Analysis of neonicotinoid pesticides and their metabolites by liquid chromatography-electrospray ionization-tandem mass spectrometry.

Neonicotinoid insecticides were first introduced in the 1990s as an alternative to older, more toxic types such as organophosphates, carbamates, and chlorinated hydrocarbons and have since become the most widely used class of insecticides globally. However, their use comes at a significant cost due to high toxicity to pollinators such as bees, beneficial predatory insects, and even insectivorous birds. To further the study of neonicotinoid effects, we developed a method for the LC-ESI-MS/MS (liquid chromatography-electrospray ionization-tandem mass spectrometry) analysis of imidacloprid, clothianidin, and nitenpyram. Standards enabled optimization of tandem quadrupole (MS/MS) detection for specific multiple reaction monitoring (MRM) method fragmentations and separation was achieved by UPLC (ultra-high performance liquid chromatography). However, mammalian samples that tested positive for neonicotinoids on enzyme-linked immunosorbent assays (ELISAs) disclosed none of the parent compounds by the LC-ESI-MS/MS method. Upon researcher request, neonicotinoid metabolites 6-hydroxynicotinic acid; 2-chloro-1,3-thiazole-5-carboxylic acid; 2-imidazolidone; 6-chloronicotinic acid; imidacloprid urea; imidacloprid olefin; desnitro-imidacloprid; and 5-hydroxyimidacloprid were included. The method was validated for linear response, limits of detection/quantitation, retention time stability, instrumental carryover, intraday- and interday-precision, standard curve stability, accuracy, and specificity. Limits of quantitation were measured at 0.1 ppb for the majority of compounds, although desnitro-imidacloprid and 5-hydroxyimidacloprid ranged above 1 ppb. The method was applied to extracted produce samples as a pilot study to test the method's utility. Red grapes contained imidacloprid, while pickles contained imidacloprid and clothianidin. Of concern to researchers studying the collapse of honeybee colonies, two of four samples of honey contained the imidacloprid metabolite 6-hydroxynicotinic acid at levels above 5 ppb.