Ultrasound-assisted extraction based on QuEChERS of pesticide residues in honeybees and determination by LC-MS/MS and GC-MS/MS.

In this work, 260 pesticide residues, including insecticides, acaricides, fungicides, and herbicides, were extracted from honeybees using the QuEChERS methodology modified by applying an ultrasonic probe, which avoided the homogenization step and reduced the extraction time. Gas and liquid chromatography, both coupled to triple-quadrupole mass spectrometry, allowed the determination of the pesticide residues extracted from the samples. The optimization of the main ultrasonic conditions (sonication amplitude, number of cycles, and time of each cycle) was performed using a Box-Behnken experimental design involving 15 experimental samples. The results obtained with this approach showed that the recoveries were not affected by these experimental parameters for 95 pesticide residues whereas the sonication amplitude was the main factor affecting the recoveries of 107 pesticide residues. The extraction time and the number of cycles affected four and one pesticide residues, respectively. The effectiveness of the ultrasonic-assisted extraction without homogenization of the honeybee samples compared favorably with that for the conventional QuEChERS methodology applied to the same previously homogenized samples. The proposed methodology was validated according to the SANTE/11945/2015 guidelines, with a 5 µg/kg limit of quantitation. Recoveries between 70 and 120% and relative standard deviations lower than 20% were obtained for most analytes. Thirty honeybee samples taken from Spanish apiaries were analyzed using this new methodology. The results revealed the presence of 30 different pesticide residues in the honeybee samples, the highest concentration levels corresponding to certain insecticides/acaricides used by beekeepers to control Varroa destructor. Permethrin, thiabendazole, carbendazim, and coumaphos were the most frequently detected pesticide residues in the selected samples.