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.