Identification and determination of deoxynivalenol (DON) and deepoxy-deoxynivalenol (DOM-1) in pig colostrum and serum using liquid chromatography in combination with high resolution mass spectrometry (LC-MS/MS (HR))

Abstract

Deoxynivalenol (DON) is one of the most common mycotoxins produced by field fungi (especially Fusarium). Contamination of livestock feed is a significant risk factor, especially for pigs that are highly susceptible to the toxic effects of deoxynivalenol. In this study, validated ultra-high performance liquid chromatography (U-HPLC) combined with a HR-Orbitrap-MS analysis method is described for the identification and quantitative determination of the mycotoxin compounds (DON and deepoxy-deoxynivalenol (DOM-1)) in pig colostrum (milk) and serum. Pre-treatment of the samples involved a deproteinisation step with methanol followed by a purification step by solid phase extraction (HLB cartridges). The chromatographic separation was performed on a C18 column with 1.7 μm-particle size using a water-methanol mobile phase. Detection of analytes was achieved on the tandem hybrid mass spectrometer Q Exactive, with a heated electrospray ionisation probe measured in positive mode (H-ESI+). For the confirmation of identification, a mass spectrometer was utilized in the full scan mode with resolving power (PR) = 140,000 (FWHM) and for quantification analysis, it was utilized in the parallel reaction monitoring mode (PRM). The method has been fully validated according to the requirements of Commission Decision 2002/657/EC for confirmatory analyses, plus the addition of a mass accuracy (MA) parameter. For the confirmation of the presence of these analytes in pig colostrum and serum, matching of the retention time with mass accuracy for the precursor ion from MS and product ions from MS/MS was used. A deuterium isotopically labelled internal standard and a matrix-matched calibration curve were employed for quantification. The linear range of quantification was 0.5-20 μg L^-1 and the correlation coefficient (R²) was >0.999 for all calibrations. The limit of detection for DON and DOM-1 in colostrum was 0.48 μg L^-1 and 0.54 μg L^-1, respectively, and in serum 0.24 μg L^-1 and 0.36 μg L^-1, respectively. The limit of quantification for DON and DOM-1 in colostrum was 0.80 μg L^-1 and 0.89 μg L^-1, respectively, and in serum 0.39 μg L^-1 and 0.60 μg L^-1, respectively. The method was successfully evaluated using the obtained samples of pig colostrum and serum.

Keywords: Colostrum; Deoxynivalenol; LC-MS (HR); Mycotoxins; Pig; Serum.