*2.3. Sample Preparation*

Samples were prepared following the QuEChERS (Quick Easy Cheap Effective Rugged Safe) approach using the slightly modified method reported by Anastassiades et al. [17]. To obtain proper homogenization and extraction, the samples were previously pulverized with a crushing mill

(A11 basic IKA-Werke GmbH & Co. KG, Staufen, Germany) cooled with liquid nitrogen. The samples were processed in duplicate as they were subsequently analyzed with both LC and GC techniques. For extraction, one gram of pollen and vegetable matrices, two grams of bees and wax or 5 g of honey, were weighed into a centrifuge tube and 10 mL of water was added. The mixture was vortexed for 5 min, acetonitrile with 0.1% acetic acid (10 mL) was added, vortexed for 20 min and cooled at −20 ◦C for 15 min. To perform the partitioning step, QuEChERS salts EN method (sodium citrate 1 g, sodium hydrogen citrate sesquihydrate 0.5 g, magnesium sulphate 4 g and sodium chloride 1 g) were added and vigorously shaken up and down for 1 min. The mixture was centrifuged and 7 mL of supernatant was transferred to a tube containing purification dispersive SPE Fatty Samples EN salts (magnesium sulphate 900 mg, PSA 150 mg and C18 150 mg). The solution was vortexed for 1 min and centrifuged, and 4 mL of the supernatant was transferred to a clean tube and evaporated to dryness under vacuum at 45 ◦C. The residue was dissolved in 0.5 mL of reconstitution solution, composed of 5 mM ammonium formiate in water with 0.1% formic acid and 5 mM ammonium formiate in methanol with 0.1% formic acid (1:1 *v*/*v*), and PTFE filtered (0.45 μm pore size) for analysis by UPLC-MS/MS (Ultra Pressure Liquid Chromatography coupled with tandem mass spectrometry). Samples analyzed using GC-MS/MS were reconstituted with 0.5 mL of heptane and PTFE filtered (0.45 μm pore size). Both instruments were programmed in MRM (multiple reaction monitor) mode with two selected transitions per molecule.
