Simultaneous Analysis of Biomarkers in Human Hair for Evaluating Chronic Tobacco Smoke Exposure and Stress/Relaxation Using Online In-Tube Solid-Phase Microextraction Coupled with Liquid Chromatography–Tandem Mass Spectrometry

Tobacco smoke exposure not only increases the risks of lung cancer and cardiovascular disease, but can be a stressor contributing to mental illness. It is important to clarify the relationship between chronic tobacco smoke exposure and mental stress from the perspective of disease prevention. We developed a simple and highly sensitive method for simultaneously analyzing nine biomarkers: nicotine and cotinine (tobacco smoke exposure markers); cortisol, testosterone, and dehydroepiandrosterone (stress-related markers); and serotonin, melatonin, dopamine, and oxytocin (relaxation-related markers). Biomarkers were extracted and concentrated by in-tube solid-phase microextraction with a Supel-Q PLOT capillary, followed by separation and detection within 7 min using liquid chromatography-tandem mass spectrometry on a Discovery HS F5 column. Calibration curves using stable isotope-labeled internal standards showed good linearity (0.005-100 ng mL^-1) with detection limits of 0.09-13.5 pg mL^-1. Intra-day and inter-day precision had relative standard deviations below 7.2% and 15.5% (<i>n</i> = 6), respectively, with recovery rates of 84.0-108.8%. The automated method requires only ultrafiltration of hair methanol extract, enabling non-invasive pg-level analysis using just a few milligrams of hair. Hair analysis reflects an association between chronic tobacco smoke exposure and stress. This method is effective for analyzing the relationship between long-term tobacco smoke exposure and chronic stress.