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.

Determination of urinary deanol-N-oxide excretion profiles after ingestion of nutritional supplements containing deanol.

2-(Dimethylamino)ethan-1-ol (Deanol) is a widely produced chemical used by both industry and consumers in a variety of applications. Meclofenoxate, a stimulant classified on the World Anti-Doping Agency Prohibited List, metabolizes into deanol and, presumably, its main metabolite deanol-N-oxide. Hence, using liquid chromatography-tandem mass spectrometry, a quantitative detection method for deanol-N-oxide in urine was developed.

Comparison of in vitro approaches for predicting the metabolism of the selective androgen receptor modulator RAD140.

The identification of metabolites allows for the expansion of possible targets for anti-doping analysis. Especially for novel substances such as selective androgen receptor modulators (SARMs), information on metabolic fate is scarce. Novel approaches such as the organ on a chip technology may provide a metabolic profile that resembles human in vivo samples more closely than approaches that rely on human liver fractions only.

Dried blood spots for doping controls-Development of a comprehensive initial testing procedure with fully automated sample preparation.

Currently, primarily urine, whole blood and serum samples are analyzed for doping-relevant substances in professional sports, but recently dried blood spots (DBS) have been introduced as complementary matrix, offering advantageous features, e.g. a minimally invasive sampling procedure.

Mass spectrometry in sports drug testing-Analytical approaches and the athletes' exposome.

Test methods in anti-doping, most of which rely on the most modern mass spectrometric instrumentation, undergo continuous optimization in order to accommodate growing demands as to comprehensiveness, sensitivity, retrospectivity, cost-effectiveness, turnaround times, etc. While developing and improving analytical approaches is vital for appropriate sports drug testing programs, the combination of today's excellent analytical potential and the inevitable exposure of humans to complex environmental factors, specifically chemicals and drugs at the lowest levels, has necessitated dedicated research, particularly into the elite athlete's exposome. Being subjected to routine doping controls, athletes frequently undergo blood and/or urine tests for a plethora of drugs, chemicals, corresponding metabolic products, and various biomarkers.

Human In Vivo Metabolism and Elimination Behavior of Micro-Dosed Selective Androgen Receptor Modulator RAD140 for Doping Control Purposes.

RAD140 is a selective androgen receptor modulator which has been abused in sporting competitions. Its use is prohibited by the World Anti-Doping Agency (WADA) for athletes at all times. In addition to its illicit use, adverse analytical findings of RAD140 in doping control samples might result from other scenarios, e.

Investigations into the elimination profiles and metabolite ratios of micro-dosed selective androgen receptor modulator LGD-4033 for doping control purposes.

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).

Organ-on-a-chip: Determine feasibility of a human liver microphysiological model to assess long-term steroid metabolites in sports drug testing.

A fundamental challenge in preventive doping research is the study of metabolic pathways of substances banned in sport. However, the pharmacological predictions obtained by conventional in vitro or in vivo animal studies are occasionally of limited transferability to humans according to an inability of in vitro models to mimic higher order system physiology or due to various species-specific differences using animal models. A more recently established technology for simulating human physiology is the "organ-on-a-chip" principle.

Meldonium residues in milk: A possible scenario for inadvertent doping in sports?

Lately, the veterinary drug Emidonol® has been discussed as a possible scenario for inadvertent doping in sports. Emidonol® is approved for use in livestock breeding, exhibiting antihypoxic and weak sedative effects. The veterinary drug rapidly dissociates into meldonium, a substance prohibited in sports, and is excreted largely in its unchanged form into urine.

Mass spectrometric characterization of urinary hydrafinil metabolites for routine doping control purposes.

Little information on the human metabolism and urinary elimination of hydrafinil (9-fluorenol) exists. In order to support preventive anti-doping activities concerning compounds such as hydrafinil, a pilot elimination study was conducted with three healthy male volunteers receiving a single oral dose of 50 mg of hydrafinil. Urine samples were collected prior to and up to 72-h post-administration and were subjected to both gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry, which allowed for the identification of the intact drug as well as Phase I and Phase II metabolites, primarily hydroxylated and/or glucuronidated or sulfo-conjugated hydrafinil.

Stanozolol-N-glucuronide metabolites in human urine samples as suitable targets in terms of routine anti-doping analysis.

