Human biomonitoring of multiple mycotoxins in biological samples of Portuguese ‘earlyMYCO‘ mother-and-child pairs

Early-life exposure to hazardous compounds is an emerging research field that urgently needs to be investigated, as an attempt to contribute to exposome research. Mycotoxins are naturally-occurring food contaminants and recent studies reported that Portuguese children (< 3 years) as well as the adult population are exposed to multiple mycotoxins through food consumption constituting a potential health threat. Considering the above, a project designated by earlyMYCO (Early-life exposure to MYCOtoxins and its impact on health) is being developed to assess the risk of early-life exposure to mycotoxins in biological samples. The earlyMYCO project included a mother-and-child longitudinal study, with three moments of observation, starting from pregnancy (24-28 weeks) and going through the child’ first months of life (1-6 months). This study was conducted in the Primary Health Care Group Lisboa Central (ACES) and aimed to determine multiple mycotoxins and its biomarkers in breast milk and urine samples from mother-and-child pairs. The sample preparation procedure for breast milk samples (n=9) consisted of a solid-phase extraction (SPE) purification followed by a liquid-liquid extraction (LLE); urine samples (n=50) were prepared by applying the QuEChERS technique. Liquid chromatography coupled to ESI+ Xevo TQ-S mass spectrometry (UHPLC-ESI-MS/MS) was used to determine multiple mycotoxin biomarkers of exposure in a targeted approach, namely aflatoxins B1, G1, B2, G2 and M1 (AFB1, AFG1, AFB2, AFG2, AFM1), ochratoxin A and alpha (OTA, OT-α), deoxynivalenol (DON), nivalenol (NIV), zearalenone (ZEN), alpha and beta zearalenol (α-ZEL, β-ZEL), fumonisins B1, B2, B3 and hydrolyzed B1 (FB1, FB2, FB3, HFB1), sterigmatocystin (STE), and aflatoxicol (AFL) using a previously optimized and validated method. All the breast milk samples analysed contained multiple mycotoxins to whom the children were exposed to. Unfortunately, due to covid-19 the number of samples was restricted. All the breastmilk samples resulted to be positive for FB1 and FB2 (exception for one sample) with concentrations between 0.16 and 34.76 ng/mL and 0.14 and 11.90 ng/mL, respectively; 5 samples resulted positive also for FB3 (1.09-4.97 ng/mL). DON was detected in one sample as well as NIV with concentrations of 0.75 and 3.26 ng/mL, respectively. Moreover, AFB1 and AFG1 were detected respectively in three samples (0.36-0.64 ng/mL) and one sample (below the LOD), while in two samples OTA was present below the LOD. Besides the targeted approach on urine samples, an untargeted LC-HRMS approach was launched on both sample matrices.This work was funded by FCT/MCTES through national funds, to earlyMYCO (PTDC/MED-TOX/28762/2017), and CESAM (UIDP/50017/2020+UIDB/50017/2020).N/