Comprehensive metabolic investigation of dopamine reuptake inhibitor HDMP-28 in equine liver microsomes and Cunninghamella elegans for doping control.

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.

Doping control approach: Identification of equine in vitro metabolites of voxelotor (GBT440), a hemoglobin S polymerization inhibitor.

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.

Electrospray ionization mass spectrometry adduct formation by mobile phase additives: A case study using nitrile functional groups containing selective androgen receptor modulators.

RATIONALE: The formation of mass adducts is common during electrospray ionization mass spectrometry (ESI-MS). However, the mechanism that leads to adduct formation is poorly understood and difficult to control. Multiplication of mass adducts at once will adversely impact the sensitivity of mass analysis and cause misinterpretation of the level of detection. Prior studies on selective androgen receptor modulators (SARMs) revealed an immense mass adduct formation in both positive and negative ESI modes. METHODS: In this study, additives in the mobile phases are investigated as a potential means of controlling mass adduct formation in various SARMs. RESULTS: The first evidence of chloride adduct formation when SARMs are detected via ESI-MS has been reported in this research. A series of mobile phase combinations were tested to achieve the optimal condition for HPLC-MS. A comparison was also made between adduct formation on various grades of water used for preparing the mobile phase. A validation study using equine urine and plasma was also conducted to assess the suitability of the developed method. CONCLUSION: The results of this study will allow for a more accurate identification of SARMs, which will make it easier to investigate their illicit use in horse racing.