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.
Highlights
Characterizing complex environmental exposures and their combined effects on toxicity and human health has become a priority recently and is often referred to in the context of the ‘exposome’ paradigm [1e3]
Mycotoxin standards were purchased from Romer Labs Diagnostic GmbH Tulln, Austria: nivalenol (NIV), 13C-NIV, deoxynivalenol (DON), 13C-DON, deepoxyDON (DOM-1), ochratoxin A (OTA), 13C-OTA, aflatoxin M1 (AFM1), 13C- AFM1, citrinin (CIT), fumonisin B1 (FB1), 13C-FB1, 13C-zearalenone (ZEN)) or Sigma, Vienna, Austria (ZEN, a- and b-zearalenol (ZEL))
Based on their broad analyte coverage allowing for potential addition of analytes in the future, we focused on solid phase extraction (SPE) columns for urine clean-up rather than highly specific and cost-intensive IAC columns
Summary
Characterizing complex environmental exposures and their combined effects on toxicity and human health has become a priority recently and is often referred to in the context of the ‘exposome’ paradigm [1e3]. Mycotoxins are a major class of natural contaminants that humans are typically exposed to throughout their life. This chemically diverse group of toxic secondary metabolites are produced by filamentous fungi and frequently occur in our diet [4]. For some mycotoxins including the carcinogenic aflatoxins, permitted levels are typically very low(e.g. aflatoxin M1 (AFM1) in milk: 50 pg mLÀ1). It is known from biomarker driven research that combined exposures to mycotoxins are common [10e13]. There is growing scientific evidence that mixtures of co-occurring mycotoxins [14e16] and mycotoxins with other xenobiotics (bioactive food constituents, drugs etc.) [17,18] have the potential to cause an additional threat through combinatory effects that legislation does not take into account to date
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