Exploring methandienone metabolites generated via homogenized camel liver: Advancements for anti-doping applications through High Resolution-Liquid Chromatography Mass Spectrometry analysis.

RATIONALE: Anabolic steroids, also known as anabolic-androgenic steroids (AAS), encompass steroidal androgens such as testosterone, as well as synthetic counterparts with similar structures and effects. The misuse of AAS has increased over the years, leading to ethical and welfare concerns in sports. The World Anti-Doping Agency (WADA) and the International Federation for Equestrian Sports (FEI) have banned AAS in relevant sports. Methandienone is one of the most identified anabolic androgenic steroids in sports drug testing, Therefore, reliable detection methods are crucial for effective doping control and maintaining the integrity of the sports. METHODS: This study explores the use of homogenized camel liver for detecting methandienone metabolites in camels. The biotransformation pathways of methandienone in homogenized camel liver tissues are analyzed using Liquid Chromatography-High Resolution Mass Spectrometry (LC-HRMS) to identify and characterize the phase I and phase II metabolites. Chromatographic separation was achieved using a Thermo-Hypersil C18 column. RESULTS: The study has identified 11 methandienone metabolites (M1-M11), this includes 10 phase I and one phase II metabolite. A glucuronic acid conjugate of methandienone was observed in this study, but no sulfonic acid conjugations were found. The metabolites and their possible chemical structures, along with their fragmentation patterns are confirmed using MSMS (MS2) experiments in data-independent acquisition (DIA) mode. CONCLUSIONS: These findings serve as a vital tool for the rapid detection of methandienone, combating its illicit use in camel racing. Comprehensive screenings covering both the parent drug and its metabolites are recommended to improve detection accuracy and ensure regulatory compliance in sports doping. Future research should explore methandienone's metabolite profile in administered camel samples.

A randomized placebo controlled trial demonstrates the effect of dl-methylephedrine on brain functions is weaker than that of pseudoephedrine.

Intellectual drug doping in athletics by using stimulants that affect central nervous system functions has been diversified. Stimulants are regulated by the World Anti-Doping Agency according to their levels of urinary concentration. Positron emission tomography could evaluate how stimulants affect central nervous system functions. We aimed to evaluate the effect of stimulants on brain function by examining the difference in brain dopamine transporter occupancy by PET after administration of dl-methylephedrine or pseudoephedrine at the clinical maximum daily dose. Four PET scans without and with drug administration (placebo, dl-methylephedrine 150 mg and pseudoephedrine 240 mg) were performed. The concentrations of dl-methylephedrine and pseudoephedrine in plasma and urine were measured. DAT occupancies in the striatum with placebo, dl-methylephedrine and pseudoephedrine were calculated by PET images. The urinary concentration of dl-methylephedrine (12.7 µg/mL) exceeded the prohibited concentration (10 µg/mL), but the DAT occupancy with dl-methylephedrine (6.1%) did not differ (p = 0.92) from that with placebo (6.2%). By contrast, although the urinary concentration of pseudoephedrine (144.8 µg/mL) was below the prohibited concentration (150 µg/mL), DAT occupancy with pseudoephedrine was 18.4%, which was higher than that with placebo (p = 0.009). At the maximum clinical dose, dl-methylephedrine was shown to have weaker effects on brain function than pseudoephedrine.

Analysis of the polymeric mixture in hypoxen, using high-performance liquid chromatography coupled with high-resolution mass spectrometry.

RATIONALE: Hypoxen is a medication known for providing individuals with a "second wind," by lowering the threshold for muscle fatigue and enhancing the body's efficiency under challenging conditions. Athletes who have used this medication report enhanced training outcomes and increased physical endurance. It is crucial to emphasize that hypoxen is not categorized as a prohibited substance as yet and is thus assumed safe for use in competitive sports. However, the polymeric nature of hypoxen presents challenges in detection and identification. METHODS: To prevent the potential misuse of this substance as a doping agent, doping control laboratories must therefore develop a detection method. This study aimed to address this gap by developing a comprehensive detection method for the polymeric mixtures within hypoxen, employing liquid chromatography-electrospray ionization-mass spectrometry. RESULTS: Among the different columns tested, the Accucore and Syncronis HILIC columns demonstrated exceptional performance, yielding excellent separation with high-quality results. The study found that the hypoxen consisted of between 1 and 6 repetitions of 2,4-dihydroxyphenylene units. It was crucial to highlight that each unit contained multiple isomers due to the incorporation of the sulfonic acid group at both the -SH and -OH positions. CONCLUSIONS: The results of this study will significantly contribute to the precise identification of hypoxen use, thereby aiding in the scrutiny of its abuse in competitive sports.

Quantification of osilodrostat in horse urine using LC/ESI-HRMS to establish an elimination profile for doping control.

