All project protocols were approved by the University of Southern Maine Office of Research Compliance and Institutional Review Board. Dr. Vincent Markowski and Dr. Richard Donahue, the project’s Principal Investigators, provided oversight of the study methodology, data collection, laboratory testing, and data analyses.
The 13 participants in this pilot survey were selected for diversity in occupations, geography, age, and gender. Trained research assistants met with potential subjects to review project goals and methodologies, answer questions, and complete formal consent documents, including a biographical and demographic questionnaire to provide information about their residences, occupations, diet, and potential toxic exposures.
Samples were collected in June, July and August of 2006 using containers and procedures supplied by the analytical laboratories and NorDx clinical laboratories. Phlebotomists at professional collection centers drew blood samples into vacutainers. Approximately 125 ml of blood was collected from each participant following all necessary safety and sample collection protocols. After clotting, serum was obtained by centrifuging tubes and pouring off or pipetting serum into storage vials. One vacutainer of whole blood was maintained for each participant for lead testing. Staff were present to ensure proper hydration. Samples were processed as necessary, frozen, placed upright in appropriate containers with ice packs, and mailed via overnight courier to the analytical laboratories.
Participants provided first morning void urine samples for phthalate, arsenic and creatinine clearance testing. Urine samples were collected in appropriate containers. Samples were transferred to chemically clean 60 ml glass jars for analysis of phthalates and sterile plastic containers for arsenic testing. Urine samples were refrigerated and mailed overnight by the medical facility to the analyzing laboratory. The analytical laboratories provided collection materials and shipping instructions. Hair samples, used to measure long-term mercury exposure, were cut from the base of the scalp (or beyond, if necessary).
All samples were coded to preserve anonymity of the participants. All samples collected were used solely for this project and will be destroyed at its conclusion.
AXYS Analytical Services, LTD, a private laboratory in Victoria, British Columbia that specializes in trace and ultra-trace detection of environmental contaminants, analyzed urine samples for phthalates and blood serum samples for PBDEs, perfluorinated chemicals, and BPA.
Phthalates. Urine samples were analyzed for phthalate monoesters by AXYS Method MLA-059, Analysis of Bisphenol A and Phthalate Metabolites in Urine by LC/MS/MS. Because phthalate esters in humans are metabolized to their respective monoesters, which in turn may be glucuronidated, urine samples were enzymatically hydrolyzed prior to extraction to convert any monoester glucuronides to their respective free monoesters. Samples were extracted on SPE cartridges, eluted, and analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). 1 mL samples were buffered with ammonium acetate, and spiked with 13C-labeled phthalate monoesters, 13C-labeled 4-methylumbelliferone, native 4-methylumbelliferone glucuronide, and β-glucuronidase enzyme. The treated samples were then incubated to hydrolyze the glucuronides (the completeness of hydrolysis was monitored by the ratio of native to labeled 4-methylumbelliferone). The incubated urine was diluted with high purity water, pH adjusted, and loaded onto pre-conditioned Waters Oasis HLB SPE cartridges, which were washed and then eluted with methanol. Extracts were reduced in volume and spiked with a 13C-labeled recovery standard. Analysis was performed on a Micromass Quattro Ultima MS/MS coupled to a Waters 2795 HPLC equipped with a reverse-phase C18 column (7.5 cm, 2.1 mm i.d., 3.5µm particle size). The LC/MS/MS was operated in the MRM mode at unit resolution, using Negative Ion Electrospray ionization. Phthalate monoester concentrations were determined by the isotope dilution method.
PBDEs. Serum samples were analyzed for polybrominated diphenyl ethers (PBDE) via EPA Method 1614, an HRGC/HRMS method that uses isotope dilution internal standard quantification. 6 mL samples were spiked with 13C-labeled PBDE surrogates and extracted with formic acid. The extracts were loaded onto pre-conditioned Waters Oasis HLB SPE cartridges, which were washed and then eluted with DCM. Extracts were further cleaned-up on silica, reduced in volume, and spiked with 13C-PCB recovery standards. Analysis of the extracts was performed on a Micromass Ultima or VG70 mass spectrometer (MS) coupled to a Hewlett Packard 5890 or 6890 gas chromatograph equipped with a DB-5HT chromatography column (30 m, 0.25 mm i.d., 0.10 µm film thickness). The HRMS was operated at a static (5000) mass resolution in the electron ionization (EI) mode using voltage selected ion recording. PBDE concentrations were determined by isotope dilution or internal standard quantification against the labeled surrogates added at the beginning of analysis using Micromass OPUSQUAN software.
