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BackgroundAminorex, (RS)-5- Phenyl-4,5-dihydro-1,3-oxazol-2-amine, is an amphetamine-like anorectic and in the United States a Drug Enforcement Administration [DEA] Schedule 1 controlled substance. Aminorex in horse urine is usually present as a metabolite of Levamisole, an equine anthelmintic and immune stimulant. Recently, Aminorex identifications have been reported in horse urine with no history or evidence of Levamisole administration. Analysis of the urine samples suggested a botanical source, directing attention to the Brassicaceae plant family, with their contained GlucoBarbarin and Barbarin as possible sources of Aminorex. Since horsepersons face up to a 1 year suspension and a $10,000.00 fine for an Aminorex identification, the existence of natural sources of Aminorex precursors in equine feedstuffs is of importance to both individual horsepersons and the industry worldwide.ResultsTesting the hypothesis that Brassicaceae plants could give rise to Aminorex identifications in equine urine we botanically identified and harvested flowering Kentucky Barbarea vulgaris, (“Yellow Rocket”) in May 2018 in Kentucky and administered the plant orally to two horses. Analysis of post-administration urine samples yielded Aminorex, showing that consumption of Kentucky Barbarea vulgaris can give rise to Aminorex identifications in equine urine.ConclusionsAminorex has been identified in post administration urine samples from horses fed freshly harvested flowering Kentucky Barbarea vulgaris, colloquially “Yellow Rocket”. These identifications are consistent with occasional low concentration identifications of Aminorex in equine samples submitted for drug testing. The source of these Aminorex identifications is believed to be the chemically related Barbarin, found as its precursor GlucoBarbarin in Kentucky Barbarea vulgaris and related Brassicaceae plants worldwide.

Aminorex is a US DEA Schedule 1 controlled substance occasionally detected in racing horses. A number of aminorex identifications in sport horses were thought to have been caused by exposure to plant sources of aminorex. Glucobarbarin, found in plants of the Brassicaceae family, has been suggested as a potential proximate chemi

Synthesis, purification, and chemical characterization of 20-dihydro-6methylprednisone, an isomeric metabolite of methylprednisolone in the horse, for use as an analytical standardTo the Editor Methylprednisolone acetate, pregna-1,4-diene-3,20-dione, 21-(acetoxy)-11,17-dihydro-6-methyl-,(6α,11β)-, is a synthetic glucocorticoid pro-drug widely used in equine medicine. [1] It is recognized as a therapeutic medication by many equine organizations, including the American Association of Equine Practitioners (AAEP), the Racing Medication and Testing Consortium (RMTC), the Association of Racing Commissioners International (ARCI), and numerous racing and other regulatory authorities in the United States and elsewhere. [2,3] As such, methylprednisolone is widely used on horses in training, usually as the long-acting Depo-medrol formulation administered by intra-articular or intra-muscular injection. With respect to racing and other equine performance events, its use as a controlled therapeutic medication on horses in training is regulated by the testing of plasma and/or urine, but usually by application of a quantitative regulatory threshold in plasma. For example, the RMTC lists methylprednisolone as a 'controlled therapeutic medication' and sets forth that a 100 pg/mL methylprednisolone threshold in plasma or serum corresponds to a seven-day 'restricted administration time' (withdrawal time) following intra-articular administration of a '100 mg total in one intra-articular space'. [4] Approximately 15 metabolites of methylprednisolone have been reported in mammalian systems, though several of them have not been fully characterized due to a lack of authentic reference standards. [5][6][7][8][9] Of these metabolites, at least three isobaric compounds have been identified in rat urine [6] and four in human urine. [8] It was observed, after reviewing our liquid chromatography-tandem mass spectrometry (LC-MS/MS) drug screening data and the relevant scientific literature, that standard equine analytical procedures commonly used to screen for or analyze methylprednisolone may occasionally have difficulty distinguishing between parent methylprednisolone and isobaric equine metabolites. One isomeric compound which was found in plasma extracts in conjunction with methylprednisolone (Figure 1), after examining mass spectral data, was thought to be 20-dihydro-6-methylprednisone (DHMP), an isomeric metabolite of methylprednisolone previously reported though not unequivocally identified in equine urine. [5] This metabolite has a close structural relationship to methylprednisolone (Figure 2), and has, presuming a similar chromatographic retention time, the potential to interfere with the forensic identification and quantitation of methylprednisolone in equine racing samples. Given these possibilities, an authentic research quality reference standard of DHMP is required to unequivocally determine the identity and quantity of this metabolite present in post-administration equine samples, and to insure that the analytical method is struc...

Based on structural similarities and equine administration experiments, Barbarin, 5-phenyl-2-oxazolidinethione from Brassicaceae plants, is a possible source of equine urinary identifications of aminorex, (R,S)-5-phenyl-4,5-dihydro-1,3-oxazol-2-amine, an amphetamine-related US Drug Enforcement Administration (DEA) controlled substance considered illegal in sport horses. We now report the synthesis and certification of d 5 -barbarin to facilitate research on the relationship between plant barbarin and such aminorex identifications. D 5 -barbarin synthesis commenced with production of d 5 -2-oxo-2-phenylacetaldehyde oxime (d 5 -oxime) from d 5acetophenone via butylnitrite in an ethoxide/ethanol solution. This d 5 -oxime was then reduced with lithium aluminum hydride (LiAlH 4 ) to produce the corresponding d 5 -2-amino-1-phenylethan-1-ol (d 5 -phenylethanolamine). Final ring closure of the d 5phenylethanolamine was performed by the addition of carbon disulfide (CS 2 ) with pyridine. The reaction product was purified by recrystallization and presented as a stable white crystalline powder. Proton NMR spectroscopy revealed a triplet at 5.88 ppm for one proton, a double doublet at 3.71 ppm for one proton, and double doublet at 4.11 ppm for one proton, confirming d 5 -barbarin as the product. Further characterization by high resolution mass spectrometry supports the successful synthesis of d 5 -barbarin. Purity of the recrystallized product was ascertained by High Performance Liquid Chromatography (HPLC) to be greater than 98%. Together, we have developed the synthesis and full characterization of d 5 -barbarin for use as an internal standard in barbarin-related and equine forensic research.