Abstract
A simple and effective approach for HPLC-MS/MS based multi-mycotoxin analysis in human urine samples was developed by application of dried urine spots (DUS) as alternative on-site sampling strategy. The newly developed method enables the detection and quantitation of 14 relevant mycotoxins and mycotoxin metabolites, including citrinin (CIT), dihydrocitrinone (DH-CIT), deoxynivalenol (DON), fumonisin B1 (FB1), T-2 Toxin (T-2), HT-2 Toxin (HT-2), ochratoxin A (OTA), 2′R-ochratoxin A (2′R-OTA), ochratoxin α (OTα), tenuazonic acid and allo-tenuazonic acid (TeA + allo-TeA), zearalenone (ZEN), zearalanone (ZAN), α-zearalenol (α-ZEL), and β-zearalenol (β-ZEL). Besides the spotting procedure, sample preparation includes enzymatic cleavage of glucuronic acid conjugates and stable isotope dilution analysis. Method validation revealed low limits of detection in the range of pg/mL urine and excellent apparent recovery rates for most analytes. Stability investigation of DUS displayed no or only slight decrease of the analyte concentration over a period of 28 days at room temperature. The new method was applied to the analysis of a set of urine samples (n = 91) from a Swedish cohort. The four analytes, DH-CIT, DON, OTA, and TeA + allo-TeA, could be detected and quantified in amounts ranging from 0.06 to 0.97 ng/mL, 3.03 to 136 ng/mL, 0.013 to 0.434 ng/mL and from 0.36 to 47 ng/mL in 38.5%, 70.3%, 68.1%, and 94.5% of the samples, respectively. Additional analysis of these urine samples with an established dilute and shoot (DaS) approach displayed a high consistency of the results obtained with both methods. However, due to higher sensitivity, a larger number of positive samples were observed using the DUS method consequently providing a suitable approach for human biomonitoring of mycotoxin exposure.
Highlights
Mycotoxins are toxic secondary metabolites of mould fungi
Method development was motivated by enabling a separation between on-site sample preparation and sample analysis in a dedicated HPLC-MS laboratory on the one hand and improving the sensitivity of human biomonitoring on the other hand
The newly developed approach allows the analysis of 14 mycotoxins and mycotoxin phase I metabolites, namely CIT, DH-CIT, DON, FB1, T-2, HT-2, ochratoxin A (OTA), 2′R-ochratoxin A (2′R-OTA), ochratoxin α (OTα), TeA + allo-TeA, ZAN, ZEN, α-ZEL and β-ZEL as well as their glucuronides after enzymatic hydrolysis
Summary
Mycotoxins are toxic secondary metabolites of mould fungi. Mould infestation and formation of mycotoxins is influenced by climatic factors like temperature and humidity and can occur at all stages of the production process as well as during storage (Vidal et al 2018). Regardless of the sample preparation, liquid urine samples are necessary for the approaches mentioned above, resulting in biological hazards through sample leakage and high expenses for safe and timely shipping of the frozen samples (Zimmer et al 2013). For this reason, shipping costs can be rather high and sometimes even exceed the costs of the analytical method itself. Dried matrix spots (DMS) obtained by application of physiological samples onto filter paper are a suitable alternative (Sadones et al 2014). Because some drugs are almost entirely metabolised into their conjugated analogues, Michely et al (2017) implemented an on-spot conjugate cleavage in order to obtain higher concentrations of the corresponding phase I metabolites
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