Development and validation of new HSS-UPLC-ESI-MS/MS method for simultaneous quantification of organophosphate and pyrethroid pesticide metabolites in human urine

Organophosphates and pyrethroids are widely used pesticides in agriculture, and might severely affect human health. For the biomonitoring of pesticides, sensitive and accurate analytical methods are essential, yet methods for simultaneous detection and quantification are limited. We present a newly developed and validated Ultra-Performance Liquid Chromatography tandem Mass Spectrometry (UPLC-MS/MS) method that leverages the capabilities of the UPLC HSS T 3 column to accurately quantify, in human urine, metabolites of commonly used pesticides with different polarities. The targeted metabolites include malathion dicarboxylic acid (MDA), 3,5,6-trichloro-2-pyridinol (TCPy), 3-phenoxybenzoic acid (3-PBA), and 4-fluoro-3-phenoxybenzoic acid (4-F-3-PBA) . Analytes were identified by selecting specific ion transitions within a defined retention time window. Solid-phase extraction was employed for sample clean-up, utilizing Oasis HLB cartridges as the sorbent and acetonitrile as the elution solvent. Method validation included the assessment of selectivity, linearity, sensitivity, accuracy, precision, recovery, and matrix effect. The optimization resulted in recovery rates ranging from 60% to 113%, accuracy within 80–120%, and a Relative Standard Deviation below 20% at the lower limit of quantification (LLOQ) for the majority of analytes. The established LLOQs were 0.5 μg L −1 for MDA and 3-PBA, and 0.25 μg L −1 for TCPy and 4-F-3-PBA. Finally, the method was applied to a batch of 26 urine samples from Moroccan farmworkers, resulting in detection frequencies that exceeded 80% for all metabolites. Our method demonstrated high sensitivity in simultaneous quantification of pesticide metabolites, and showed successful applicability to field urine samples, supporting its potential use in human biomonitoring studies. Its application to field samples highlights great robustness for large-scale biomonitoring studies and supports extended risk assessment in human populations.