Development of Normal Phase - High Performance Liquid Chromatography (NP-HPLC) fractionation for the isolation of active compounds in sediment sample
Résumé
Aquatic ecosystems are the sink of a wide diversity of compounds, among them endocrine disrupting compounds (EDCs). Many chemicals as hydrophobic compounds are trapped in sediments. Physical disturbance and bioturbation release chemicals into water column allowing them to be bioavailable for aquatic organisms. Investigations were performed on a sediment sample from a small river subjected to agricultural and urbain pressure. Previous studies reported the occurence of multiple endocrine disrupting activities of the crude extract sample. In order to isolate the active chemicals and to identify them, Effect-Directed Analysis (EDA) method was used. This approach aims at progressive isolation of active chemicals detected by bioassays using sequential reduction of complex mixture by fractionation process (solid phase extraction (SPE) and HPLC). The diversity of contaminants (with different physico-chemical properties) present in sediment sample requires multi-step fractionation methods. A preliminary fractionation of the sediment sample by SPE using increasing solvant polarity led to 4 fractions (F1-F4). Each fraction was tested using in vitro cell lines based on luciferase gene reporter. The four fraction have been a biological effect. F2 and F3 were hyperfractionted by reverse phase - HPLC (RPHPLC). But, a part of observed biological activies of F2 was lost with RP-HPLC hyperfractionation. Considering this, normal phase - HPLC (NP-HPLC) hyperfractionation was developed as complementary process of RP-HPLC. This hyperfractionation was performed using NH2 column. The gradient used was from 100% pentane to 100% dichloromethane, and then to 100% acetonitrile at a flow rate of 1 mL/min. This fractionation procedure has been calibrated using a mixture of 17 chemicals with different physico-chemical properties and biological effects. The environmental application is under progress using sediment fractions (non polar fraction F1 and F2). Biological activies of their hyperfractions will be presented as well as first identifications of toxicity-associated compounds.