Katja Walpurgis’s research while affiliated with Deutsche Sporthochschule Köln and other places

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.

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) β‐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 β‐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.

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‐R 3 ‐IGF‐I, R 3 ‐IGF‐I, des 1–3 ‐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.

The authenticity of a doping control sample is a key element of sports drug testing programmes. Doping control sample manipulation by providing another individual's urine or blood (instead of the tested athlete's sample) has been observed in the past and is an unequivocal violation of the World Anti‐Doping Agency anti‐doping rules. To determine attempts of manipulations by sample swapping, the utility of a single nucleotide polymorphism (SNP)‐based sample authentication with a multi‐target SNP panel was assessed. The panel comprises detection assays for 44 different SNPs, 3 gender markers and 5 quality control markers for DNA‐profile determination. Sample analysis is based on a multiplex polymerase chain reaction step followed by a multiplex single base extension (SBE) reaction and subsequent SBE‐product detection by MALDI‐TOF MS. Panel performance was evaluated for urine and dried blood spot (DBS) samples. Urine (8 ml) and DBS (20 μl) test samples were reliably typed and matched to whole blood reference samples, while efficient typing of urine samples correlated with sample quality and input amounts. Robust profiling of urine doping control specimens was confirmed with an assay input of 12 ml. Samples can be processed in a high‐throughput format with an overall assay turnaround time of approximately 11 h. SNP‐based DNA typing via MALDI‐TOF MS thus represents a high throughput‐capable possibility for doping control sample authentication. SNP profiling of samples could offer the opportunity to complement existing steroid profile analytics to substantiate sample manipulations and to support quality control processes in high throughput routine settings.

The manipulation of blood and blood components in sports is prohibited at all times, and besides blood transfusions, also hemoglobin‐based oxygen carriers (HBOCs) can be employed to artificially improve the oxygen transport capacity of the blood. But while most drug candidates based on stabilized hemoglobin (Hb) were found to be characterized by serious side effects, the natural giant extracellular Hb from the marine invertebrate Arenicola marina (lugworm) could be another candidate for transfusion medicine and cheating athletes, as it was found to be well tolerated in preclinical animal studies. Within this research project, lugworm Hb was implemented into the existing doping control detection method for bovine HBOCs based on ultrafiltration, tryptic digestion, and liquid chromatography coupled with high‐resolution tandem mass spectrometry (LC‐HRMS/MS). For the mass spectrometric identification of lugworm Hb, two precursor–product ion pairs for a total of four tryptic peptides originating from subunits hbA2 (T 6 ), hbB1 (T 3 and T 6 ), and the linker chain (T 16 ) were employed. The modified approach was comprehensively characterized and found to allow for the specific and sensitive detection of lugworm Hb down to concentrations of 10 μg/mL from 50 μL of serum/plasma. Therefore, it can serve as confirmation procedure for lugworm Hb following visual or electrophoretic screening. Moreover, a proof‐of‐concept rat administration study was conducted, and the observed detection windows of at least 4 (dose: 200 mg/kg) and 8 h (dose: 600 mg/kg) suggest that the approach can be readily employed to efficiently test in‐competition doping control samples for the presence of the drug candidate.

Gene doping has been classified as a prohibited method by the World Anti-Doping Agency (WADA) and the International Olympic Committee (IOC) for over two decades. As gene therapeutic approaches improve and, concomitantly, safety concerns regarding clinical applications decline, apprehensions about their illicit use in elite sports continue to grow. Two products available via Internet-based providers and advertised as EPO-gene- and IGF1-gene-containing materials were analyzed for the presence of potential gene doping agents using a newly developed analytical approach, allowing for the detection of transgenic DNA corresponding to seven potential targets (EPO, FST, GH1, MSTN (Propeptide), IGF1, VEGFA, and VEGFD). Panel detection was based on a 20-plex polymerase chain reaction (PCR) followed by a single base extension (SBE) reaction and subsequent SBE product analyses via matrix-assisted time-of-flight laser desorption/ionization mass spectrometry (MALDI-TOF MS). Extracts of both products were found to contain transgenic EPO-DNA, while transgenic DNA for IGF-1 was not detected. The results were confirmed using SYBR Green qPCR with primer sets directed against EPO and IGF1 cDNA, and the CMV promotor sequence. In this case study, the detection of authentic (whilst low concentrated) transgenes, potentially intended for gene doping practices in readily available products, is reported for the first time.

