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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. [ABSTRACT FROM AUTHOR]

Rationale: Phosphodiesterase 4 (PDE4) inhibitors are a newer class of drugs that induce bronchodilation and have anti‐inflammatory effects, making them susceptible to misuse as performance enhancers in competitive sports. Methods: This study explores the metabolic conversion of PDE4 inhibitor ibudilast in thoroughbred horses after oral administration and in vitro using equine liver microsomes and Cunninghamella elegans. A liquid chromatography‐high resolution mass spectrometry method was used to postulate the plausible structures of the detected metabolites. Results: A total of 20 in vivo metabolites were identified under experimental conditions, including 12 Phase I and 8 Phase II conjugated metabolites. Phase I metabolites were predominantly formed through hydroxylation (mono‐, di‐, and tri‐hydroxylation). Demethylated metabolites were also identified during this investigation. Additionally, the research detected Phase II metabolites conjugated with glucuronic and sulfonic acids. Conclusions: The data presented here can assist in detecting the PDE4 inhibitor ibudilast and uncover its illicit use in competitive sports. [ABSTRACT FROM AUTHOR]

A dopamine reuptake inhibitor is a type of medication or substance that works by blocking the reuptake of dopamine in the brain. Dopamine reuptake inhibitors offer multiple effects, including increased alertness, improved mood, and therapeutic potential for conditions like depression, ADHD, and Parkinson's disease. HDMP‐28, or methylnaphthidate, is a potent synthetic stimulant from the phenyltropane class. It surpasses methylphenidate in both dopamine reuptake inhibition and half‐life. As a dopamine reuptake inhibitor, it boosts dopamine levels by hindering reuptake into nerve cells, resulting in heightened stimulation and increased energy. In order to comprehensively address both the tangible and potential repercussions of the unauthorized utilization of the aforementioned substance in sports, it is imperative to establish analytical methodologies for the identification of the parent drug and its primary metabolites. Additionally, a comprehensive analysis of the metabolic characteristics of HDMP‐28 in both human and animal subjects has yet to be published. This study explores the metabolic conversion of HDMP‐28 mediated by equine liver microsomes and Cunninghamella elegans. An extraction and detection method was developed, optimized, and validated for doping assessment in equine urine and plasma. Liquid chromatography–high‐resolution mass spectrometry was employed to determine metabolite structures. The study identified 31 (22 phase I and 9 phase II) metabolites of HDMP‐28, including hydroxylated, hydrogenated, and hydrolyzed analogs. Glucuronic acid‐conjugated metabolites were also detected. This manuscript describes metabolites based on the in vitro studies, which might not be the same in vivo. These findings aid in the detection and understanding of the illicit use of HDMP‐28 in equestrian sports. [ABSTRACT FROM AUTHOR]

Rationale: Sickle cell disease, a debilitating genetic disorder affecting numerous newborns globally, has historically received limited attention in pharmaceutical research. However, recent years have witnessed a notable shift, with the Food and Drug Administration approving three innovative disease‐modifying medications. Voxelotor, also known as GBT440, is a promising compound that effectively prevents sickling, providing a safe approach to alleviate chronic hemolytic anemia in sickle cell disease. It is a novel, orally bioavailable small molecule that inhibits hemoglobin S polymerization by enhancing oxygen affinity to hemoglobin. The investigation demonstrated that voxelotor led to an unintended elevation of hemoglobin levels in healthy individuals by increasing serum erythropoietin levels. Methods: Voxelotor and its metabolites in an in vitro setting utilizing equine liver microsomes were discussed. Plausible structures of the identified metabolites were inferred through the application of liquid chromatography in conjunction with high‐resolution mass spectrometry. Results: Under the experimental conditions, a total of 31 metabolites were detected, including 16 phase I metabolites, two phase II metabolites, and 13 conjugates of phase I metabolites. The principal phase I metabolites were generated through processes such as hydroxylation, reduction, and dissociation. The presence of glucuronide and sulfate conjugates of the parent drug were also observed, along with hydroxylated, reduced, and dissociated analogs. Conclusions: The data acquired will accelerate the identification of voxelotor and related compounds, aiding in the detection of their illicit use in competitive sports. It is crucial to emphasize that the metabolites detailed in this manuscript were identified through in vitro experiments and their detection in an in vivo study may not be guaranteed. [ABSTRACT FROM AUTHOR]

Nonsteroidal selective androgen receptor modulators (SARMs) are a novel class of compounds that have not yet been clinically approved; however, they appear to have a better anabolic/androgenic ratio than steroids and cause slighter side effects. Sports drug testing laboratories are required to maintain continuously updated doping control analytical methods in light of the widespread misuse of SARMs in elite and amateur sports. This paper describes the metabolic conversion of SARM GSK2881078 in thoroughbred horses following oral administration and in vitro with equine liver microsomes. A liquid chromatography–high‐resolution mass spectrometry method was used to postulate the plausible structures of the detected metabolites. A total of five (M1–M5) in vivo metabolites and six (M1–M6) in vitro metabolites were detected under experimental conditions. Phase I metabolites mainly result from hydroxylation. Methoxylated and side‐chain dissociated metabolites were also detected. Neither sulfonic acid nor glucuronic acid conjugated metabolites were observed in this study. Data reported here could aid in the detection of nonsteroidal SARM GSK2881078 and reveal its illicit use in competitive sports. [ABSTRACT FROM AUTHOR]

