Quantification of emerging micropollutants in an amphipod crustacean by nanoliquid chromatography coupled with mass spectrometry in MRM3 mode
Résumé
Over the last decades, human activities (industries, agriculture, transports, consumption, urbanization…) and their impacts on the environment have not ceased to increase. Many molecules related to these activities are released to the environment and their occurrences within aquatic ecosystem become an important issue. In particular, the dose-response relationships of emerging pollutants on organisms at lower trophic levels remain little-known. In order to conduct biomonitoring studies, it is necessary to acquire knowledge about the kinetics of bioconcentration and depuration of these molecules within species, taking into account the inter-individual variability. In this context, an innovative analytical method was developed to quantify, in an amphipod crustacean (Gammarus fossarum), three emerging micropollutants known as anthropic pollution markers: carbamazepine, oxazepam and testosterone which are respectively an anticonvulsant, an anxiolytic and an endogenous hormone. A salting out liquid-liquid extraction, known as QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) was miniaturized and optimized. The miniaturization of the method made it possible to adapt to the low mass sample (approximatively 5 mg dry weight) and to perform the analysis at the scale of a single organism. Beside an efficient sample preparation, ultra-trace analysis of compounds in such matrices of small size requires a good sensitivity. Therefore, an original coupling was developed. It consisted of a separation by nanoliquid chromatography (nanoLC) coupled to tandem mass spectrometry using multiple reaction monitoring cubed mode (MRM3). For the nanoLC separation, a preconcentration system by on-column trapping was optimized to increase the injection volume. In order to keep both sensitivity and selectivity, detection is usually performed by mass spectrometry in multiple reaction monitoring mode (MRM). Nevertheless, proteomic studies showed that sensitivity and selectivity could be improved using MRM3 mode. This technic was developed in this project and compared to the classic MRM. In the best of our knowledge, this was the first time that MRM3was used for small molecule ( The analytical strategy developed allowed to obtain limits of quantification lower than 1 ng.g-1 (wet weight) and to establish the kinetics of bioconcentration of contaminants within G. fossarum.