Analysis of pesticide residues in honeybees, pollen and beeswax by QuECHERS extraction and LC-MS/MS determination

Abstract

Pesticides affect several non-target beneficial insect, such as honeybees, being not only able to kill them but also to contaminate beehive products. This study aimed at developing multiresidue methods for monitoring 62 pesticides and degradation products in honeybees, pollen and beeswax by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Pollen and honeybee samples (5 g) were extracted following a modified QuEChERS protocol using acetonitrile (10 mL) followed by a dispersive solid phase extraction clean up with PSA and C18 (50 mg) to remove sugars, fatty acids and nonpolar interferences [1, 2]. Beeswax samples were also extracted with acetonitrile by melting wax in a water bath at 80 ºC and then, the extract was cool (−18 °C, for at least 2 h) for precipitaaon of the wax. Finally, 25 mg PSA and 25 mg C18 were added for clean-up and pH was adjusted to 5 [3]. The resulted extracts were then analysed by LC-ESIMS/MS in positive mode. Separation was carried out on a Luna C18 column (150 × 2.0 mm, 3 µm) using a gradient elution profile with mobile phase consisting of water-methanol both, 10 mM ammonium formate. The two most intense precursor ion → product ion transiaons were monitored to obtain unambiguous confirmation of the compound identity. The sensitivity of the methods was estimated by establishing the limits of detection (LODs) and quantification (LOQs). In pollen matrix LODs were lower than 2 ng·g-1 and LOQs were below 5 ng·g1 for all pesticides. Honeybees matrix showed LODs from 0.3 to 3 ng·g-1 whereas LOQs ranged from 1 to 10 ng·g-1. In beeswax, LODs were from 0.3 to 4.2 ng·g-1, whereas LOQs ranged from 1 to 12.5 ng·g-1. Precision, expressed as relative standard deviation (RSD), was < 20% in most pesticides analyzed. Matrix effects were mostly suppressive in the three matrices and ranged from -60 to 50% over the response of the standards prepared in solvent. The average recoveries values were 89, 70 and 83% for pollen, honeybees and beeswax, respectively. Wax and pollen were the most contaminated matrices and exhibited a wide contamination by pyrethroids and organophosphates. Acaricides used in beekeeping, such as coumaphos, chlorfenvinphos, amitraz and fluvalinate were the most frequently detected pesticides in wax. Neonicotinoid acetamiprid and organophosphates chlorpyrifos and dimethoate were detected in pollen samples. Honeybee samples were less contaminated, although some acaricides and insecticides were found in this matrix