1. Environmental Studies - Spring 2005 HPLC Lab (Environ.- & Occu. Med.) The job and the problem You want to do this by using a test (an assay) that responds to hormones and hormone-like substances (the CALUX assay) Consider this: You want to determine the hormone-like effect of xenobiotic compounds found in blood serum Xenobiotic compound: A substance foreign to a living system. 2,3,7,8-TCDD (”Dioxin”) Xenobiotic substance O O Cl Problem 2: The test gives a false positive if hormones are present. Problem 1: Your serum contains a mixture of both hormones and xenobiotic compounds Estradiol (Female sex hormone) Naturally occuring hormone

2. Environmental Studies - Spring 2005 HPLC Lab (Environ.- & Occu. Med.) Sample preparation strategy Two steps are necessary: Removal of interfering substances from serum sample Removal of hormones from the clean serum sample

3. Environmental Studies - Spring 2005 HPLC Lab (Environ.- & Occu. Med.) Sample Waste Wash Water Waste Elute Methanol Serum TCDD Estradiol Removal of interfering substances from serum sample To remove water, salts and proteins from serum This is called sample preparation What is left after this step is a sample of hormones and ”other things” Solid phase extraction (SPE)

4. Environmental Studies - Spring 2005 HPLC Lab (Environ.- & Occu. Med.) Removal of hormones from the clean serum sample Imagine that we have a continous flow of liquid through the tube Flow Because of the flow the mixture moves down the tube Interaction is not equally strong for all components Imagine a tube filled with tiny particles like grains of sand – completely packed The particles in the tube interacts with the mixture High pressure liquid chromatography (HPLC) Now we put a mixture of TCDD and Estradiol on top of the tube Sample with a mixture of TCDD and Estradiol Sample

5. Environmental Studies - Spring 2005 HPLC Lab (Environ.- & Occu. Med.) Removal of hormones from the clean serum sample Imagine a tube filled with tiny particles like grains of sand – completely packed Imagine that we have a continous flow of liquid through the tube The particles in the tube interacts with the mixture Flow Now we put a mixture of TCDD and Estradiol on top of the tube Sample with a mixture of TCDD and Estradiol TCDD Estradiol Separation complete Because of the flow the mixture moves down the tube Interaction is not equally strong for all components Some components moves slower High pressure liquid chromatography (HPLC)

6. Environmental Studies - Spring 2005 HPLC Lab (Environ.- & Occu. Med.) Thank you