Ultra-sensitive, stable isotope assisted quantification of multiple urinary mycotoxin exposure biomarkers

Highlights: • An ultra-sensitive method for urinary biomarkers of mycotoxin exposure was established. • Simultaneous biomonitoring of regulated and emerging mycotoxins at trace levels by a single analytical method. • First multiple stable isotope assisted quantification method for mycotoxin exposure biomarkers validated. • Applicability in realistic chronic low dose exposure to mycotoxins in large-scale cohort. - Abstract: There is a critical need to better understand the patterns, levels and combinatory effects of exposures we are facing through our diet and environment. Mycotoxin mixtures are of particular concern due to chronic low dose exposures caused by naturally contaminated food. To facilitate new insights into their role in chronic disease, mycotoxins and their metabolites are quantified in bio-fluids as biomarkers of exposure. Here, we describe a highly sensitive urinary assay based on ultra-high performance liquid chromatography - tandem mass spectrometer (UHPLC-MS/MS) and ¹³C-labelled or deuterated internal standards covering the most relevant regulated and emerging mycotoxins. Utilizing enzymatic pre-treatment, solid phase extraction and UHPLC separation, the sensitivity of the method was significantly higher (10-160x lower LODs) than in a previously described method used for comparison purpose, and stable isotopes provided compensation for challenging matrix effects. This method was in-house validated and applied to re-assess mycotoxin exposure in urine samples obtained from Nigerian children, adolescent and adults, naturally exposed through their regular diet. Owing to the methods high sensitivity, biomarkers were detected in all samples. The mycoestrogen zearalenone was the most frequently detected contaminant (82%) but also ochratoxin A (76%), aflatoxin M₁ (73%) and fumonisin B₁ (71%) were quantified in a large share of urines. Overall, 57% of 120 urines were contaminated with both, aflatoxin M₁ and fumonisin B₁, and other co-exposures were frequent. These results clearly demonstrate the advanced performance of the method to assess lowest background exposures (pg mL^-1 range) using a single, highly robust assay that will allow for the systematic investigation of low dose effects on human health.