SPE–UHPLC–MS/MS Method for Simultaneous Quantification of 50 Pesticide Biomarkers Across Nine Current-Use Chemical Classes in Human Urine

A comprehensive ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed for the simultaneous quantification of 50 pesticide biomarkers across nine current-use chemical classes in human urine. These classes include organophosphorus insecticides (which encompass dialkyl phosphates and specific metabolites), pyrethroid insecticides, fungicides, neonicotinoid insecticides, herbicides, insect repellents, organochlorine pesticide metabolites, and plant growth regulators. The method employs solid-phase extraction (SPE) for sample preparation, requiring only 0.2 mL of urine. Chromatographic separation was optimized using a Hypersil Gold AQ column, achieving a total run time of 18 min. Mass spectrometric detection utilized polarity switching in electrospray ionization mode with multiple reaction monitoring. Method validation demonstrated satisfactory linearity (R2 > 0.99), high sensitivity with limits of detection ranging from 0.01 to 0.88 ng/mL, and extraction efficiencies between 85% and 113%. Precision and accuracy were within acceptable ranges, with relative standard deviations generally below 15%. The method’s robustness was confirmed through participation in external quality assessment schemes. Application to real samples revealed significant inter-individual variability in pesticide biomarker concentrations, with total measured biomarker levels ranging from 89 to 1242 ng/mL across the 10 individuals analyzed. This method offers comprehensive coverage of current-use pesticide chemical classes, including 30 biomarkers from the U.S. National Health and Nutrition Examination Survey (NHANES) biomonitoring program, and demonstrates improved sensitivity and broader analyte coverage compared to existing methods. The developed assay provides a valuable tool for large-scale biomonitoring studies and environmental health research.