Because of its influence on carbohydrate metabolism and, at the same time, anti-catabolic effects, the misuse of the peptide hormone insulin and its synthetic analogs is prohibited in sports at all times according to the regulations of the World Anti-Doping Agency (WADA). The biological effects of insulin and its analogs are mediated through binding to the insulin receptor, which was also found to be activated by different peptides structurally largely unrelated to insulin. Such insulin-mimetic peptides or selective-insulin receptor modulators (SIRMs) represent a novel class of potential performance-enhancing agents, which is currently not explicitly mentioned on the WADA Prohibited List. Within this research project, advanced solid-phase extraction (SPE) and liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS) were employed to develop a fast, reliable, and specific assay for the detection of the insulin-mimetic peptides S597 and S519 from plasma. Method validation demonstrated a detection limit of 0.5 ng/mL and successfully illustrated the applicability of the approach to routine sports drug testing programs. Moreover, sophisticated and comprehensive in vitro metabolism experiments were conducted, and several metabolic degradation products were identified, which will enhance the information generated from future analyses of doping control samples.

Capromorelin is a growth hormone secretagogue. Despite promising results to alleviate muscle-wasting in the elderly, it has not advanced further in human development. Subsequent studies demonstrated capromorelin's ability to increase food intake in animals, leading to approval in the United States and Europe as an appetite stimulant for cats (Elura) and dogs (Entyce). Capromorelin is prohibited in sports due to its ability to stimulate growth hormone production and enhance performance. However, given that its veterinary preparation is formulated as a highly concentrated solution (20 or 30 mg/mL) delivered orally, incidental ingestion or dermal absorption may result in an adverse analytical finding (AAF) by way of direct exposure during oral administration to a pet. An administration study was conducted by either oral or transdermal application of capromorelin solution to mimic the scenario of inadvertent exposure to the drug. Ingestion of 30 μg of capromorelin orally (equivalent to 1 μL of Entyce) resulted in detectable amounts of capromorelin in urine for up to 48 h after administration with a maximum urinary concentration of 7 ng/mL. Importantly, when applied directly to the skin on the hands in larger quantities mimicking a pet administration exposure scenario (30 mg or 1 mL of Entyce), capromorelin was also detected reaching a maximum urinary concentration of 0.7 ng/mL. Athletes and testing authorities should be aware of the risk of an AAF arising due to incidental exposure to veterinary preparations of capromorelin. To our knowledge, before 2022, no positive test for capromorelin had ever been reported.

Across species, skeletal muscle mass is negatively regulated by the TGF-β cytokine myostatin (MSTN). Inhibitors of this growth factor and its signaling pathways are therefore not only promising therapeutics for muscular diseases but also potential performance-enhancing agents in sports. Within this study, protein precipitation and liquid chromatography high-resolution tandem mass spectrometry (LC-HRMS/MS) were employed to develop a detection method for six novel MSTN inhibitory peptides derived from the regulatory MSTN propeptide and the natural MSTN inhibitor follistatin (FST) from doping control serum samples. The approach was comprehensively characterized and found to allow for a specific detection down to concentrations of 3-9 ng/mL. Moreover, several potential metabolites of the drug candidates referred to as DF-3, DF-25, and Peptide 7 were identified as valuable complementary analytical targets for doping control analytical assays. Overall, the acquired data pave the way for an implementation of MSTN inhibitory peptides into routine sports drug testing. Even though no drug candidate has obtained clinical approval yet, a proactive development of detection assays is of utmost importance to deter athletes from misusing such compounds, which are readily available for research purposes and on the black market.

For decades, blood testing has been an integral part of routine doping controls. The breadth of information contained in blood samples has become considerably more accessible for anti-doping purposes over the last 10 years through technological advancements regarding analytical instrumentation as well as enhanced sample collection systems. Particularly, microsampling of whole blood and serum, for instance as dried blood spots (DBS), has opened new avenues in sports drug testing and substantially increased the availability and cost-effectiveness of doping control specimens. Thus, microvolume blood specimens possess the potential to improve monitoring of blood hormone and drug levels, support evaluation of circulating drug concentrations in competition, and enhance the stability of labile markers and target analytes in blood passport analyses as well as peptide hormone and steroid ester detection. Further, the availability of the fraction of lysed erythrocytes for anti-doping purposes warrants additional investigation, considering the sequestering capability of red blood cells (RBCs) for certain substances, as a complementary approach in support of the clean sport.