In this study, we aimed to develop a method for determination of five neonicotinoids in pollen. We chose acetamiprid, imidacloprid, clothianidin, thiacloprid and thiamethoxam. Determination was performed with HPLC-DAD, which has been previously optimised for analysis of neonicotinoids, we have only checked if it is still viable. Solid-phase extraction (SPE) was used as a pre-treatment method of the sample and needed to be optimised. We have proved that acetone and acetonitrile have significant impact on the recovery of extraction if present in at least 10% (v/v) in starting solution of sample. Limit of detection and limit of quantification of the developed method SPE-HPLC-DAD were estimated by lowering concentrations of standard solutions. Chromatograms of pollen samples were very complex, which means we have not succeeded to extract only neonicotinoids. Nevertheless, we have confirmed a successful extraction with reference UV spectra of standard solutions in artificially polluted samples with theoretical mass percent 2 μg/g in 0.5 μg/g. Maximum recovery was 96.1% and was achieved for acetamiprid at theoretical mass percent 0.5 μg/g. In the very end, we have adjusted the subject of this study to present a smaller part of it as a matter of a lesson in high school. First part of the lesson consists of getting acquainted with chromatography by TLC experiment, which is followed by introduction of new knowledge or revising prior knowledge from the topic Introduction to safe experimental work.
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