A robust and sensitive multiresidue analytical method based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was developed and validated for the simultaneous determination of 40 fungicides from diverse chemical classes, including strobilurins, triazoles, imidazoles, benzimidazoles, carbamates, dicarboximides, pyrimidines, and anilinopyrimidines. Target analytes included commonly used active substances such as azoxystrobin, boscalid, carbendazim, cyazofamid, prochloraz, and tebuconazole. These fungicides are widely applied in agriculture for fungal disease control but require strict monitoring due to health and environmental concerns.

Sample preparation utilized an optimized QuEChERS extraction protocol combined with dispersive solid-phase extraction (d-SPE) cleanup to reduce matrix interferences and ensure high analyte recovery. Chromatographic separation was achieved on a reversed-phase C18 column using gradient elution of water and methanol/acetonitrile with 0.1% formic acid, with occasional addition of ammonium formate to enhance peak shape and ionization efficiency.

Detection was performed using electrospray ionization (ESI) in both positive and negative ion modes with scheduled multiple reaction monitoring (sMRM), enabling selective and sensitive quantification of structurally diverse fungicides in complex matrices.

Method validation followed SANTE/11312/2021 guidelines, showing excellent linearity (R² > 0.99), recoveries between 70% and 120%, repeatability with relative standard deviations (RSD) below 20%, and limits of quantification (LOQ) below 10 µg/kg for most analytes. Matrix effects were evaluated and compensated using matrix-matched calibration.

This validated LC-MS/MS method provides a powerful tool for routine high-throughput monitoring of fungicide residues in food and environmental samples, supporting regulatory compliance with maximum residue limits (MRLs) set by the European Union and other authorities.