Few human data on exposure and toxicity are available on neonicotinoids and neonicotinoid-like compounds (NNIs), an important group of insecticides worldwide. Specifically, exposure assessment of humans by biomonitoring remains a challenge due to the lack of appropriate biomarkers. We investigated the human metabolism and metabolite excretion in urine of acetamiprid (ACE), clothianidin (CLO), flupyradifurone (FLUP), imidacloprid (IMI), sulfoxaflor (SULF), thiacloprid (THIAC) and thiamethoxam (THIAM) after single oral dosages at the currently acceptable daily intake levels of the European Food Safety Authority. Consecutive post-dose urine samples were collected up to 48 h. Suspect screening of tentative metabolites was carried out by liquid chromatography–high-resolution mass spectrometry. Screening hits were identified based on their accurate mass, isotope signal masses and ratios, product ion spectra, and excretion kinetics. We found, with the exception of SULF, extensive metabolization of NNIs to specific metabolites which were excreted next to the parent compounds. Overall, 24 metabolites were detected with signal intensities indicative of high metabolic relevance. Phase-I metabolites were predominantly derived by mono-oxidation (such as hydroxy-FLUP, -IMI, and -THIAC) and by oxidative N-desalkylation (such as N-desdifluoroethyl-FLUP and N-desmethyl-ACE, -CLO and -THIAM). IMI-olefin, obtained by dehydration of hydroxylated IMI, was identified as a major metabolite of IMI. SULF was excreted unchanged in urine. Previously reported metabolites of NNIs such as 6-chloronicotinic acid or 2-chlorothiazole-4-carboxylic acid and their glycine derivatives were detected either at low signal intensities or not at all and seem less relevant for human biomonitoring. Our highly controlled approach provides specific insight into the human metabolism of NNIs and suggests suitable biomarkers for future exposure assessment at environmentally relevant exposures.

The analgesic paracetamol/acetaminophen (N-acetyl-4-aminophenol, APAP) is commonly used to relieve pain, fever and malaise. While sales have increased worldwide, a growing body of experimental and epidemiological evidence has suggested APAP as a possible risk factor for various health disorders in humans. To perform internal exposure-based risk assessment, the use of accurate and optimized biomonitoring methods is critical. However, retrospectively assessing pharmaceutical use of APAP in humans is challenging because of its short half-life. The objective of this study was to address the key issue of potential underestimation of APAP use using current standard analytical methods based on urinary analyses of free APAP and its phase II conjugates. The question we address is whether investigating additional metabolites than direct phase II conjugates could improve the monitoring of APAP. Using non-targeted analyses based on high-resolution mass spectrometry, we identified, in a controlled longitudinal exposure study with male volunteers, overlooked APAP metabolites with delayed formation and excretion rates. We postulate that these metabolites are formed via the thiomethyl shunt after the enterohepatic circulation as already observed in rodents. Importantly, these conjugated thiomethyl metabolites were (i) of comparable diagnostic sensitivity as the free APAP and its phase II conjugates detected by current methods; (ii) had delayed peak levels in blood and urine compared to other APAP metabolites and therefore potentially extend the window of exposure assessment; and (iii) provide relevant information regarding metabolic pathways of interest from a toxicological point of view. Including these metabolites in future APAP biomonitoring methods therefore provides an option to decrease potential underestimation of APAP use. Moreover, our data challenge the notion that the standard methods in biomonitoring based exclusively on the parent compound and its phase II metabolites are adequate for human biomonitoring of a non-persistent chemical such as APAP.