Integrating dispersive liquid-liquid microextraction with HPLC-MS/MS for simultaneously determining urinary tobacco...

Tobacco smoke exposure (TSE) during early life is a major public health concern, as the thousands of toxic and neurotoxic compounds present in cigarette smoke can critically disrupt brain development. Dysregulation of neurotransmitter levels has been linked to oxidative damage and an increased risk of neurodevelopmental and neurodegenerative disorders. Despite its clinical relevance, simultaneous quantification of neurotransmitters and tobacco metabolites in urine remains challenging due to their broad polarity range and the complexity of the biological matrix. To address this gap, we developed a novel hydrophilic interaction ultra-high-performance liquid chromatography coupled with tandem mass spectrometry (HILIC-MS/MS) method for the simultaneous quantification of 21 urinary biomarkers, including three tobacco-specific biomarkers, sixteen neurotransmitters and related metabolites, and two oxidative stress biomarkers. Sample preparation combines a clean-up step prior to the metabolite extraction by dispersive liquid-liquid microextraction from only 1 mL of urine, effectively reducing matrix effects to below 10% without enzymatic hydrolysis or derivatisation. The method achieved limits of detection as low as 0.004 ng/mL for NNAL, with extraction recoveries of 83-92% for tobacco biomarkers, 69-111% for neurotransmitters, and 83-94% for oxidative stress biomarkers. Repeatability and reproducibility were below 5% for all compounds, confirming the robustness of the method. The method was successfully applied to urine samples from children living with smoking adults, revealing possible neurotransmitters and oxidative stress alterations linked to the exposure. These findings demonstrate the suitability of the proposed approach as a reliable, sensitive, and high-throughput analytical tool for large-scale biomonitoring studies investigating the early biochemical effects of tobacco smoke exposure on neurodevelopmental health outcomes.