The exogenous anabolic-androgenic steroid (AAS) stanozolol stays one of the most detected substances in professional sports. Its detection is a fundamental part of doping analysis, and the analysis of this steroid has been intensively investigated for a long time. This contribution to the detection of stanozolol doping describes for the first time the unambiguous proof for the existence of 17-epistanozolol-1'N-glucuronide and 17-epistanozolol-2'N-glucuronide in stanozolol-positive human urine samples due to the access to high-quality reference standards.

Elimination profiles of microdosed ostarine mimicking contaminated products ingestion.

The possibility of nutritional supplement contamination with minute amounts of the selective androgen receptor modulator (SARM) ostarine has become a major concern for athletes and result managing authorities. In case of an adverse analytical finding (AAF), affected athletes need to provide conclusive information, demonstrating that the test result originates from a contamination scenario rather than doping. The aim of this research project was to study the elimination profiles of microdosed ostarine and characterize the time-dependent urinary excretion of the drug and selected metabolites.

Implementation of the HIF activator IOX-2 in routine doping controls - Pilot study data.

Early in 2020, racehorse doping cases revolved around the hypoxia-inducible factor (HIF) activator IOX-2. While the composition of IOX-2 has also been known and monitored in human doping controls for several years, the testing capability of routine sports drug testing methods was revisited for this newly surfaced doping agent. IOX-2 and the analytically well-established HIF activator roxadustat (FG-4592) share identical precursor/product ion pairs, enabling their co-detection in existing initial testing procedures in routine doping controls for the intact unconjugated analytes.

Paper spray mass spectrometry - A potential complementary technique for the detection of polar compounds in sports drug testing.

In this proof-of-concept study, paper spray mass spectrometry was investigated as a high-throughput and fully automated technique for the initial testing of particularly polar compounds that are prohibited in sports. The technique allows the ionization of analytes from complex sample matrices such as blood and urine when spotted onto a paper strip. By minimizing sample preparation and omitting chromatographic separation, paper spray mass spectrometry benefits from considerable cost- and time-savings compared with conventional high performance liquid chromatography/tandem mass spectrometry, especially in cases where conventional reversed-phase liquid chromatography offers limited applicability.

Pilot study on the effects of intravesical oxybutynin hydrochloride instillations on the validity of doping control urine samples.

According to class M2.1 of the World Anti-Doping Agency (WADA) Prohibited List, the manipulation of doping control urine samples to alter their integrity and validity is prohibited both in- and out-of-competition. However, some paraplegic athletes with an overactive bladder need to be regularly treated with anti-cholinergic and anti-spasmodic drugs such as oxybutynin, which are often administered intravesically to reduce the substantial side effects observed after oral application.

Recent improvements in sports drug testing concerning the initial testing for peptidic drugs (< 2 kDa) - sample preparation, mass spectrometric detection, and data review.

In this work, a novel initial testing assay based on liquid chromatography-mass spectrometry is presented, enabling the detection of peptidic drugs and drug candidates (< 2 kDa) prohibited in sports. The assay covers representatives and metabolites of gonadotropin releasing hormone and its analogs (GnRHs), growth hormone secretagogues (GHS), growth hormone releasing peptides (GHRPs), and the Vasopressin-analog Desmopressin. The general objective of this work was to reduce sample preparation efforts to a minimum while preserving highest possible sensitivity and specificity of the assay, demonstrating limits of detection between 50 and 200 pg/mL.

The atypical excretion profile of meldonium: Comparison of urinary detection windows after single- and multiple-dose application in healthy volunteers.

Following a one-year monitoring program providing unequivocal analytical evidence for a high prevalence in international elite sports, meldonium has been included in the World Anti-Doping Agency's (WADA) list of prohibited substances that came into effect on 1 January 2016. Despite of the polar and hydrophilic nature of the molecule, an unusual long detection window was observed in pilot elimination studies. Consequently, in the present study, urinary excretion profiles after single-dose (5 volunteers, 1×500mg) and multiple-dose oral application (5 volunteers; 2×500mg/day for 6days) were determined in order to facilitate the result management concerning meldonium findings in doping controls.

Simplifying and expanding analytical capabilities for various classes of doping agents by means of direct urine injection high performance liquid chromatography high resolution/high accuracy mass spectrometry.

