Identification of crotonyl glycine in urine of sheep after 48h road transport

Thousands of metabolites are excreted in urine, and potentially can be detected in NMR spectra. Currently, NMR spectral information for about one thousand metabolites has been deposited in publicly available sources, limiting the identification of chemical compounds that are potential biomarkers for clinical and subclinical applications. This study reports the identification of crotonyl glycine, one of the key metabolites detected by H NMR as excreted in the urine of sheep after 48h road transport and during the subsequent 72h recovery period. This metabolite was important in separating the metabolic responses as expressed in the urine from animals undergoing shorter road transport treatments. At the time of the metabonomic analysis, the NMR signals from this metabolite were designated as unassigned as no match was found in public databases or the literature. Selected sheep urine samples containing the metabolite were resolved by reversed phase HPLC reducing the sample complexity. Subsequent H NMR spectra of the collected fractions revealed that the unknown metabolite was present in a single HPLC fraction. High-resolution 1D and 2D H NMR spectra of this fraction followed by mass determination of the parent ion and its fragments by nanoESI-TOF-MS/MS revealed the identity of the compound as crotonyl glycine (N-but-(E)-2-enoyl glycine). The HPLC fraction was subsequently spiked with synthetic crotonyl glycine which confirmed identification