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 Appendix A - Methodology Minimize

All project protocols were approved by the University of Southern Maine Institutional Review Board. John Krueger, MS, ChE, the project’s Principal Investigator, provided oversight of the study methodology, data collection, laboratory testing and data analysis. The twenty-five participants in this project were selected for diversity in their towns and occupations. Fifteen of the participants were selected in part because they are either pregnant or have young children, representing two groups most vulnerable to phthalates exposure.

The Principal Investigator and two trained research assistants met with each participant in December 2013 to review project goals and methodologies, answer questions, obtain formal consent, and conduct a biographical and demographic survey. Each participant provided a urine sample using containers and procedures supplied by the Washington Environmental Biomonitoring Survey (WEBS), a project of the Washington State Department of Health. Samples were immediately frozen and then placed upright in an appropriate box with dry ice and shipped overnight to the laboratory. All samples were coded with two separate numbers to preserve anonymity of the participants. All samples collected were used solely for this project and were destroyed at its conclusion.

Through WEBS, the Washington State Public Health Laboratories measured levels of phthalates in the twenty-five urine samples using methods developed and approved by the U.S. Centers for Disease Control and Prevention. Each batch of samples was analyzed for Quality Control Standards before and after analysis, as part of the lab’s Quality Management Plan approved by the College of American Pathologists and Clinical Laboratory Improvement Act.

The parent compounds people are exposed to are phthalate diesters, which are metabolized in humans to their respective monoesters, which in turn may be glucuronidated. Therefore, urine samples were enzymatically hydrolyzed prior to extraction to convert any monoester glucuronides to their respective free monoesters. The test protocol uses addition of standards, spiking solutions, incubation, extraction by solid phase extraction (SPE) cartridges, elution, and analysis by high performance liquid chromatography - electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) - for the quantitative detection of the phthalate metabolites in urine.

The concentration of the individual analytes in each sample is calculated using the calibration curve derived from the known standard mixtures. The final urinary concentrations of phthalate metabolites are adjusted for creatinine, a urinary biomarker. Creatinine correction helps eliminate any dilution bias introduced as a result of some people being more hydrated than others at the time of sampling. The urinary concentrations of phthalate monoesters obtained using this analytical method provide an estimate of recent exposure to phthalates. However, the metabolism of each phthalate is unique and the proportion of monoester metabolite and oxidative metabolites is different for each phthalate. Therefore, similar metabolite concentrations from different phthalates may not reflect similar exposure levels to the parent phthalate compound.

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