Green Extraction and Targeted LC-MS Analysis of Biopesticides in Honey Using Natural Deep Eutectic Solvents

Natural Deep Eutectic Solvents (NADES) were synthesized from food-grade components and evaluated as green extractants for the simultaneous recovery and liquid chromatography coupled to quadrupole-Orbitrap mass spectrometry (LC-Q-Orbitrap-MS) analysis of biopesticide residues in a complex matrix like honey. Conventional solid-liquid extraction (SLE) was applied, initially using choline chloride-2,3-butanediol (1:4, molar ratio) as the NADES extractant solvent, before systematically evaluating other NADES formulations. Extraction parameters, such as time (10 min, 20 min, and 30 min), technique (rotary mixing vs. sonication), and NADES composition, namely lactic acid-glucose-water (LGH, 5:1:9, molar ratio), lactic acid-glycerol-water (LGLH, 1:1:3, molar ratio), urea-glycerol-water (UGLH, 1:1:2, molar ratio), and choline chloride-2,3-butanediol (ChClBt, 1:4, molar ratio), were systematically optimized. Rotating agitation for 10 min yielded the highest overall recoveries and was therefore selected as the optimal extraction time. Rotary shaking was chosen over sonication due to its superior performance across both simple and complex matrices. Among the NADES tested, UGLH proved to be the most effective composition for the honey matrix. The analytical method was validated for the honey matrix. Linearity showed excellent performance across the tested concentration range, with R² values above 0.95 for all analytes. Matrix effects were within ±20% for nearly half of the compounds, while a few exhibited moderate matrix enhancement. Recoveries ranged from 50.1% to 120.5% at 500 µg/kg and 1000 µg/kg, demonstrating acceptable extraction performance. Intra-day and inter-day precision showed relative standard deviations (RSDs) below 20% for most analytes. Limits of quantification (LOQs) were established at 500 µg/kg for eight compounds based on recovery and precision criteria. These results confirm the suitability of the proposed NADES-based method for sensitive and reliable analysis of biopesticide residues in honey. When compared to conventional extraction methods, the proposed NADES-based protocol proved to be a greener alternative, achieving the highest AGREEprep score due to its use of non-toxic solvents, lower waste generation, and overall sustainability.