Metabolomics approach to anabolic steroid urine profiling of bovines treated with prohormones.

In livestock production, illegal use of natural steroids is hard to prove because metabolites are either unknown or not significantly above highly fluctuating endogenous levels. In this work we outlined for the first time a metabolomics based strategy for anabolic steroid urine profiling. Urine profiles of controls and bovines treated with the prohormones dehydroepiandrosterone (DHEA) and pregnenolone were analyzed with ultraperformance liquid chromatography in combination with time-of-flight accurate mass spectrometry (UPLC-TOFMS). The obtained full scan urinary profiles were compared using sophisticated preprocessing and alignment software (MetAlign) and multivariate statistics, revealing hundreds of mass signals which were differential between untreated control and prohormone-treated animals. Moreover, statistical testing of the individual accurate mass signals showed that several mass peak loadings could be used as biomarkers for DHEA and pregnenolone abuse. In addition, accurate mass derived elemental composition analysis and verification by standards or Orbitrap mass spectrometry demonstrated that the observed differential masses are most likely steroid phase I and glucuronide metabolites excreted as a direct result from the DHEA and pregnenolone administration, thus underlining the relevance of the findings from this untargeted metabolomics approach. It is envisaged that this approach can be used as a holistic screening tool for anabolic steroid abuse in bovines and possibly in sports doping as well.

The chain length of lignan macromolecule from flaxseed hulls is determined by the incorporation of coumaric acid glucosides and ferulic acid glucosides.

Lignan macromolecule from flaxseed hulls is composed of secoisolariciresinol diglucoside (SDG) and herbacetin diglucoside (HDG) moieties ester-linked by 3-hydroxy-3-methylglutaric acid (HMGA), and of p-coumaric acid glucoside (CouAG) and ferulic acid glucoside (FeAG) moieties ester-linked directly to SDG. The linker molecule HMGA was found to account for 11% (w/w) of the lignan macromolecule. Based on the extinction coefficients and RP-HPLC data, it was determined that SDG contributes for 62.0% (w/w) to the lignan macromolecule, while CouAG, FeAG, and HDG contribute for 12.2, 9.0, and 5.7% (w/w), respectively. Analysis of fractions of lignan macromolecule showed that the higher the molecular mass, the higher the proportion of SDG was. An inverse relation between the molecular mass and the proportion (%) CouAG+FeAG was found. Together with the structural information of oligomers of lignan macromolecule obtained after partial saponification, it is hypothesized that the amount of CouAG+FeAG present during biosynthesis determines the chain length of lignan macromolecule. Furthermore, the chain length was estimated from a model describing lignan macromolecule based on structural and compositional data. The average chain length of the lignan macromolceule was calculated to be three SDG moieties with CouAG or FeAG at each of the terminal positions, with a variation between one and seven SDG moieties.