Abstract
In the course of assessing the human exposure to mycotoxins, biomarker-based approaches have proven to be important tools. Low concentration levels, complex matrix compositions, structurally diverse analytes, and the large size of sample cohorts are the main challenges of analytical procedures. For that reason, an online solid phase extraction-ultra high-performance liquid chromatography-tandem mass spectrometry (online SPE-UHPLC-MS/MS) method was developed, allowing for the sensitive, robust, and rapid analysis of 11 relevant mycotoxins and mycotoxin metabolites in human urine. The included spectrum of analytes comprises aflatoxin M1 (AFM1), altenuene (ALT), alternariol monomethyl ether (AME), alternariol (AOH), citrinin (CIT) and its metabolite dihydrocitrinone (DH-CIT), fumonisin B1 (FB1), ochratoxin A (OTA), and zearalenone (ZEN) as well as α- and β-zearalenol (α- and β-ZEL). Reliable quantitation was achieved by means of stable isotope dilution, except for ALT, AME and AOH using matrix calibrations. The evaluation of method performance displayed low limits of detection in the range of pg/mL urine, satisfactory apparent recovery rates as well as high accuracy and precision during intra- and interday repeatability. Within the analysis of Zimbabwean urine samples (n = 50), the applicability of the newly developed method was shown. In addition to FB1 being quantifiable in all analyzed samples, six other mycotoxin biomarkers were detected. Compared to the occurrence rates obtained after analyzing the same sample set using an established dilute and shoot (DaS) approach, a considerably higher number of positive samples was observed when applying the online SPE method. Owing to the increased sensitivity, less need of sample handling, and low time effort, the herein presented online SPE approach provides a valuable contribution to human biomonitoring of mycotoxin exposure.
Highlights
Mycotoxins are secondary metabolites of fungal origin produced by various genera.The infestation of agricultural crops such as grains, fruits, nuts, or spices by filamentous fungi as well as the formation of mycotoxins is influenced by climatic factors and can occur both pre- and post-harvesting [1,2]
Crucial points of method development included a significant lowering of the limit of detection (LOD) and limit of quantitation (LOQ) compared to frequently used approaches for the analysis of urine samples while comprising short analysis times and enabling a high sample throughput
Sample preparation prior to injection comprised spiking the urine sample with a mixture of the stable isotope-labelled standards followed by centrifugation to prevent the online solid phase extraction (SPE)-UHPLC system from clogging with particles
Summary
Mycotoxins are secondary metabolites of fungal origin produced by various genera. The infestation of agricultural crops such as grains, fruits, nuts, or spices by filamentous fungi as well as the formation of mycotoxins is influenced by climatic factors and can occur both pre- and post-harvesting [1,2]. As a result of carry-over effects, plant-based and foodstuffs of animal origin may be contaminated [4]. Owing to multiple toxicological effects related to mycotoxins such as carcinogenicity, renal toxicity, immunosuppression, or estrogenic effects potentially occurring with chronic ingestion, the exposure assessment is of decisive importance [5]. While wealthier countries have legally specified maximum levels for contaminants in foodstuffs, especially impoverished regions are affected by dietary mycotoxin exposure [3,5]
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