1. Method for screening organic xenoestrogens in biological samples from pregnant women Scientific coordinators: Sef lucr. Dr. CROITORU Mircea Sef lucr. Dr. FÜLÖP Ibolya Author: ZECHERU Ioana Luciana Co-authors: MODROIU Adriana HADMA Roxana Maria

2. Motivation Xenoestrogens are widely used industrial compounds They have estrogenic effects on a living organism even though their chemical structure differ from the estrogenic substances produced by the endocrine system

3. Objectives Developing a HPLC method suitable for determination of the xenoestrogens from pregnant womens'urine and placental blood Identifying xenoestrogens from cosmetic care products and consumer goods in order to test the method.

4. Presentation’s structure 1.Chemical classification 2.Sources of xenoestogens 3.Laws regarding acceptable daily intake 4. Mechanism of toxicity 1.Material şi metodă 1. Material and Method 2. Results 3.Conclusions I. General presentation II. Experimental procedures

5. I.2. Sources of xenoestrogens I.1. Chemical classification Phtalic acid esthers P-aminobenzoic acid esters Phenol derivatives

6. I.3.Legislation U.E. Legislation is highly permissive BPA was forbidden in U.E. back in 2009 but in 2010 this law was repealed Phtalates were forbidden in U.E. only for plastics intended for children use. There are no restrictions on the use of parabens in cosmetics so far.

8. I.4.Toxicity Amiotrophic lateral sclerosis Increased obesity prevalence Decrease in anogenital distance among male infants with prenatal phthalate exposure. Insulin resistance (IR) Increased breast cancer prevalence Erectile dysfunction

9. II.1. Material and method Xenoestrogens were identified using a reverse phase HPLC method with UV detection. We diluted samples using methanol. Before injecting, samples were diluted with high purified water 1:10.

10. Final method Mobile phase: -Aqueous phase pH=2.5 with glacial acetic acid - Acetonitrile(ACN) - Injection volumes : 10 µl - Spectrum : 200-600 nm - Analysis time: 46 mins - We analized chromatograms at 205 i 234 nm wavelength Time(min)% Sol pH=2.5% ACNinlow ml/min 085151.000 3370301.000 3540601.000 4220801.000 4520801.000 4685151.000

11. II.2.Results

12. Developing analitical method Propilparaben 2,5 diclorfenol 2,4 diclorfenol Monobutil ftalalt

13. Developing analitical method pH=2.5 pH=4.0 pH=6.0

14. Identifications

16. -All of them have environmental sources of xenoestrogens -All of them drink water from PET (polyethylenterephtalate)

17. Conclusions We developed a HPLC method suitable for 12 xenoestrogens Those 12 xenoestrogens will be determined from urine colected monthly during IInd and IIIrd trimester of pregnancy and placental blood from 40 volunteers, pregnant women with male fetuses We expect to find some correlation between intrauterine exposure to xenoestrogens and anogenital distance in new born boys. Pregnant women are highly exposed to main xenoestrogens without even knowing. All the subjects considered useful special warnings on the label.

20. Thank you for your attention! Special thanks for support Dr. Andrei Sorin!