Metabolism and excretion of the benzodiazepine analogue etizolam in the horse

• Published in: Drug testing and analysis 2021-03, Vol.13 (3), p.583-594
• Main Authors: Johnson, Erin, Heemst, Jasper, Benavides, Jeshurun, Gray, Bobby
• Format: Article
• Language: English
• Subjects: Drugs; equine; etizolam; excretion; Horse racing; Horses; in vitro; Metabolism; Metabolites; Urine
• ISSN: 1942-7603; 1942-7611
• DOI: 10.1002/dta.2967

Etizolam is a benzodiazepine analogue that is approved for use in Japan, Italy and India but has recently appeared as a nonapproved product on the illicit drug market in Europe and North America. Etizolam was identified in a crystalline material seized at a Kentucky racetrack, raising concerns that this drug may have been used in racing. The aim of this study was to characterize the metabolism and excretion of etizolam in horses to generate information on its disposition and to incorporate the correct urinary and serum target analytes into anti‐doping screening procedures. Etizolam was administered both intravenous and orally at a dose of 0.1 mg/kg of body weight to three horses using a two‐way crossover design. Pre‐administration and post‐administration serum and urine samples were collected and experiments conducted to identify potential metabolites in these samples. Additionally, in vitro metabolism studies using horse liver S9 were undertaken to complement the in vivo metabolism studies. Numerous metabolites were id1entified in both serum and urine in additional to parent drug, with α‐hydroxy‐etizolam producing the most abundant analytical signal (in terms of signal intensity and duration of detection) of the identified metabolites in both matrices. Therefore, α‐hydroxy‐etizolam is considered to be the most appropriate analyte for detection for anti‐doping purposes. Analytical methods were developed and validated and then applied to post‐administration samples to generate concentrations of etizolam and its major metabolites in serum and urine, resulting in excretion profiles that can be used to guide approaches to detecting the use of the drug. The in vitro and in vivo metabolism of etizolam in the thoroughbred horse is investigated and discussed. The excretion of etizolam and metabolites in serum and urine following both oral and IV administration is determined using validated methods and the results applied to animal sport doping control.