Human exposure to volatile organic compounds (VOCs) poses significant health risks, contributing to cardiovascular disease, pulmonary disease, and cancer. Measurement of VOC metabolites (VOCm) in urine by liquid chromatography-mass spectrometry (LC-MS) is a preferred method for VOCm analysis; however, existing methods encounter challenges related to sensitivity, throughput, and analyte coverage. In addition to VOCm, the measurement of tobacco alkaloids (TAm) is critical to account for tobacco use in population-based studies. A method is needed that is highly sensitive, offers higher throughput, and can analyze VOCm and TAm in a single run. Herein, we present a robust dilute-and-shoot method aimed at overcoming these analytical challenges and expanding the targeted analysis to include 35 urinary VOCm and TAm and their metabolites. By leveraging high-speed polarity switching and optimized chromatographic parameters, our method achieved comprehensive analyte coverage and enhanced sensitivity, enabling reliable individual level VOC exposure assessment. Validation demonstrates robust linearity, sensitivity, accuracy, and precision, with minimal matrix effects. This high-throughput UPLC-MS/MS method significantly enhances VOC exposure assessment by enabling simultaneous measurement of 35 urinary VOC and TAm with high sensitivity and efficiency. Multiple metabolites from single parent xenobiotics are included in one run, expanding biomarker specificity. Our data indicate the method effectively accounts for tobacco consumption as a confounder in population-based studies, ensuring accurate VOC exposure assessment.
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