Development and validation of a gas chromatography–mass spectrometry method for determination of deoxynivalenol and its metabolites in human urine

The determination of deoxynivalenol (DON) and its metabolites such as deepoxy-deoxynivalenol (DOM-1) in human urine is complicated due its low levels (ng/mL) and the complexity of the matrix. A gas chromatography–mass spectrometry method was optimized and validated for the confirmation analysis of DON and its metabolites in urine samples using 13C isotopic-labeled DON as internal standard. In the sample preparation the type and amount of β-glucuronidase for enzymatic hydrolysis was investigated as well as the cleanup procedure, being compared the immunoaffinity column with solid-phase extraction (SPE). As far as we know, SPE C18 cleanup procedure was applied for the first time in the analysis of DON and its metabolites in human urine. Using this analytical methodology the detection and quantification limits achieved ranged from 0.06 to 0.30 ng/mL and from 0.2 to 1.0 ng/mL, respectively. Recoveries were higher than 73% for fortification levels between 25 and 100 ng/mL and repeatability were lower than 13%. The natural occurrence of DON and its metabolites in human urine samples from the north zone of Portugal was studied. Free DON was detected in 15% of the samples whereas total (free + conjugated) DON was detected in 69% of the samples. Deepoxy-deoxynivalenol, 3-acetyldeoxynivalenol and 15-acetyldeoxynivalenol were not detected in any of the samples analyzed.