Pesticides are commonly used in agriculture to control pests and improve crop yields, but they pose significant risks to human health and the environment due to potential residues in biological matrices. This study presents an innovative ultrasonicator-aided cartridge-based extraction method specifically designed for detecting pesticides in simulated biological samples, utilizing ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). To address the need for reliable analytical techniques in forensic science, we developed a robust method for determining Tetramethrin (TMT) and Dimethoate (DMT) in simulated gastric fluid and urine samples using UPLC-MS/MS. While various methods have been employed to analyze biological materials with different solvents, salt combinations, and solid-phase extraction (SPE) sorbents, there is limited research focused on optimizing conditions for detecting TMT and DMT using ultra sonicator aided cartridge based extraction method by following design of experiments. Moreover, no previous studies have reported on their detection in simulated urine and gastric fluid. Our approach involved ultrasound-facilitated HLB cartridge-based solid-phase extraction (SPE) to effectively extract the pesticides across a concentration range of 2.5 to 160 ng/mL and showed high sensitivity, precision, and accuracy, meeting the validation criteria established by the Scientific Working Group of Toxicology (SWGTOX). Its practicality and efficiency make it a valuable tool for analyzing pyrethroid and organophosphorus pesticides in forensic matrices, due to its efficient, rapid and straightforward sample preparation process.

This study presents an analytical method for detection and identification of pesticides in simulated biological matrices, i.e., simulated gastric fluid (SGF) and simulated urine sample (SUS). The approach combines the QuEChERS (quick, easy, cheap, effective, rugged and safe) sample preparation technique with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) for pesticide analysis. A modified QuEChERS extraction protocol was developed to enhance pesticide recovery, improving both accuracy and reproducibility in pesticide quantification in seven different concentrations ranging from 2.5 ng/mL to 160 ng/mL. This combined methodology offers a robust tool for the precise detection and characterization of pesticide residues in biological fluids, with promising applications in toxicological analysis and forensic investigations. The developed method demonstrates the ability to reliably detect low concentrations of pesticide residues, establishing a strong foundation for comprehensive pesticide detection in complex biological matrices. As a supporting analysis, the molecular docking simulations were employed to explore the interaction dynamics of selected pesticides with key proteins unique to each matrix–pepsin (PEP) in SGF and Tamm-Horsfall (THF) protein in SUS. These simulations revealed the binding affinities and interaction strengths of the pesticides, providing further insight into their stability and persistence within different biological environments. This molecular perspective enhances the interpretation of pesticide residue behaviour, complementing the analytical results and deepening our understanding of pesticide dynamics in biological systems.