Optimization of the first extraction protocol for metabolomic studies of Brucella abortus.

Brucellosis is a zoonosis prevalent worldwide and very recurrent in less developed or developing regions. This zoonosis affects livestock, generating high financial losses to producers, in addition to transmitting diseases to humans through meat consumption or handling contaminated products and animals. In this study, five extraction methods for Brucella abortus intracellular metabolites, using different solvent compositions and cell membrane disruption procedures, were evaluated. Derivatized extracts were analyzed by GC-HRMS. Raw data were processed in XCMS Online and the results were evaluated through multivariate statistical analysis using the MetaboAnalyst platform. The identification of the extracted metabolites was performed by the Unknowns software using the NIST 17.L library. The extraction performance of each method was evaluated for thirteen representative metabolites, comprising four different chemical classes. Most of these compounds are reported in the cell membrane composition of Gram-negative bacteria. The method based on extraction with methanol/chloroform/water presented the best performance in the evaluation of the extracted compounds and in the statistical results. Therefore, this method was selected for extracting intracellular metabolites from cultures of Brucella abortus for untargeted metabolomics analysis.

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.

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.

Application of QuEChERS method and gas chromatography-mass spectrometry for the analysis of cypermethrin residue in milk.

Analytical methods for the isolation and determination of cypermethrin in milk, based on the solid-phase microextraction (SPME) and QuEChERS methods (Quick, Easy, Cheap, Effective, Rugged, and Safe) are presented. The SPME technique was not appropriate to analyse cypermethrin in milk, even establishing the best extraction conditions, polydimethylsiloxane fiber, 60 min time extraction, 60 °C temperature extraction, addition of salt (NaCl) and stirring rate. The extraction efficiency was low probably because of the matrix constituents. The QuEChERS method involves the extraction of the analyte with acetonitrile and simultaneous liquid-liquid partitioning formed by adding anhydrous MgSO(4) plus NaCl, followed by the removal of residual water and cleanup using a procedure called dispersive solid-phase extraction, in which anhydrous MgSO(4) plus PSA and C18 are mixed with 1 mL of acetonitrile extract. The detection and quantification limits were 0.01 and 0.04 mg kg(-1), respectively, and the percentage recovery obtained ranged from 92 to 105% with relative standard deviations below 7%.