Rationale: According to previous research, aminorex is the major metabolite of levamisole; however, in the screening of levamisole‐positive racehorse urine and plasma samples, aminorex could only be detected in trace amounts or not at all. In forensic laboratories, hydroxy levamisole and its phase II conjugates make it easier to confirm levamisole misuse and to differentiate between the abuse of levamisole and aminorex. This study aimed to identify the major levamisole metabolites that can be detected along with the parent drug. Methods: The study describes levamisole and its metabolites in thoroughbred horses following oral administration and in vitro with equine liver microsomes. The plausible structures of the detected metabolites were postulated using liquid chromatography combined with high‐resolution mass spectrometry. Results: Under the experimental conditions 26 metabolites (17 phase I, 2 phase II, and 7 conjugates of phase I metabolites) were detected (M1–M26). The major phase I metabolites identified were formed by hydroxylation. In phase II, the glucuronic acid conjugates of levamisole and hydroxy levamisole were detected as the major metabolites. In plasma, the parent drug and major metabolites are detectable for up to eight days, while in urine, they are detectable for up to twenty days. Levamisole levels rapidly increased at 45 min following administration, then declined gradually until detectable levels were reached approximately 8 days after administration, according to a pharmacokinetics study. Conclusions: A prolonged elimination profile and relatively high concentration of hydroxy metabolites suggest that the detection of hydroxy metabolites is imperative for investigating levamisole doping in horses. [ABSTRACT FROM AUTHOR]

Performance‐enhancing substances and methods have become a serious problem in competitive sports. The hypoxia‐inducible factor (HIF) stabilizers can enhance the organism's capacity for molecular oxygen transport and are likely to be abused as performance‐enhancing agents in sports. This paper describes the metabolic conversion of the popular hypoxia inducible factor‐prolyl hydroxylase (HIF‐PH) inhibitors, namely, daprodustat, desidustat, and vadadustat using equine liver microsomes, determined on a QExactive high‐resolution mass spectrometer. During this study, a total of 10 metabolites for daprodustat (all are Phase I), 10 metabolites for desidustat (five each for Phase I and Phase II), and 15 metabolites for vadadustat (six for Phase I and nine for Phase II) were detected. The important findings of the current research are as follows: (1) All the three HIF‐PH inhibitor drug candidates are prone to oxidation, which results in corresponding hydroxylated metabolites; (2) in desidustat, hydrolysis and dissociation of oxime linkage also observed; (3) the glucuronic acid conjugate (except daprodustat) of the parent drugs as well as the monohydroxylated analogs were observed; (4) sulfonic acid conjugated metabolites were observed only for vadadustat. [ABSTRACT FROM AUTHOR]

ACP‐105 is a novel nonsteroidal selective androgen receptor modulator (SARM) with a tissue‐specific agonist effect and does not have side effects associated with the use of common androgens. This research reports a comprehensive study for the detection of ACP‐105 and its metabolites in racehorses after oral administration (in vivo) and postulating its structures using mass spectrometric techniques. To obtain the metabolic profile of ACP‐105, a selective and reliable LC‐MS/MS method was developed. The chemical structures of the metabolites were determined based on their fragmentation pattern, accurate mass, and retention time. Under the current experimental condition, a total of 19 metabolites were detected in ACP‐105 drug administered equine urine samples. The study results suggest the following: (1) ACP‐105 is prone to oxidation, which gives corresponding monohydroxylated, dihydroxylated, and trihydroxylated metabolites; (2) along with oxidation, there is a possibility of elimination of water molecule (dehydration) from the third position of the tropine moiety, resulting in the dehydrated analogs of corresponding monohydroxylated, dihydroxylated, and trihydroxylated metabolites; (3) from the study on the metabolites using LC‐MS/MS, it is clear that the fragmentation pattern is identical and a great number of fragment ions are common in all the metabolites and the parent drug. (4) The ACP‐105 and its metabolites were detected for up to 72 h; thus, the result is a valuable tool for evaluating its use and/or misuse in sport. [ABSTRACT FROM AUTHOR]

The phosphodiesterase 4 (PDE4) inhibitors constitute a relatively modern class of medications that are known for inducing bronchodilation and exhibiting anti-inflammatory properties within the body. Due to these properties, there is concern regarding their potential misuse as performance-enhancing substances in competitive sports. This study delves into the metabolic conversion of roflumilast in thoroughbred horses following oral administration and in vitro experimentation using equine liver microsomes and Cunninghamella elegans. High-performance liquid chromatography coupled with a Q Exactive Orbitrap mass spectrometer (HPLC-HRMS) was employed for analysis. The investigation identified 10 metabolites of roflumilast, including six phase I and four phase II metabolites from in vivo studies, and 11 metabolites from in vitro studies, consisting of eight phase I and three phase II metabolites. The identified biotransformation products encompassed processes such as hydroxylation, chlorine substitution, methylation, N-oxide formation, and even the dissociation of methylenecyclopropane and difluoromethane. Furthermore, the study identified three glucuronic acid and one sulfonic acid conjugated phase II metabolites of the investigated drug candidate. The aforementioned findings contribute to the detection and comprehension of the unauthorized utilization of roflumilast in equestrian sports., (© 2024 John Wiley & Sons Ltd.)