A validated LLE–LTP–LC–MS/MS workflow for pesticide biomonitoring in human plasma: Application to occupational exposure assessment in rural workers

Low-temperature partitioning liquid–liquid extraction (LLE–LTP) is a promising alternative for preparing samples for liquid chromatography-tandem mass spectrometry (LC–MS/MS) to determine multiresidue pesticides in biological samples. This technique enables simultaneous extraction and cleanup through phase separation at temperatures below freezing (−18 °C). This study reports the development, optimization, miniaturization, and validation of an LLE–LTP method for quantifying 30 pesticides in human plasma using LC–MS/MS. Initially, a factorial screening design was used to identify the most influential variables, followed by a central composite design (CCD) to optimize the volumes of acetonitrile (ACN), ultrapure water, and plasma. A reduced quadratic model was fitted to the experimental data, and statistically significant terms were selected through confidence interval analysis. The optimal extraction conditions were determined to be 500 μL of water, 1000 μL of ACN, and 250 μL of plasma. This was confirmed through constrained numerical optimization, response surface plots, and experimental validation. The method adheres to the validation guidelines set by Brazilian regulations (INMETRO DOQ-CGCRE-008) and international standards (EU SANTE/11312/2021). The developed methodology exhibited a matrix effect (ME < ±20%; p > 0.05), excellent linearity ( r > 0.995), a limit of quantification (LOQ) ranging from 0.01 to 30 ng mL −1 , accuracy of 80%–120%, and precision (RSD < 15%). A direct comparison with the micro-QuEChERS AOAC 2007.1 workflow showed equivalent or improved recovery and precision for most analytes. Finally, the LLE–LTP–LC–MS/MS method was applied to analyze real plasma samples from agricultural workers in Goiás, Brazil. Remarkably, 11 of 21 workers had at least one detectable pesticide signal, with chlorpyrifos quantified at 14 ± 4.6 ng mL −1 in one individual. Therefore, the validated LLE–LTP–LC–MS/MS workflow combines analytical rigor, operational simplicity, and sustainability, making it a practical tool for assessing occupational exposure and biomonitoring. • Development of a robust and environmentally sustainable LLE–LTP method for pesticide biomonitoring. • Successful application of LLE–LTP for multiresidue extraction in human plasma. • Use of multivariate experimental design for method optimization. • Satisfactory analytical performance with recoveries of 80–120%, RSD ≤ 15%, and limits of quantification (LOQs) ranging from 0.01 to 30 ng mL −1 . • Practical application of the method for occupational exposure assessment.