Automated sample preparation reduces variation caused by human factors and improves efficiency, throughput, and reliability, making it especially important in large-scale epidemiological biomonitoring applications. In this study, we demonstrated an automated liquid-liquid extraction platform that streamlines sample preparation for human biomonitoring of urinary phthalate metabolites. This platform integrates temperature-controlled enzymatic hydrolysis, adds extraction solvents, conducts shaking extraction, performs centrifugal separation, and transfers liquids. We optimized extraction solvents for liquid-liquid extraction of urinary phthalate metabolites and compared the extraction efficiency between manual and automated methods. The analytical performance of the platform was validated and compared with those obtained by manual liquid-liquid extraction and solid-phase extraction methods. We applied the automated liquid-liquid platform for determining urinary phthalate metabolites in the human biomonitoring of 232 health participants and evaluated their association with oxidative stress levels. Urinary phthalate metabolite concentrations showed a clear declining trend with increasing age. Males had significantly higher total urinary concentrations of phthalate metabolites than females. Monobutyl phthalate was the dominant metabolite in urine samples, followed by mono-isobutyl phthalate and monoethyl phthalate, with minor gender differences observed among individual metabolites. Trend tests and Bayesian Kernel Machine Regression analysis showed a significant positive association between urinary phthalate metabolites and the oxidative stress markers 8-hydroxyguanosine and 8-hydroxy-2'-deoxyguanosine, and monobutyl phthalate was identified as the most significant metabolite for the elevated 8-hydroxy-2'-deoxyguanosine levels. The automated liquid-liquid extraction platform exhibited high efficiency and reliability in preparing urinary samples for phthalate metabolite analysis, showing great promise in large-scale sample preparation of human biomonitoring applications.
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