Abstract
A comprehensive ultra-high-performance liquid chromatography–high-resolution mass spectrometry (UHPLC–HRMS) method was developed and validated for the simultaneous detection and quantification of 171 pesticide residues in wildlife blood samples. The method employed a modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) extraction procedure, optimized for efficient recovery of analytes while minimizing matrix interferences. Chromatographic separation was achieved by optimizing the mobile phase, flow rate, and gradient programme, which significantly enhanced peak separation and stability. The mass spectrometric analysis was performed using a full scan-ddMS² mode in an Orbitrap mass spectrometer, with three intense precursor-to-fragment ion transitions for each pesticide. The method was validated according to SANTE guidelines, assessing linearity, recovery, precision, accuracy, matrix effect and limit of quantification (LOQ). The calibration curves exhibited strong linearity (R² = 0.990–0.999) over the concentration range of 1–250 µg L⁻¹. Mean recoveries for 109 pesticides ranged from 70% to 119% at 25 µg L⁻¹ spiking level, with relative standard deviations (RSD) below 20% for most compounds. LOQs ranged from 5.0 to 59.64 µg L⁻¹. The matrix effect was evaluated and addressed through matrix-matched calibration. The developed UHPLC–HRMS method enables sensitive, selective, and reliable quantification of a wide range of pesticides in blood, supporting long-term monitoring of pesticide exposure in wildlife and aiding conservation efforts.
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Acknowledgements
The Research was supported by GZRRC-VANTARA. The authors are greatly thankful to the Director, GZRRC – VANTARA for support. We are thankful to our HOD, Central Lab; Toxicology lab team who supported us. Further, we extend our gratitude to Research Guiding Team.
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Ethical concerns are not needed since all samples were leftover diagnostic samples and no animals were sampled specifically for this study.
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Patil, K.B., Soni, R.V. & Ahire, T.R. Comprehensive method validation for detecting multiple pesticide residues in blood using ultra high performance liquid chromatography-high resolution mass spectrometry.
Sci Rep (2026). https://doi.org/10.1038/s41598-026-49063-4
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DOI: https://doi.org/10.1038/s41598-026-49063-4
Keywords
- UHPLC-HRMS
- Pesticide residues
- Blood matrix
- QuEChERS extraction
- Method validation
Source: Ecology - nature.com
