Monitoring Early-Life Mycotoxin Exposures via LC-MS/MS Breast Milk Analysis

Infants are particularly susceptible towards the toxic effects of food contaminants including mycotoxins. However, multi-mycotoxin exposure assessment in breast milk has received very limited attention so far, resulting in a poor understanding of co-exposures during early-life. Here, we present the development and application of a highly sensitive, specific and quantitative assay assessing up to 28 mycotoxins including regulated (aflatoxins, ochratoxin A, deoxynivalenol, zearalenone) and emerging mycotoxins as well as key metabolites by LC-MS/MS. After careful optimization of the sample preparation procedure, a QuEChERS (quick, easy, cheap, effective, rugged and safe) protocol combined with a freeze-out step was utilized for method validation. The limits of quantification varied between 0.009 and 2.9 ng/mL, and for most analytes extraction recovery (74-116%) and intermediate precision (2-20%) were satisfactory. The method was applied to examine multiple breast milk samples obtained from 22 women (n=75 in total) from Ogun State, Nigeria. Most samples were either entirely free of mycotoxins or contaminated to a minimal extent with beauvericin (56%), enniatins B (9%), ochratoxin A (15%) and aflatoxin M1 (1%). The most abundant mycotoxin was beauvericin, which was not reported in this biological fluid before, with concentrations up to 0.019 ng/mL. In conclusion, the method demonstrated to be fit for purpose to determine and quantify low background contaminations in human breast milk. Based on the high sensitivity of the novel analytical method, it was possible to deduce that tolerable daily intake values were not exceeded by breastfeeding in the examined infants.