Use of in vitro technologies to study phase II conjugation in equine sports drug surveillance.

Background: Within equine drug surveillance, there is significant interest in analyzing intact phase II conjugates of drugs in urine, but progress has been limited by a lack of reference material.

Method: In this study, in vitro techniques using equine liver fractions were employed to produce glucuronide and sulfate conjugates of stanozolol, 16β-hydroxystanozolol and nandrolone, the glucuronide conjugate of morphine and the glutathione metabolite of chlordinitrobenzene for the first time in equine sports drug surveillance.

Results: The glucuronide conjugate of the synthetic progestagen altrenogest was also produced in vitro, removing the requirement for sample hydrolysis during routine urinalyses.

The use of in vitro technologies coupled with high resolution accurate mass LC-MS for studying drug metabolism in equine drug surveillance.

The detection of drug abuse in horseracing often requires knowledge of drug metabolism, especially if urine is the matrix of choice. In this study, equine liver/lung microsomes/S9 tissue fractions were used to study the phase I metabolism of eight drugs of relevance to equine drug surveillance (acepromazine, azaperone, celecoxib, fentanyl, fluphenazine, mepivacaine, methylphenidate and tripelennamine). In vitro samples were analyzed qualitatively alongside samples originating from in vivo administrations using LC-MS on a high resolution accurate mass Thermo Orbitrap Discovery instrument and by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap.

The application of in vitro technologies to study the metabolism of the androgenic/anabolic steroid stanozolol in the equine.

In this study, the use of equine liver/lung microsomes and S9 tissue fractions were used to study the metabolism of the androgenic/anabolic steroid stanozolol as an example of the potential of in vitro technologies in sports drug surveillance. In vitro incubates were analysed qualitatively alongside urine samples originating from in vivo stanozolol administrations using LC-MS on a high-resolution accurate mass Thermo Orbitrap Discovery instrument, by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap and by GC-MS/MS on an Agilent 7000A. Using high-resolution accurate mass full scan analysis on the Orbitrap, equine liver microsome and S9 in vitro fractions were found to generate all the major phase-1 metabolites observed following in vivo administrations.

Epidermal (infundibular) and dermoid cysts in the dorsal midline of a three-year-old thoroughbred-cross gelding.

Epidermal (infundibular) and dermoid cysts are unusual in the horse in contrast with other species. The diagnosis and treatment of six lesions in the dorsal midline of a three-year-old Thoroughbred-cross gelding is described. The lesions were believed to be congenital and presented asymptomatically but required attention because five of them were in the saddle region, thus preventing ridden exercise.