Within the Human Biomonitoring for Europe initiative (HBM4EU), a study to determine new biomarkers of exposure to pesticides and to assess exposure patterns was conducted. Human urine samples (N=2,088) were collected from five European regions in two different seasons. The objective of the study was to identify pesticides and their metabolites in collected urine samples with a harmonized suspect screening approach based on liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS) applied in five laboratories. A combined data processing workflow included comprehensive data reduction, correction of mass error and retention time (RT) drifts, isotopic pattern analysis, adduct and elemental composition annotation, finalized by a mining of the elemental compositions for possible annotations of pesticide metabolites. The obtained tentative annotations (n=498) were used for acquiring representative data-dependent tandem mass spectra (MS²) and verified by spectral comparison to reference spectra generated from commercially available reference standards or produced through human liver S9 in-vitro incubation experiments. 14 parent pesticides and 71 metabolites (including 16 glucuronide and 11 sulfate conjugates) were detected. Collectively these related to 46 unique pesticides.

For the remaining tentative annotations either (i) no data-dependent MS² spectra could be acquired, (ii) the spectral purity was too low for sufficient matching, or (iii) RTs indicated a wrong annotation, leaving potential for more pesticides and/or their metabolites being confirmed in further studies. Thus, the reported results are reflecting only a part of the possible pesticide exposure.