Despite the fact that a chromatographic peak was detected within the Title Loaded From File extracted ion chromatogram (ESI unfavorable mode) at the precise mass in the deprotonated 6-CNA-gly (m/z 213.0071), the excretion profile was inconclusive when compared with the other metabolites (Figure S1a). Furthermore, the mass signals within the dd-MS2 product-ion spectra couldn‘t be attributed to the expected mass signals (Figure S2a). Finally, the anticipated 35Cl and 37 Cl isotopolog mass signals and their ratios could not be observed, resulting inside a less conclusive 35Cl/37Cl isotopecorrelation (Figure S3a). General, a self-confidence amount of 5 may very well be assigned to 6-CNA-gly only. The solution ion spectra of your observed peak for hydroxylated IMI (tR = 7.7 min), the observed neutral loss of water and the discovering of IMI-olefin pointed towards the formation of hydroxylated IMI in the 4- and/or 5-position of your 1-H-imidazole moiety rather than at the 6-chloronicotinoyl moiety or the methylene bridge. The retention time was also identical to those of commercially readily available standards of 4- and 5-OH-IMI, therefore additional strengthening our conclusion that IMI has been hydroxylated at the 4- and/or 5-position of the 1-H-imidazole moiety. Applying an exploratory isocratic chromatographic approach, we had been in a position to separate 4- and 5-OHIMI and could even show the formation of each metabolites in our samples. The formation of 5-OH-IMI has been previously reported in metabolism research of rodents (Ford and Casida 2006). Furthermore, its formation also has been observed in humans (Taira et al. 2013; Ospina et al. 2019; Song et al. 2021; K ferlein et al. 2021; Wrobel et al. 2020). A dihydroxylated metabolite of IMI like 4,5-OHIMI couldn‘t be detected in our study albeit it has been reported previously in a qualitative profiling study (TairaArchives of Toxicology (2022) 96:121et al. 2013). Similarly, desnitro-IMI (also known as IMIguanidine) which has lately been analyzed in urine samples from China (Wang et al. 2020) couldn‘t be identified in our experiments. Moreover, desnitro-IMI-olefin has been previously recommended for examination in human urine determined by the fact that this metabolite has been found in lizard brain following oral exposure to IMI (Wang et al. 2018, 2020). In our study, we certainly had been in a position to detect a chromatographic peak in the exact mass of desnitro-IMIolefin in ESI optimistic ionization mode. On the other hand, this peak had the identical retention time of IMI (tR = 8.5 min) and was also observed when analyzing a neat common remedy of 50 /L IMI. Accordingly, we concluded that the presence of desnitro-IMI-olefin is much more probably due to in-source fragmentation involving the neutral loss of nitrous acid (as previously described in the gas-phase fragmentation of IMIolefin (Fusetto et al. 2016)) as an alternative to being formed as a metabolite of IMI in vivo (Figure S4). 6-CNA has been previously recommended as biomarker for NNIs which contain a 6-chloro-3-nicotinoyl moiety (Uroz et al. 2001; Kavvalakis et al. 2013; Tao et al.In our study is also in line with all the benefits reported by Harada et al.