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VIII SIMPOSIO INTERNAZIONALE ICPBR HAZARDS
OF PESTICIDES TO BEES Effetti dell’imidacloprid sulle api Imidacloprid determination using HPLC and GC/MS in several matrices Simona Rossi (1), Anna Gloria Sabatini (1), Severino Ghini (2), Stefano Girotti (2), Roberto Cenciarini (3), Marcello Succi (3) and Pierpaolo Tentoni (3) (1)Istituto
Nazionale di Apicoltura, via di Saliceto 80, 40128 Bologna, Italy. E-mail:
rossi.simona@alma.unibo.it Imidacloprid is a new insecticide-acting active principle with a wide range of action. In fact, it is used not only to kill sucking insects –such as whiteflies and aphids– and chewing insects, but also to control termites and insects infesting domestic animals. Honey bees are very sensitive to this substance, so, in order to verify its possible presence in cultivated lands, two techniques allowing to detect it in several matrices both of animal and vegetable origins have been developed. According to the first one, honey bees (dehydrated or in their natural state) are extracted with dichloromethane, by ultrasonic extraction; the extract is concentrated in a rotavapor concentrator and purified in a small column containing florisilâ . Then, an elution is carried out with two solutions: ethyl acetate/n-hexane (80/20) and acetonitrile that is dried in a rotavapor concentrator and to which 2 ml of acetonitrile are added. The extract obtained is analyzed by HPLC with a UV detector. The quantification of imidacloprid is determined through the external standard method, with a detection limit of 50 ppb. However, developing another technique has been necessary because, as widely reported in the literature, imidacloprid can be often found in the environment not as a simple molecule but as a group of degradation products, many of which are toxic. These observations have led to the application of a method able to quantify all these species as oxidation products. Thanks to the results achieved, it is now possible to determine the initial quantity of imidacloprid through a conversion factor. According to this method, imidacloprid and its degradation products are extracted from the matrix (filters made from paper, bees, grass, flowers, etc.) by using a water-methanol mixture in acid conditions by sulphuric acid. Then, if necessary, the extract is cleaned of the lipidic fraction by hexane extraction. The sample is further cleaned up in a column filled with XAD 4 resin. Then, it is oxidized at a high temperature by potassium permanganate in basic conditions by sodium hydroxide. In these conditions, both imidacloprid and its degradation products are completely oxidized to 6-chloronicotinic acid that is then extracted with methyl tert - butyl ether (MTBE), dried on a rotavapor and to which is added acetonitrile. With the gas chromatography-mass spectrometry detection it is necessary to derivatize the molecule with MSTFA (N-Methyl-N-(trimethylsilyl)trifluoroacetamide). The compound obtained 6-chloronicotinic acid trimethylsilylester can be easily identified in the chromatogram due to the characteristic fragmentation. In this process, the quantification is carried out by the external standard method, as well. The chromatographic analysis could detect quantities of 10 ppb derivatized 6-chloro nicotinic acid. The linearity was excellent (R=0,999) over a wide concentration range (10 ppm ÷ 10 ppb). The data obtained
from the analyses of several matrices (filters made of paper applied
to the sowing machine, grass, flowers, bees, etc.) have allowed us to
determine imidacloprid dispersion in the environment during the period
of the sowing of imidacloprid-treated corn.
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