues that were attributable to scenarios where the prohibited substances were not administered consciously but rather by the unconscious ingestion of or exposure to contaminated products. These scenarios and their doubtless clarification represent a great challenge. Here, too, modern MS systems and their applications can provide good insights in the interpretation of dose-related metabolism of prohibited substances. In addition to the development of new instruments itself, software-assisted analysis of the sometimes highly complex data is playing an increasingly important role and facilitating the work of doping control laboratories. EXPERT OPINION: The sensitive analysis and evaluation of a higher number of samples in a shorter time is made possible by the ongoing developments in mass spectrometry.

Chromatographic-mass spectrometric analysis of peptidic analytes (2-10 kDa) in doping control urine samples.

Peptides with a molecular mass between 2 and 10 kDa that are prohibited in elite sports usually require dedicated sample preparation and mass spectrometric detection that commonly cannot be combined with other (lower molecular mass) substances. In most instances, the physicochemical differences are too significant to allow for a generic analytical procedure. A simplification of established and comparably complex analytical approaches is therefore desirable and has been accomplished in the context of this study. With urine samples representing still the most frequently collected doping control specimens, efficient extraction of peptidic analytes from this matrix was a major goal of this method, as demonstrated for the included compounds such as insulins (human, lispro, aspart, glulisine, tresiba, glargine metabolite, bovine insulin, porcine insulin), growth hormone-releasing hormones (sermorelin, CJC-1295, tesamorelin) incl. their respective metabolites, insulin-like-growth factors (long-R3 -IGF-I, R3 -IGF-I, des1-3 -IGF-I), synacthen, gonadorelin and mechano growth factors (human MGF, MGF-Goldspink). Sample preparation and detection are controlled by five internal standards, covering all five included peptide drug categories. Nearly all requirements of the recent technical documents from the World Anti-Doping Agency (WADA) considering their minimum required performance levels (MRPL) are fulfilled, and the method was validated for its utilisation as initial testing procedure in doping controls. Finally, the approach was applied to authentic post-administration study urine samples (for insulins and gonadorelin) in order to provide proof of principle.