Aminorex identified in horse urine following consumption of Barbarea vulgaris; a preliminary report

Abstract

Background: Aminorex, (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.

Results: Testing 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.

Conclusions: Aminorex 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.

Keywords: Aminorex; Barbarea vulgaris; Brassicaceae; Drug testing; Horse; Urine.

Fig. 1

Conversion of GlucoBarbarin (via Myrosinase) to Barbarin and Aminorex structure

Fig. 2

Yellow Rocket, Barbarea vulgaris growing in Ithaca, NY, May 21th 2018

Fig. 3

a: Chromatogram of a post-8-h Yellow Rocket administration urine extract scanned from 100 to 500 am with the Aminorex peak identified. Aminorex peak determined as such by the presence of the protonated ion of Aminorex at 163 am and the major daughter ion at 120 amu, as shown in Fig. 3b. b: The extracted protonated ions, mass 163 (Aminorex parent ion) and mass 120 (major Aminorex daughter ion) which were used to identify Aminorex in the complex urine matrix of Fig. 3a

Fig. 4

The 6470 Triple Quadrupole LC/MS data for the determination of Aminorex in pre- and post- Yellow Rocket administration urines. The transition for Selected Ion Monitoring of Aminorex was 163.1 > 103.1. a shows the chromatogram of the pre-administration urine extract, b shows the chromatogram of a 4 h post–administration urine extract, c shows the chromatogram of a 8 h post–administration urine extract, d shows the chromatogram of a 12 h post–administration urine extract, e shows the chromatogram of a pre-administration urine extract demonstrating no carry-over, and f shows the chromatogram of the Aminorex reference standard. No evidence for the presence of Barbarin in these post B. vulgaris administration blood or urine samples has been identified to date

Fig. 5

Distribution of Yellow Rocket, Barbarea vulgaris, in the United States; Data re-constructed from USDA reports [13]. The British Isles show similar distribution, with only parts of Scotland having none reported as of May, 2019 ([14]