So far, in sports drug testing compounds of different classes are processed and measured using different screening procedures. The constantly increasing number of samples in doping analysis, as well as the large number of substances with doping related, pharmacological effects require the development of even more powerful assays than those already employed in sports drug testing, indispensably with reduced sample preparation procedures. The analysis of native urine samples after direct injection provides a promising analytical approach, which thereby possesses a broad applicability to many different compounds and their metabolites, without a time-consuming sample preparation.

Detection of the diuretic hydrochlorothiazide in a doping control urine sample as the result of a non-steroidal anti-inflammatory drug (NSAID) tablet contamination.

Hydrochlorothiazide (HCTZ, 6-chloro-3,4-dihydro-2H-1,2,4-benzothiadiazine-7-sulfonamide-1,1-dioxide) belongs to the class of diuretic agents that represent one of today's cornerstones of the treatment of hypertensive patients. In addition to its clinical relevance, HCTZ is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA) at all times and has frequently been detected in sports drug testing urine samples worldwide since its ban was introduced in 1988. Despite these facts, the adverse analytical finding concerning HCTZ in an in-competition routine doping control sample collected in December 2014 was further investigated, particularly motivated by the comparably low urinary concentration of the drug accounting for approximately 5ng/mL.

Mitragynine (Kratom) - monitoring in sports drug testing.

In 2014, mitragynine (Kratom) was placed on the Monitoring List of the World Anti-Doping Agency to gain information of its current use in professional sports. Therefore, analytical strategies in sports drug testing are presented and the first Kratom case in professional sports is described. It is outlined that thorough monitoring by anti-doping laboratories is of utmost importance to obtain data on Kratom's misuse and to protect athletes from potential health hazards.

Simplifying and expanding the screening for peptides <2 kDa by direct urine injection, liquid chromatography, and ion mobility mass spectrometry.

The analysis of low-molecular-mass peptides in doping controls has become a mandatory aspect in sports drug testing and, thus, the number of samples that has to be tested for these analytes has been steadily increasing. Several peptides <2 kDa with performance-enhancing properties are covered by the list of prohibited substances of the World Anti-Doping Agency including Desmopressin, LH-RH, Buserelin, Triptorelin, Leuprolide, GHRP-1, GHRP-2, GHRP-3, GHRP-4, GHRP-5,GHRP-6, Alexamorelin, Ipamorelin, Hexarelin, ARA-290, AOD-9604, TB-500 and Anamorelin. With the presented method employing direct urine injection into a liquid chromatograph followed by ion-mobility time-of-flight mass spectrometry, a facile, specific and sensitive assay for the aforementioned peptidic compounds is provided.

"Dilute-and-inject" multi-target screening assay for highly polar doping agents using hydrophilic interaction liquid chromatography high resolution/high accuracy mass spectrometry for sports drug testing.

In the field of LC-MS, reversed phase liquid chromatography is the predominant method of choice for the separation of prohibited substances from various classes in sports drug testing. However, highly polar and charged compounds still represent a challenging task in liquid chromatography due to their difficult chromatographic behavior using reversed phase materials. A very promising approach for the separation of hydrophilic compounds is hydrophilic interaction liquid chromatography (HILIC).

Mildronate (Meldonium) in professional sports - monitoring doping control urine samples using hydrophilic interaction liquid chromatography - high resolution/high accuracy mass spectrometry.

To date, substances such as Mildronate (Meldonium) are not on the radar of anti-doping laboratories as the compound is not explicitly classified as prohibited. However, the anti-ischemic drug Mildronate demonstrates an increase in endurance performance of athletes, improved rehabilitation after exercise, protection against stress, and enhanced activations of central nervous system (CNS) functions. In the present study, the existing evidence of Mildronate's usage in sport, which is arguably not (exclusively) based on medicinal reasons, is corroborated by unequivocal analytical data allowing the estimation of the prevalence and extent of misuse in professional sports.

Screening and confirmation of myo-inositol trispyrophosphate (ITPP) in human urine by hydrophilic interaction liquid chromatography high resolution / high accuracy mass spectrometry for doping control purposes.

Myo-inositol trispyrophosphate (ITPP) is a novel allosteric effector of haemoglobin with high permeation selectivity across the red blood cell plasma membrane. Due to its potential to reduce the oxygen affinity of haemoglobin, ITPP application results in an enhanced oxygen release in hypoxic tissues. Therefore, ITPP is being examined for the treatment of numerous illnesses that involve hypoxia, such as cardiovascular diseases, cancer or Alzheimer's disease.