Beeswax Pesticide Extraction and Purification

Beeswax samples were free of beebread, honey, cocoons, and brood before their analysis. The beeswax extraction method was based on Niell et al. (2014 (link)). Briefly, beeswax pieces of 15 cm² were cut as small as possible and mixed to make a homogenised sample. Then, 1 g of beeswax and 5 mL of acetonitrile were added to a PP (polypropylene) centrifuge tube and the tube was heated in a water bath at 80 °C. When the beeswax had melted, the contents of the tube were homogenised by vortexing for 30 s, followed by sonication for 5 min in an ultrasound water bath at 60 °C. These procedures were repeated five times to ensure an efficient extraction of the pesticides. The sample was then centrifuged at -4 °C and 5000 rpm for 15 min. The supernatant was collected in a PP tube and stored in a freezer at -20 °C overnight, followed by centrifugation to ensure a good separation of the beeswax and the solvent. The supernatant was diluted with acetonitrile (1:1, v/v). An aliquot of the extract was then purified with PSA and C18 (50 mg of each sorbent per ml of extract). The tube containing the sorbents was vortexed for 3 min and centrifuged at 5000 rpm for 10 min. Finally, the supernatant was filtered through a 0.22 µm nylon filter and 1 mL of the filtrate was mixed with 0.01 mL of a solution of acetonitrile with 5% of formic acid in a vial before chromatographic analysis.