Aim: The use of osilodrostat, developed as a medication for Cushing's disease but categorized as an anabolic agent, is banned in horses by both the International Federation of Horseracing Authorities and the Fédération Equestre Internationale. For doping control purposes, elimination profiles of hydrolyzed osilodrostat in horse urine were established and the detectability of free forms of osilodrostat and its major metabolite, mono-hydroxylated osilodrostat (M1c), was investigated.Materials & methods: Post-administration urine samples obtained from a gelding and three mares were analyzed to establish the elimination profiles of osilodrostat using a validated method involving efficient enzymatic hydrolysis followed by LC/ESI-HRMS analysis.Results: Applying the validated quantification method with an LLOQ of 0.05 ng/ml, hydrolyzed osilodrostat could be quantified in post-administration urine samples from 48 to 72 h post-administration; by contrast, both hydrolyzed osilodrostat and M1c were detected up to 2 weeks. In addition, confirmatory analysis identified the presence of hydrolyzed osilodrostat for up to 72 h post-administration.Conclusion: For doping control purposes, we recommend monitoring both hydrolyzed M1c and osilodrostat because of the greater detectability of M1c and the availability of a reference material of osilodrostat, which is essential for confirmatory analysis.

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Two-dimensional high performance liquid chromatography purification of underivatized urinary prednisone and prednisolone for compound-specific stable carbon isotope analysis.

The gas chromatography-combustion isotope ratio mass spectrometry (GC/C/IRMS) confirmation procedure for prednisone (PS) and prednisolone (PSL) is still a great challenge for the doping control laboratory due to the many structurally similar steroids present in urinary matrices. This study aims to establish an innovative online two-dimensional high performance liquid chromatography (2D-HPLC) purification method for measuring the carbon isotope ratios (CIRs) and achieving the identification of the synthetic forms of these two endogenous anabolic androgenic steroids (EAASs). Initially, the one-dimensional chromatographic column was used to separate and purify endogenous reference compounds (ERCs), and the co-elution fluids containing PS and PSL were switched to a two-dimensional chromatographic column for further purification through an online transfer system. Then the purified compounds were analyzed using GC/C/IRMS after sample pretreatments. The results showed that the minimum detection concentration of PS and PSL reached 30 ng mL-1, and no isotope fractionation occurred during the entire collection and preparation process. This method has been validated with the WADA technical document and showed good sensitivity and selectivity, demonstrating its practical applicability for urine samples in doping control laboratories.

Motivations for Using Dietary Supplements in Elite Ice Hockey-Controlling Weight and Enhancing Performance.

Background: Excessive or improper use of dietary supplements (DSs) by athletes may cause adverse effects, such as impaired performance or failing a doping test, making it important for athletes to mitigate risk and make well-informed choices when using supplements. Methods: This study used focus group interviews to examine the attitudes, motivations, and practices related to DSs among male elite ice hockey players. Results: The players used a wide range of products, ranging from vitamins to multi-ingredient pre-workout supplements. Consuming DSs was considered as a practical and convenient way to ingest sufficient calories to gain or maintain the body weight and muscle mass needed to meet the physical requirements of the sport. The athletes demonstrated a lenient and ignorant attitude when acquiring and using supplements, with a non-critical trust in the guidance provided to them by the coach or physician. Having completed basic anti-doping education in the form of an e-learning program did not appear to result in taking a more careful approach to using DSs. Conclusions: Through their DS practices, elite ice hockey players may put themselves at risk for anti-doping rule violations. A comprehensive approach is needed when aiming to prevent unintentional doping in this athlete cohort.

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 offenses and unintentional contamination of urine by seminal fluid. To this end, 480 seminal fluids from nonathletes were analyzed 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 nonapproved proliferator-activated receptor delta agonist GW1516) in a total of 81 samples, with 91% of these suspected cases being verified by the confirmation method. In addition to 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.

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.

Hydrophilic/hydrophobic modified microchip for detecting multiple gene doping candidates using CRISPR-Cas12a and RPA.

With significant advancements in understanding gene functions and therapy, the potential misuse of gene technologies, particularly in the context of sports through gene doping (GD), has come to the forefront. This raises concerns regarding the need for point-of-care testing of various GD candidates to counter illicit practices in sports. However, current GD detection techniques, such as PCR, lack the portability required for on-site multiplexed detection. In this study, we introduce an integrated microfluidics-based chip for multiplexed gene doping detection, termed MGD-Chip. Through the strategic design of hydrophilic and hydrophobic channels, MGD-Chip enables the RPA and CRISPR-Cas12a assays to be sequentially performed on the device, ensuring minimal interference and cross-contamination. Six potential GD candidates were selected and successfully tested simultaneously on the platform within 1 h. Demonstrating exceptional specificity, the platform achieved a detection sensitivity of 0.1 nM for unamplified target plasmids and 1 aM for amplified ones. Validation using mouse models established by injecting IGFI and EPO transgenes confirmed the platform's efficacy in detecting gene doping in real samples. This technology, capable of detecting multiple targets using portable elements, holds promise for real-time GD detection at sports events, offering a rapid, highly sensitive, and user-friendly solution to uphold the integrity of sports competitions.

