Mutistep fractionation based on normal phase SPE and reverse phase HPLC (RP-HPLC) for isolation of endocrine disrupting chemicals in environmental extracts
Résumé
Effected Directed Analysis (EDA) approach aims to identify adverse pollutants by reducing the complexcity of environmental matrices. Single hyperfractionation combined to bioassays is usefull to isolate known active chemicals and to direct chemical analyses to these "classical'pollutants. However, although the emergence of promising chemical tools (e.g. Orbitrap), identification of unknown active chemicals is still time and cost consuming due to the complexity of each active fraction (e.g. mixture effect). Hence, further fractionation steps are often needed. The aims of this study was to develop and to test the use of a first pre-fractionation step on SPE that will be followed by a RP-HPLC fractionation. First the separation of 12 EDCs have been evaluated with several elution conditions. Silica cartridges with 4 step elution - heptane, heptane/dichloromethane (50/50, v/v), ethyl-acetate and methanol/water (50/50, v/v) - allowing the best and reproductible isolation of chemicals, have been chosen for further investigations. For these conditions, recoveries were assessed for the mixture alone and for a blank sediment extract spiked with this mixture. Finally, a natural sediment known to exert estrogenic, PXR-like, anti-androgenic and dioxin-like activity was fractionated following these conditions. Good mixture recoveries (74-110 %), were obtained. The fractionation F1 contained only the PCBs and the PAHs, while 4-tert-octylphenol, triphenyl phosphate and fenofibrate were detected only in F2. Finally, steroids, bisphenol A and clotrimazole were found in F3 while F4 contained more polar chemicals. Fractionation on natural sediment allows isolation of TCDD-like activity in F1 and F2 while PAH like activity was detected in F1, F2 and in F3. Then estrogenic compounds were only detected in F2 and F3. Interestingly, the sum of the estrogenic activity found in these 2 fractions is higher than the activity found in the crude extract, sugggesting the occurrence of anti-estrogenic chemicals. Finally, PXR-like activity was mainly detected in F3. this pre-fractionation protocol allows, in the present case study, the isolation of several biological activities. Based on this first isolation directed hyperfractionation has then been undergone, RPHPLC hyperfractionation on C18 has been calibrated for the separation of 35 EDCs with broad range of chemical properties and will be readly used for the isolation of active chemicals in the polar and semi-polar pre-fractions.