Validation d’un procédé automatisé d’extraction par SPE pour identifier et quantifier les dinophysistoxines en LC/ESI/SM/SM par piégeage d’ions

A new extraction method (SPE) for detection and quantification of dinophysistoxins (DTXs) using highperformance liquid chromatography coupled to mass spectrometry with an ion trap and electrospray interface (HPLC/ESI/MS2) was elaborated. This report presents development and validation of this method. Parameters of the ESI source and ion trap spectrum analyser were optimized providing detection of DTXs with high sensitivity. These improvements were obtained after reverse-phase separation on a C18 column, with a first elution to 0.1% TFA (75:25, v/v) in isocratic acetonitrile /water mode without flow split with a column flow rate of 200 Jl/min for 15 min. A second elution gradient step was added allowing an optimised processing of long series of analyses without signal obstruction. Validation of this new method (specificity, detection and quantitation limits, linearity, accuracy) was performed on two distinct matrices: mussel digestive glands analysed after a classical liquid/liquid extraction procedure and natural phytoplankton analysed after extraction in solid phase on silica cartridges. This new method reduced quantification limits from 0.5 ng (using classical spectrofluorescence detection with HPLC (HPLC/F)) to 0.05 ng injected, i.e. allowing detection of 0.011 Jg okadaic acid (OA) per gram of crude mussel digestive gland extract. As well, 2 pg.cell-1 OA can be detected for a crude phytoplankton extract of Dinophysis spp with 50 cells/litre,. Combination of HPLC techniques and Paul ion trap mass spectrometer evidences toxin traces analogs to DTX1. Their abundance increases during DTX1 ageing, unlike observations performed on AO toxin. It is noteworthy that observed consecutive losses of H2O from the protonated toxin present a fingerprint similar to that observed during non-ageing DTX1 analyses suggesting that they could be isomer of DTX1.