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Abstract

Neonicotinoids and neonicotinoid-like compounds (NNIs) are widely used insecticides and their ubiquitous occurrence in the environment requires methods for exposure assessment in humans. The majority of the NNIs can be divided into 6-chloropyridinyl- and 2-chlorothiazolyl-containing compounds, suggesting the formation of the group-specific metabolites 6-chloronicotinic acid (6-CNA), 2-chloro-1,3-thiazole-5-carboxylic acid (2-CTA), and their respective glycine derivatives (6-CNA-gly, 2-CTA-gly). Here, we developed and validated an analytical method based on gas chromatography coupled to mass spectrometry (GC-MS/MS) to simultaneously analyze these four metabolites in human urine. As analytical standards for the glycine conjugates were not commercially available, we synthesized 6-CNA-gly, 2-CTA-gly, and their 13C2,15N-labeled analogs for internal standardization and quantitation by stable isotope dilution. We also ensured chromatographic separation of 6-CNA and its isomer 2-CNA. Enzymatic cleavage during sample preparation was proven unnecessary. The limits of quantitation were between 0.1 (6-CNA) and 0.4 μg/L (2-CTA-gly) and the repeatability was satisfactory (coefficient of variation was <19% over the calibration range). We analyzed 38 spot urine samples from the general population and were able to quantify 6-CNA-gly in 58% of the samples (median 0.2 μg/L). In contrast, no 6-CNA could be detected. The results are in line with well-known metabolic pathways specific in humans, that, compared to rodents, favor the formation and excretion of phase–II–metabolites (glycine derivatives) rather than phase-I metabolites (free carboxylic acids). Nevertheless, the exact source of exposure (i.e., the specific NNI) remains elusive in the general population, may even vary quantitatively between different NNIs, and also might be regional specific based on the respective use of individual NNIs. In sum, we developed a robust and sensitive analytical method for the determination of four group-specific NNI metabolites.