Supplemental data.docx

This study introduces an innovative approach for the highly selective extraction and quantification of norcotamine (NCOT), a key biomarker of the carcinogenic N′-nitrosonornicotine (NNN) metabolism, in the urine of smokers. A smart ionic liquid, (S)-1-(4-hydroxypyridin-3-yl)-1-methyl-2-oxopyrrolidinium chloride ([Hpyr][Cl]), was rationally designed based on NCOT’s molecular structure to enhance extraction efficiency and selectivity. The synthesized ionic liquid was thoroughly characterized using FTIR, NMR, UV-Vis, and CHN elemental analysis. Additionally, UV-Vis spectroscopy was employed to assess spectral changes during the complex formation between NCOT and [Hpyr][Cl]. Density Functional Theory (DFT) calculations further elucidated the nature of these interactions at the molecular level. Following extraction, the resulting complex was magnetized, allowing efficient separation from the aqueous phase using a strong external magnet, eliminating the need for centrifugation. The method’s analytical performance was evaluated through key validation parameters, yielding a limit of detection of 0.10 ng mL-1, limit of quantification of 0.19 ng mL-1, and a linear dynamic range of 1.0-300 ng mL-1. The method demonstrated excellent precision, with inter-day and intra-day relative standard deviations (RSDs) of 2.7% and 1.6%, respectively. Finally, the developed system was successfully applied to real urine samples from smokers and non-smokers, as well as environmental water samples with complex matrices, achieving an accuracy range of 98.1-99.9%.