Laboratory medicine and sports: where are we now?

Laboratory medicine in sport and exercise has significantly developed during the last decades with the awareness that physical activity contributes to improved health status, and is present in monitoring both professional and recreational athletes. Training and competitions can modify concentrations of a variety of laboratory parameters, so the accurate laboratory data interpretation includes controlled and known preanalytical and analytical variables to prevent misleading interpretations. The paper represents a comprehensive summary of the lectures presented during the 35th Annual Symposium of the Croatian Society of Medical Biochemistry and Laboratory Medicine. It describes management of frequent sport injuries and sums up current knowledge of selected areas in laboratory medicine and sports including biological variation, changes in biochemical parameters and glycemic status. Additionally, the paper polemicizes sex hormone disorders in sports, encourages and comments research in recreational sports and laboratory medicine. In order to give the wider view, the connection of legal training protocols as well as monitoring prohibited substances in training is also considered through the eyes of laboratory medicine.

Magnetic molecularly imprinted polymer based on coordination for the determination of trace banned substance furosemide in human urine.

Furosemide (FUR), banned in sports events by the World Anti-Doping Agency, is a key target in drug tests, necessitating a pretreatment material capable of selectively, rapidly, and sufficiently separating/enriching analytes from complex matrices. Herein, a metal-mediated magnetic molecularly imprinted polymer (mMIP) was rationally designed and synthesized for the specific capture of FUR. The preparations involved the utilization of chromium (III) as the binding pivot, (3-aminopropyl)triethoxysilane as functional monomer, and Fe3O4 as core, all assembled via free radical polymerization. Both the morphologies and adsorptive properties of the mMIP were characterized using multiple methods. The resulting Cr(III)-mediated mMIP (ChM-mMIP) presented excellent selectivity and specificity toward FUR. Under optimized conditions, the adsorption capacity reached 128.50 mg/g within 10 min, and the imprinting factor was 10.41. Moreover, it was also successfully applied as a dispersive solid-phase extraction material, enabling the detection of FUR concentration as low as 20 ng/mL in human urine samples when coupled with a high-performance liquid chromatography/photodiode array. Overall, this study offers a valuable strategy for the development of novel recognition material.

Detection of sildenafil and its 9 metabolites in a post-race horse urine sample: A case report.

The use of prohibited substances in horse racing is a major concern that jeopardizes both the fairness of competitions and the health of horses. This problem can stem from the use of licensed drugs for animal health, as well as unlicensed substances. Horse doping laboratories monitor the potential use of these substances in racehorses within the framework of regulations set by the International Federation of Horse Racing Authority. In this context, sildenafil and its major metabolite n-desmethyl sildenafil were detected in a post-race horse urine sample sent to the Pendik Veterinary Control Institute Doping Control Laboratory through a screening analysis performed with Liquid Chromatography Triple Quadrupole Mass Spectrometry. These results were confirmed by Q Exactive Orbitrap Mass Spectrometry and follow-up analyses were performed. As a result of these analyses; simultaneous detection of 9 metabolites in horse urine was reported, two of them for the first time. In addition, the pioneer and comprehensive data resulting from this study provide preliminary data for future studies and anti-doping analyses.

Investigating the detection of the novel doping­relevant peptide kisspeptin­10 in urine using liquid chromatography high­resolution mass spectrometry.

Kisspeptin-10 is a peptide hormone capable of increasing circulating follicle-stimulating hormone, luteinizing hormone and testosterone levels in humans. Clinically, these effects suggest its use as a treatment for infertility. However, its testosterone-increasing effect indicates potential misuse in sports. As such, it is included in the 2024 World Anti-Doping Agency Prohibited List. This work describes the successful validation of an initial testing procedure (screening) and a confirmation procedure for kisspeptin-10 in urine using liquid chromatography-mass spectrometry. Additionally, kisspeptin-10 was incubated in human serum to mimic endogenous metabolism to improve method sensitivity, as previous research had demonstrated a rapid elimination time of only 30 min after injection (in rats). Four metabolites, corresponding to peptide fragments y9, y8, y7 and y5, were found and added to the ITP in full scan mode. A degradation product discovered during early experimentation was found to probably be caused by oxidation of the tryptophan residue into a kynurenine residue. Further research should elucidate the kinetic parameters of the reaction to improve product stability. Using the validated confirmation procedure, a black-market vial of kisspeptin-10 was analysed. The product contained no unexpected impurities, although it appeared to have undergone more degradation than the purchased reference standard.

