Simultaneous quantification of fourteen urinary tobacco biomarkers via UHPLC-Q-Orbitrap HRMS and isotope dilution

This study developed and validated an ultra-high-performance liquid chromatography-quadrupole-Orbitrap high-resolution mass spectrometry(UHPLC-Q-Orbitrap HRMS)method for the simultaneous quantitative 14 tobacco biomarkers in human urine. This method encompasses biomarkers including nicotine metabolites, tobacco-specific nitrosamine metabolites, and minor tobacco alkaloids. Sample preparation employed a combination of enzymatic digestion and supported liquid extraction (SLE), coupled with isotopically labeled internal standards. Analyte separation was achieved rapidly using a Gemini NX-C18 column (150 mm × 2.0 mm, 3 μm), enabling detection of all target compounds within a 15-min chromatographic run time. High-resolution mass spectrometry (HRMS) operated in Full MS and parallel reaction monitoring (PRM) modes. Precise mass screening of parent ions was performed, with confirmation achieved through isotope-labeled internal standards and secondary fragment information. This method has undergone comprehensive validation, demonstrating excellent linearity for all analytes within their respective dynamic ranges. Accuracy ranged from 82.9 % to 121 %, with within-day precision between 1.0 % and 13 % and between-day precision between 1.7 % and 25 %. The limit of detection (LOD) ranged from 0.003 to 2 μg/L, while the limit of quantification (LOQ) spanned 0.01-5 μg/L. Furthermore, matrix effects, recovery rates, and stability all complied with the requirements of bioanalytical guidelines. Ultimately, this method was applied to 123 urine samples from the China National Human Biomarker Monitoring Program (CNHBM), including 30 smokers and 93 non-smokers. The results revealed significant differences in biomarker levels between groups with varying smoking statuses, demonstrating the method's potential for application in epidemiological studies and evaluation of tobacco exposure.