Hydroxy levamisole and its phase II conjugates as potential indicators of levamisole doping in thoroughbred horses.

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.

Metabolic study of hypoxia-inducible factor stabilizers BAY 87-2243, MK-8617, and PT-2385 in equine liver microsomes for doping control.

A number of erythropoiesis stimulants are entering the final stage of clinical trials due to recent scientific progress in hypoxia-regulated erythropoiesis. Considering how erythropoiesis-stimulating compounds enhance the capacity of the organism to transport oxygen, they pose a great risk of being misused as performance enhancers. In this paper, we report the metabolic fate of three popular hypoxia-inducible factor-prolyl hydroxylase Inhibitors (HIF-PHI) compounds, namely, BAY 87-2243, MK-8617, and PT-2385 in equine liver microsomes using Q-Exactive high-resolution mass spectrometry. This study found 22 metabolites for BAY 87-2243 (19 phase I and three phase II), three metabolites for MK-8617 (all phase I), and five metabolites for PT-2385 (two phase I and three phase II). The major findings of the present study are as follows: (1) all three potential HIF-PHI drug candidates, namely, BAY 87-2243, MK-8617, and PT-2385 are susceptible to oxidation, producing their corresponding hydroxylated metabolites; (2) the ring dissociated metabolites were detected for BAY 87-2243 and PT-2385; (3) in the case of BAY 87-2243 and PT-2385, glucuronic acid conjugated metabolites were detected; and (4) none of the drugs produced sulfonic acid conjugated metabolites.

Characterization of growth hormone secretagogue small molecule ibutamoren (MK-0677) and its possible metabolites in thoroughbred horses for doping control.

RATIONALE: It is important to remember that performance-enhancing agents such as non-peptide growth hormone secretagogues present a significant risk of abuse. Ibutamoren (MK-0677) is a potent, long-acting, selective non-peptide growth hormone secretagogue that can be taken orally. METHODS: This study examines ibutamoren and its metabolites in thoroughbred horses after oral administration. Liquid chromatography/high-resolution mass spectrometry was used to determine the probable structures of the detected metabolites. RESULTS: In this study, 22 metabolites of ibutamoren were identified (17 phase I and 5 phase II). Oxidation of ibutamoren leads to hydroxylated metabolites (mono and di). The study also detected dissociated side chains (benzyl group and 2-amino-2-methylpropanamide) and hydrogenated metabolites. The glucuronic acid conjugated analogs of ibutamoren were detected during phase II of the study, but no sulfonic acid conjugated analogs were observed. The major metabolites can be detected up to 96 hours after a single dose, and ibutamoren can persist for up to 72 hours. CONCLUSIONS: These findings will aid in the detection of ibutamoren and the detection of its illegal use in competitive sports.

In vitro studies of hypoxia inducible factor-prolyl hydroxylase inhibitors daprodustat, desidustat, and vadadustat for equine doping control.

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.

Identification of Hypoxia-inducible factor (HIF) stabilizer roxadustat and its possible metabolites in thoroughbred horses for doping control.

Hypoxia-inducible factor (HIF) stabilizer belongs to a novel class of pharmacologically active substances, which are capable of inducing the endogenous erythropoietic system. The transcriptional activator HIF has been shown to significantly increase blood hemoglobin and is well set for the treatment of anemia resulting from chronic kidney disease. This research work reports a comprehensive study of the most popular HIF stabilizer roxadustat and its metabolites in thoroughbred horse urine after oral administration. The plausible structures of the detected metabolites were postulated using liquid chromatography-high-resolution mass spectrometry. Under the experimental condition 13 metabolites (7 phase I, 1 phase II, and 5 conjugates of phase I metabolism) were positively detected (M1-M13). The major phase I metabolites identified were formed by hydroxylation. Dealkylated and hydrolyzed phase I metabolites were also observed in this study. In phase II, a glucuronic acid conjugate of roxadustat was detected as the major metabolite. The sulfonic acid conjugates were observed to be formed from phase I metabolites. The characterized in vivo metabolites can potentially serve as target analytes for doping control analysis; hence, the result is an important tool for assessing its use and abuse in competitive sport.

Metabolic studies of hypoxia-inducible factor stabilisers IOX2, IOX3 and IOX4 (in vitro) for doping control.

The transcriptional activator hypoxia-inducible factor (HIF) is a vital arbitrator in the performance of cellular responses lacking oxygen supply in aerobic organisms. Because these compounds are capable of enhancing the organism's capacity for molecular oxygen transport, they possess great potential for abuse as a performance-enhancing agent in sports. A comprehensive study of the metabolic conversion of the most popular HIF stabilisers such as IOX2, IOX3 and IOX4 using equine liver microsomes (in vitro) is reported. The parents and their metabolites were identified and characterised by liquid chromatography-mass spectrometry in negative ionisation mode using a QExactive high-resolution mass spectrometer. Under the current experimental condition, a total of 10 metabolites for IOX2 (three phase I and seven phase II), nine metabolites for IOX3 (four phase I and five phase II) and five metabolites for IOX4 (three phase I and two phase II) were detected. The outcome of the present study is as follows: (1) all the three IOX candidates are prone to oxidation, results in subsequent monohydroxylated, and some dihydroxylated metabolites. (2) Besides oxidation, there is a possibility of hydrolysis and de-alkylation, which results in corresponding carboxylic acid and amide, respectively. (3) The glucuronide and sulphate conjugate of the parent drugs as well as the monohydroxylated analogues were observed in this study. The characterised in vitro metabolites can potentially serve as target analytes for doping control analysis.

Development and validation of a chiral LC-MS method for the enantiomeric resolution of (+) and (-)-medetomidine in equine plasma by using polysaccharide-based chiral stationary phases.

The detection and separation of medetomidine enantiomers from the complex biological matrices poses a great analytical challenge, especially in the field of forensic toxicology and pharmacology. Couple of researchers reported resolution of medetomidine using protein-based chiral columns, but the reported method is quiet challenging and tedious to be employed for routine analysis. This research paper reported a method that enables the enantio-separation of medetomidine by using polysaccharide cellulose chiral column. The use of chiralcel OJ-3R column was found to have the highest potential for successful chiral resolution. Ammonium hydrogen carbonate was the ideal buffer salt for chiral liquid chromatography (LC) with electrospray ionization (ESI)+ mass spectrometry (MS) detection for the successful separation and detection of racemic compound. The method was linear over the range of 0 to 20 ng/mL in equine plasma and the inter-day precisions of levomedetomidine, dexmedetomidine were 1.36% and 1.89%, respectively. The accuracy of levomedetomidine was in the range of 99.25% to 101.57% and that for dexmedetomidine was 99.17% to 100.99%. The limits of quantification for both isomers were 0.2 ng/mL. Recovery and matrix effect on the analytes were also evaluated. Under the optimized conditions, the validated method can be adapted for the identification and resolution of the medetomidine enantiomers in different matrices used for drug testing and analysis.