Pollution in 5 Extraordinary Women: Study Methodology
Sample acquisition: Environmental Working Group (EWG) scientists obtained Institutional Review Board (IRB) approval for study design and protocols from Chesapeake Research Review, Inc (CRRI) in September, 2007. Each participant submitted a signed Informed Consent document to EWG verifying their understanding of the scope and purpose of the study, and what was required of participants.
Local phlebotomy laboratories collected whole blood and serum samples from each of the 5 participants on a single day in July or August 2008, depending on the participant. Each participant independently collected a first-morning urine void on the day of their blood draw. The samples were frozen upon collection at -20 degrees Celsius and shipped from the phlebotomy labs where they to AXYS Analytical Services (Sydney, British Columbia). All samples were stored at AXYS at 4 degrees Celsius.
Sample preparation: After all samples were received at AXYS, portions of each sample were sent to secondary laboratories for analysis (musks, perchlorate, bisphenol A, and metals). Samples were stored at -20 degrees Celsius prior to extraction and analysis.
Analysis of PBDEs: Samples were analyzed in one batch alongside QC samples including three procedural blanks and a spiked matrix sample. Analysis was performed by AXYS Method MLA-033 Analytical Method for the Determination of Polybrominated Diphenylethers by High Resolution GC/MS. Samples were spiked with the isotopically labeled surrogate standards prior to liquid-liquid extraction with ethanol, saturated ammonium sulphate and hexane mixture. Extracts were cleaned up using AXYS method MLA-033 for Polybrominated diphenylether analysis using an automated fluid management system (FMS) with silica, acid/base layered silica and alumina columns. In addition, samples were given a manual alumina finishing column. Gravimetric lipid analysis was conducted using an aliquot of the extract prior to clean up and analysis and surrogated were corrected accordingly.
The final extracts were spiked with isotopically labeled recovery (internal) standards prior to instrumental analysis. Analysis was performed on a high-resolution mass spectrometer (HRMS) coupled to a high-resolution gas chromatograph (HRGC) equipped with a DB-5HT chromatography column (30 m, 0.25 mm i.d., 0.10 um film thickness). The method was carried out in accordance with the protocols described in EPA method 1614 with some additional internal AXYS guidelines applied.
Calculations for PBDEs: Target concentrations for each analysis were determined by isotope dilution or internal standard quantification procedures using Micromass OPUSQUAN software. Sample specific detection limits (DL’s) were determined from the analysis data by converting three times the height of the average noise signal to a response, using the area/height ratio of the labeled standard, and then to a concentration following the same procedures used to convert target peak responses to concentrations. If the OPUSquan or MassLynx software selected an unrepresentative area for the detection limit calculation, the data interpretation chemist or the QA chemist made corrections.
Analysis of Perfluorinated Chemicals (PFC) and Tetrabromo Bisphenol-A (TBBPA)- AXYS method MLA-049/042 and AXYS Method 4226: Blood samples were spiked with isotopically labeled PFC and TBBPA surrogate, extracted in acetonitrile, cleaned up on SPE cartridges, split into two portions (1) PFC and (2) for TBBP-A and submitted for separate instrumental analysis runs. Samples were analyzed by liquid chromatography/mass spectrometry (LC-MS/MS). Analysis of sample extracts for perfluorinated organics was performed on a high performance liquid chromatograph column (Agilent Zorbax XDB Reverse phase C18, 7.5cm, 2.1mm i.d., 3.5 um particle size (or equivalent) coupled with a triple quadrupole mass spectrometer, running MassLynx v.4.0 software. Final sample concentrations were determined by isotope dilution/internal standard quantification against matrix calibration standards carried through the analysis procedure alongside the samples.
Calculations for PFCs and TBBPA: Target concentrations for each analysis were determined by isotope dilution or internal standard quantification procedures using Micromass MassLynx software. Sample specific detection limits (DL’s) were determined from the analysis data by converting three times the height of the average noise signal to an area using the area/height ratio of the labeled standard, and then to a concentration following the same procedures used to convert target peak responses to concentrations. If the MassLynx software selected an unrepresentative area for the detection limit calculation, the data interpretation chemist or the QA chemist made corrections. Reporting limits were equal to the greater of the lowest calibration standard concentration equivalent or the sample specific detection limit (SDL).
Analysis of BPA- AXYS method MLA-059: Urine samples were spiked with isotopically labeled Bisphenol A and 4-methylumbelliferyl glucuronide solution and B-glucuronidase enzyme (for deconjugation of glucuronidated forms of the target analytes) and extracted and cleaned up using solid phase extraction (SPE). The final extracts were reconstituted with methanol and spiked with isotopically labeled recovery (internal) standards prior to instrumental analysis. Analysis of extracts was performed by LC-MS/MS on a high performance liquid chromatograph (HPLC) coupled with a triple quadrupole mass spectrometer in the Multiple Reaction Monitoring (MRM) mode using a Waters Xterra C18MS, 10.0 cm, 2.1mmi.d., 3.5 um particle with C18 opti-guard column.
