A Streamlined Automated Liquid Handling and 96-Well Solid-Phase Extraction for Single Low-Volume Urine Aliquot Multiclass Biomarker Analysis in Human Biomonitoring.

Laboratory automation has gained significant importance in large-scale biomonitoring studies, providing notable advantages in sample handling, pre-treatment, and cleanup processes. This study introduces a validated automated 96-well format method for the simultaneous extraction and analysis of various urinary biomarkers belonging to different chemical classes. These chemical classes, listed in alphabetical order, include compounds or their metabolites belonging to cannabinoids, environmental phenols, organophosphate flame retardants, non-persistent pesticides, polycyclic aromatic hydrocarbons, tobacco, and volatile organic compounds. The automated workflow employs an epMotion 5075 vtc workstation for sample preparation, featuring automated sample aliquoting, enzymatic pre-treatment, and solid-phase extraction cleanup, followed by liquid chromatography-tandem mass spectrometry analysis. Method validation was performed using urine quality control pools spiked with 0.1 to 10 ppb of each analyte. Results demonstrated excellent precision, with coefficients of variation below 20% for both native and internal standard responses across the chemical classes. Recovery rates for spiked samples consistently ranged from 80% to 120%, affirming the method's accuracy and reliability. The consistency in performance across different chemical classes and concentration ranges highlights the robustness of the automated method. The automated system effectively quantified biomarkers at low ng/mL concentrations in native (unspiked) urine samples, demonstrating sensitivity that is appropriate for comprehensive exposure assessment studies.