Diisopropylamine (DIPA), a hydrophilic chemical compound, is used as an intravenous antihypertensive agent. DIPA is prohibited for use in the horse racing industry due to its performance enhancing effects. A cyano (CN) hydrophilic interaction liquid chromatography (HILIC) column was used for the separation of DIPA from its metabolite. Ammonium formate was added to the mobile phase to increase the ionization of the basic substance. The metabolite was identified as an N-oxidized metabolite of DIPA, which eluted earlier than the parent drug and was less polar on the HILIC column. The main finding of the study was the identification of a metabolite with a mass shift of 15.9944. The in vitro experiment showed that the metabolite was produced as a result of N-oxidation processes, mainly mediated by flavin-containing monooxygenase (FMO). Methimazole was used to inhibit the FMO enzyme-mediated N-oxidation metabolism and metabolite production in a concentration-dependent manner. The metabolite was confirmed to be present in an actual horse urine sample that tested positive for DIPA. This study demonstrated that the metabolite could be screened using in vitro samples and their presence corresponded to a positive result in actual samples. This metabolite screening could therefore find application as a flexible way to test for new and modified banned substances in the racing industry.
Keywords: Diisopropylamine; Flavin-containing monooxygenases; Horse liver microsomes; Hydrophilic interaction liquid chromatography.
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