Abstract
The emergence of synthetic nicotine analogs in "tobacco-free" products, such as 6-methylnicotine (6MN; aka Metatine) in SPREE BAR, presents new regulatory and public health challenges. Alarmingly, little is known about the metabolism of 6MN, its potential biomarkers of exposure, or its toxicity. In this study, we systematically characterized oxidized metabolites of 6MN in the urine of mice exposed to 6MN (via intraperitoneal or inhalation route) using liquid chromatography-high resolution mass spectrometry. Similarly, human urine samples were analyzed for 6MN metabolites after use of the SPREE BAR (Blue Razz Ice) product. Nine 6MN metabolites were identified in mouse urine, and each metabolite corresponded with a known nicotine metabolite, albeit with increased mass (i.e. m/z + 14 Da). Although 6MN and nicotine share oxidative routes, the metabolism of 6MN was dominated via N-oxidation (likely FMO3-mediated) rather than C-oxidation (likely CYP2A6-dependent) pathways, whereas nicotine metabolism is vice versa. Six 6MN metabolites were detected in human urine after SPREE BAR use, demonstrating strong cross-species metabolic concordance. Among these 6MN human metabolites, 6-methylcotinine, 6-methyl-3'-hydroxycotinine, and 6-methylcotinine-N-oxide emerged as potential urinary biomarkers of exposure due to their prevalence. Importantly, 6MN, yet not an equimolar dose of nicotine, induced acute neurotoxic effects in mice, highlighting distinct toxicological risks of 6MN compared with nicotine. This research revealed a distinct metabolic profile of 6MN and established a framework for biomonitoring of 6MN exposure. Together, these findings advanced our understanding of the metabolism of synthetic nicotine analogs and emphasized the importance of compound-specific profiling to support regulatory oversight of emerging nicotine-like products.