Knowing the minimal detectable dose can facilitate the interpretation of a hair test result: Case example with chlortalidone.

In case of an adverse analytical finding, a low (estimate) urine concentration can be the consequence of 2 very different situations: it can be the tail end of a drug voluntarily consumed to enhance athletic performance, even by microdosing (which is not effective for all drugs), or it can be the result of a contamination, irrespective of its source. For numerous doping agents, a hair test can allow discriminating doping from contamination based on the measured concentration or even the absence of the target drug. Given hair produces incremental concentrations, its analysis offers the possibility of establishing a pattern of drug use and thus, verifying self-reported histories of exposure. In order to provide a retrospective calendar of drug use, segmental analysis of the hair strand can be performed. In doping, the usual practice is to test the substance in short segments, such as 1 cm to avoid drug dilution when using larger segments. During the last months, seven athletes have returned an adverse analytical finding for the diuretic chlortalidone, with reported urine concentrations in the range 20 to 50 ng/mL. All these athletes submitted, via their legal team, their hair for establishing a pattern of exposure. Results were always consistent with incidental contamination (hair concentration lower than 5 pg/mg), although the source of contamination was never identified. The interpretation of the findings was established in the light of the limited literature, including hair tests after microdosing and therapeutic use.

Psychosocial predictors of doping intentions and use in sport and exercise: a systematic review and meta-analysis.

OBJECTIVE: To conduct a meta-analytic review of psychosocial predictors of doping intention, doping use and inadvertent doping in sport and exercise settings. DESIGN: Systematic review and meta-analysis. DATA SOURCES: Scopus, Medline, Embase, PsychINFO, CINAHL Plus, ProQuest Dissertations/Theses and Open Grey. ELIGIBILITY CRITERIA: Studies (of any design) that measured the outcome variables of doping intention, doping use and/or inadvertent doping and at least one psychosocial determinant of those three variables. RESULTS: We included studies from 25 experiments (N=13 586) and 186 observational samples (N=3 09 130). Experimental groups reported lower doping intentions (g=-0.21, 95% CI (-0.31 to -0.12)) and doping use (g=-0.08, 95% CI (-0.14 to -0.03), but not inadvertent doping (g=-0.70, 95% CI (-1.95 to 0.55)), relative to comparators. For observational studies, protective factors were inversely associated with doping intentions (z=-0.28, 95% CI -0.31 to -0.24), doping use (z=-0.09, 95% CI -0.13 to to -0.05) and inadvertent doping (z=-0.19, 95% CI -0.32 to -0.06). Risk factors were positively associated with doping intentions (z=0.29, 95% CI 0.26 to 0.32) and use (z=0.17, 95% CI 0.15 to 0.19), but not inadvertent doping (z=0.08, 95% CI -0.06 to 0.22). Risk factors for both doping intentions and use included prodoping norms and attitudes, supplement use, body dissatisfaction and ill-being. Protective factors for both doping intentions and use included self-efficacy and positive morality. CONCLUSION: This study identified several protective and risk factors for doping intention and use that may be viable intervention targets for antidoping programmes. Protective factors were negatively associated with inadvertent doping; however, the empirical volume is limited to draw firm conclusions.

Non-Targeted Detection of Synthetic Oligonucleotides in Equine Serum Using Liquid Chromatography-High-Resolution Mass Spectrometry.

There is great concern in equine sport over the potential use of pharmaceutical agents capable of editing the genome or modifying the expression of gene products. Synthetic oligonucleotides are short, single-stranded polynucleotides that represent a class of agents capable of modifying gene expression products with a high potential for abuse in horseracing. As these substances are not covered by most routine anti-doping analytical approaches, they represent an entire class of compounds that are not readily detectable. The nucleotide sequence for each oligonucleotide is highly specific, which makes targeted analysis for these agents problematic. Accordingly, we have developed a non-targeted approach to detect the presence of specific product ions that are not naturally present in ribonucleic acids. Briefly, serum samples were extracted using solid-phase extraction with a mixed-mode cartridge following the disruption of protein interactions to isolate the oligonucleotides. Following the elution and concentration steps, chromatographic separation was achieved utilizing reversed-phase liquid chromatography. Following an introduction to a Thermo Q Exactive HF mass spectrometer using electrospray ionization, analytes were detected utilizing a combination of full-scan, parallel reaction monitoring and all ion fragmentation scan modes. The limits of detection were determined along with the accuracy, precision, stability, recovery, and matrix effects using a representative 13mer oligonucleotide. Following method optimization using the 13mer oligonucleotide, the method was applied to successfully detect the presence of specific product ions in three unique oligonucleotide sequences targeting equine-specific transcripts.