Exposure to Mycotoxin-Mixtures via Breast Milk: An Ultra-Sensitive LC-MS/MS Biomonitoring Approach

Abstract

Exposure to natural food contaminants during infancy may influence health consequences later in life. Hence, breast milk may serve as a vehicle to transport these contaminants, including mycotoxins, from mothers to their infants. Analytical methods mostly focused on single exposures in the past, thus neglecting co-occurrences and mixture effects. Here, we present a highly sensitive multi-biomarker approach by a sophisticated combination of steps during sample preparation including QuEChERS extraction followed by SPE cleanup and utilizing stable isotopes for compensating challenging matrix effects. The assay was validated in-house, reaching limits of detection (LOD) for all 34 analytes in the range of 0.1 to 300 ng/L with satisfying extraction efficiencies (75 - 109%) and stable intermediate precisions (1 - 18%) for most analytes. Compared to our first published multi-mycotoxin method LODs were decreased by a factor of 2-60x enabling the assessment of chronic low-dose exposures. The new method was applied to a small set of Nigerian breast milk samples to compare results with already published data. Concentration levels of samples determined to be contaminated before could be confirmed. The newly investigated alternariol monomethyl ether (AME) was found for the first time in this biological fluid at concentrations up to 25 ng/L. Moreover, in a pooled Austrian sample trace amounts of multiple mycotoxins confirmed co-occurrence and exposure even in countries with high food safety standards. In conclusion, the method facilitates the determination of mycotoxins at ultra-trace levels in breast milk, enabling the generation of occurrence data necessary for comprehensive co-exposure assessment.<br>

Longitudinal assessment of mycotoxin co-exposures in exclusively breastfed infants

“…In the former study, AME, BEA and OTA were the most frequently found toxins with concentrations up to 11, 12 and 68 ng/L, respectively (Braun et al unpublished; Ezekiel et al unpublished). EnnB and EnnB1 were detectable at lower concentrations (Braun et al 2018;Braun et al 2020b). In contrast to these results, high mycotoxin levels were reported by Rubert et al (2014) in breast milk obtained from Spanish mothers.…”

“…EnnB and EnnB1 in two out of 21 samples at a mean concentration of 105,000 and 96,000 ng/L, respectively. In addition, ZEN was reported in 62% of the samples in a concentration range of 2,100 to 14,000 ng/L (Rubert et al 2014), whereas Austrian or Nigerian samples were not contaminated with ZEN (LOD 16 ng/L) (Braun et al 2020b). Hence, it is not unlikely that the Spanish study over-estimated exposure levels as no internal standards were applied.…”

“…For mycotoxin analysis, the optimized protocol of Braun et al (2020b) was used. In brief, samples were thawed,homogenized and 1 mL breast milk sample was extracted with 1 mL of acidified ACN (1% formic acid).…”

“…After homogenization and ultrasonication (5 min), the samples were transferred to amber LC-vials and analyzed. Details of the LC-MS/MS method, including instrumental parameters and analytical figures thereof were reported in Braun et al (2020b). In brief, the LC-MS/MS system consisted of an Agilent 1290 Infinity II LC system coupled to a Sciex QTrap® 6500 + mass spectrometer (Darmstadt, Germany).…”

“…Recently, we developed and validated two targeted LC-MS/MS assays to simultaneously assess multiple classes of mycotoxins in breast milk. The methods proved to be fit for purpose and allow the evaluation of these natural contaminants down to the pg/L range, enabling quantitative exposure assessment for infants even in countries with high food safety standards and low mycotoxin risk (Braun et al 2018;Braun et al 2020b).…”