A novel strategy for the detection of boldenone undecylenate misuse in cattle using ultra-high performance liquid chromatography coupled to high resolution orbitrap mass spectrometry: From non-targeted to targeted.

ABSTRACT

In this work multivariate strategies were employed in order to highlight new potential biomarkers of interest to detect the exogenous treatment of steers intramuscularly treated with boldenone undecylenate. Serum samples collected from treated (n = 4) and control (n = 8) crossbred animals of varying ages and weights were extracted using a simple sample preparation procedure based on salt assisted liquid-liquid extraction. Data acquisition was performed using liquid chromatography and Q-Exactive™ Orbitrap mass spectrometry. Data processing and treatment were performed using two non-targeted workflows (1) Compound Discoverer software and (2) XCMS package on the open-source R software combined with MetaboAnalyst. Three potential biomarkers were highlighted taking into account the chromatographic shapes, the feature location on the generated s-plots, the fold change, the adjusted p values, the coefficient of variation in the QC samples and the area under the ROC curves. Predicted formulas based on mass accuracy, structural composition and spectra similarity were proposed. A robust statistical model to predict the boldenone treatment was further developed based on the weighted abundances of the selected biomarkers. The requirements for screening methods were successfully fulfilled, together with a wider detection window in comparison with the monitoring of the deconjugated metabolite boldenone, although biomarker identification studies are still ongoing.