PFCs. Serum was analyzed for PFCs by AXYS Method MLA-042, Analysis of Perfluorinated Organic Compounds (PFC) in Blood Serum by LC-MS/MS. 0.5 mL samples were spiked with 13C-labeled PFCs and extracted with formic acid. Extracts were loaded onto pre-conditioned Waters Oasis WAX SPE cartridges, which were washed and then eluted with basic methanol. The cleaned-up extracts were spiked with 13C-labeled PFC recovery standards, diluted to final volume with methanol, and analyzed by LC/MS/MS. Analysis was performed on a Micromass Quattro Ultima MS/MS coupled to a Waters 2795 HPLC equipped with a reverse-phase C18 column (7.5cm, 2.1mm i.d., 3.5µm particle size). The LC/MS/MS was operated in the MRM mode at unit resolution, using Negative Ion Electrospray ionization. PFC concentrations were determined by isotope dilution or internal standard quantification against the labeled surrogates added at the beginning of the analysis.
BPA. Serum was analyzed for Bisphenol A (BPA) and Bisphenol A diglycidyl ether (BADGE), according to procedures documented in AXYS Method MLA-0056, Analysis of Bisphenol A and Bisphenol A Diglycidyl Ether in Human Blood Serum by Liquid Chromatography–Mass Spectrometry. BPA and BADGE may be present in serum as both the free phenol and glucuronated conjugate. Samples were therefore enzymatically hydrolyzed to convert any glucuronates to the free phenol. BADGE is unstable in aqueous solutions due to hydrolytic ring opening of the two epoxide rings and for this reason BADGE was analyzed as its hydrolyzed product, BADGE-4OH. 1mL samples were spiked with deuterated BPA and 13C-labeled 4-methylumbelliferone, buffered with ammonium acetate, and further spiked with native 4-methylumbelliferone glucuronide, and β-glucuronidase enzyme. The treated samples were then incubated to hydrolyze the BPA and BADGE glucuronides (the completeness of hydrolysis was monitored by the ratio of native to labeled 4-methylumbelliferone). The incubated samples were diluted with high-purity water and loaded onto preconditioned Waters Oasis HLB SPE cartridges. The cartridges were washed with a series of solutions, and then eluted with ethyl acetate. The cleaned extracts were reduced in volume, reconstituted with methanol, filtered, spiked with recovery standard, and analyzed by LC/MS/MS. Analysis was performed on a Micromass Quattro Ultima MS/MS coupled to a Waters 2795 HPLC equipped with a reverse-phase C18 column (7.5cm, 2.1mm i.d., 3.5µm particle size). The LC/MS/MS was operated in the MRM mode at unit resolution, using Negative Ion Electrospray ionization. BPA concentrations were determined by isotope dilution, while BADGE-4OH was determined by internal standard quantification against the labeled BPA added at the beginning of the analysis.
For phthalates, PFCs, and PBDEs, medians were calculated setting non-detectable values at the detection limit divided by the square root of two. Total PBDEs were calculated in the same manner.
Lead in blood samples, methyl mercury in hair samples, and both total arsenic and arsenic species in urine samples were performed by Brooks Rand Labs, a private laboratory located in Seattle, Washington, which specializes in trace level metals analysis.
Arsenic. For total arsenic (As), urine samples were closed-vessel oven digested with nitric acid. Digests were then analyzed by Inductively Coupled Plasma-Dynamic Reactive Cell-Mass Spectrometry (ICP-DRC-MS). For inorganic arsenic species, urine samples were extracted with HCl. Aliquots for inorganic arsenic were adjusted to pH 1.5. Sample aliquots for As(III) were adjusted to pH 6. Samples were then analyzed by hydride generation with NaBH4 reduction, cryogenic trap precollection, H2/Air flame quartz furnace decomposition and atomic absorption detection.
Lead:. Whole blood samples were diluted 50x with a diluent comprised of EDTA, TMAH, ethanol, and Triton X-100 in DI water. Digests were then analyzed by Inductively Coupled Plasma-Mass Spectrometry (ICP-MS).
Methylmercury. Hair samples were cut into ~1 cm segments and then washed to remove contaminants deposited on the surface of the hair. Successive wash and filtering cycles were done with Triton-X, acetone and deionized water, followed by oven drying. Washed hair samples were then digested in a KOH/methanol solution. Digestates were then analyzed by ethylation, Tenax trap pre-concentration, gas chromatography separation, pyrolytic combustion and atomic fluorescence spectroscopy.