Calculations for BPA: Target concentrations for each analysis were determined by isotope dilution or internal standard quantification procedures using Micromass MassLynx software. Sample specific detection limits (DL’s) were determined from the analysis data by converting three times the height of the average noise signal to an area using the area/height ratio of the labeled standard, and then to a concentration following the same procedures used to convert target peak responses to concentrations. If the MassLynx software selected an unrepresentative area for the detection limit calculation, the data interpretation chemist or the QA chemist made corrections. Reporting limits were equal to the greater of the lowest calibration standard concentration equivalent or the sample specific detection limit (SDL).
Analysis of musks: A serum sample is weight into a clean glass 60 ml vial. Methanol, 0.1 M HCI and a set of internal standards (one or more for each group of chemicals) is added to the sample. The sample is extracted three times with a hexane-diethyl ether mixture and centrifuged after each extraction to separate the organic phase. The combined extracts are washed with a 1% KCI-solution and dried with anhydrous sodium sulphate. The serum extract is concentrated to a small volume and purified using a florisil clean-up procedure. The purified extracts are concentrated to a small volume and an injection standard is added. The final extracts are ana lysed with gas chromatography coupled with mass spectrometry (GC/MS) in the selected ion monitoring mode (SIM). The identification of analytes is based on correct retention times and/or qualifier ion ratios, compared to an external standard. The quantification was based on an external standard analysed together with the samples. The recovery of added internal standards (musk xylene-d 15 and tonalide-d3) were used to determine the performance ofthe analysis, but not to correct the results of the target compounds. The results are expressed in ng/g matrix. The matrix is serum and blood.
Analysis of lead: Whole blood samples were diluted 50x with a 1% HNO3. Digests are analyzed using Inductively Coupled Plasma - Mass Spectrometry (ICP-MS) for the analysis of Lead (Pb). Results were blank corrected as per Brooks Rand SOPs for EPA 1638 Modified method.
Analysis of total mercury: All samples were prepared and analyzed in accordance with the Appendix to EPA Method 1631E. Blood samples were first digested with nitric acid/sulfuric acid (HNO3/H2SO4) and further oxidized with bromine monochloride (BrCl). All samples were analyzed with stannous chloride (SnCl2) reduction, gold amalgamation and cold vapor atomic fluorescence spectroscopy (CVAFS) using a BRL Model III CVAFS Mercury Analyzer. Summarized sample results were blank corrected as described in EPA Method 1631 E.
Analysis of methyl mercury: Blood samples were prepared by potassium hydroxide/methanol (KOH/MeOH) digestion followed by distillation. All samples were analyzed by aqueous phase ethylation, Tenax trap collection, gas chromatography separation, isothermal decomposition, and cold vapor atomic fluorescence spectrometry (CVAFS). The samples were analyzed by a modification of EPA Draft Method 1630, as detailed in the BRL SOP BR-0011. All results were blank corrected as described in the method.
Analysis of perchlorate: Urine samples were spiked to a final concentration of 5ppb with an isotopically labeled perchlorate internal standard 5mL of sample was added to 0.5g of Dowex HCR-W2 cation exchange resin that was pre-washed with methanol and water and vortexed for 60s. 0.5 mL of a 10% ammonium hydroxide solution was added to the resulting liquid and the sample was passed through 3cm x 1.5cm diameter column of basic activated alumina. The resulting liquid was passed through a 0.45μm syringe filter and placed into an autosampler vial for analysis. Samples were analyzed using IC-MS/MS using Dionex AS-16 (2mm x 250mm) column with AG-16 guard column. A Quantum Discovery Max ESI-MS with HESI probe was used in the MS/MS mode for quantitation.
QA/QC: All organic analyses were conducted in accordance with AXYS’ accredited QA/QC program including regular analysis of QC samples and participation in international inter-laboratory comparison programs. Each analysis batch included a procedural blank to demonstrate cleanliness and a spiked laboratory control sample to monitor precision and recovery. The sample results were reviewed and evaluated in relation to the QA/QC samples worked up at the same time. The sample surrogate standard recoveries and detection limits, procedural blank data and the laboratory control sample data were evaluated against method criteria to ensure acceptable data quality. The laboratory flagged some values for not meeting certain analytical criteria. These related to ion abundance ratios and the method calibration limit. We used these values, but note the data quality flags in the data section of our Human Toxome website.
Four background samples were analyzed for each of the contaminants studied. Background contamination was detected only in the PBDE family.
The following criteria were applied to account for background contamination:
- Analyses reported as detections in this study exceed the 95% confidence interval and the maximum value for the 4 background samples.
- Where applicable, reported concentrations are adjusted for background concentration. Reported concentrations are taken as the difference between the sample concentration reported by the laboratory and the highest reported concentration in laboratory blank samples.
The number of chemicals detected is reported as a range due to some co-eluting chemicals in the PBDE families. The minimum value in the range considers each co-eluting group of chemicals to represent a single chemical, while the maximum value in the range represents the case where both co-eluting chemicals are present.
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