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Study methodology

Body Burden: The Pollution in Newborns: Study methodology

July 14, 2005

Introduction. For 10 children born in U.S. hospitals between August 11th and September 8th, 2004, the moment of birth was marked not only by a first cradling in parents' arms, but also by a blood draw integral to a benchmark study of industrial pollutants in newborns. In the most comprehensive tests yet conducted on human umbilical cord blood, we analyzed each child's cord blood for a broad battery of industrial chemicals, pollutants, and pesticides — 413 chemicals in total, from nine chemical classes. To our knowledge this work includes the first reported cord blood tests for 261 of the targeted chemicals. Information below describes the components of this new study, detailing the blood collection procedures, sample preparation and analysis methods, and the quality assurance and quality control provisions included in the study design.

Cord blood sample acquisition and storage. The American National Red Cross obtained ten umbilical cord blood samples from live births in U.S. hospitals in August and September 2004. Besides each child's birthday, the Environmental Working Group obtained no identifying information, either personal or geographic, regarding the samples. Samples consisted of between 79 and 121 milliliters (mL) of umbilical cord blood and 35 mL citrate-phosphate-dextrose (CPD) anticoagulant in a 250 mL Baxter Fenwal Blood-Pack unit (Baxter Healthcare Corporation, Deerfield, IL). The 35 mL of CPD anticoagulant consisted of 921 mg sodium citrate, 893 mg dextrose, 105 mg citric acid, 78 mg monobasic sodium phosphate. Samples were shipped to AXYS Analytical Services (Sydney, BC) within 24 hrs of collection in coolers with gel ice packs. Samples were stored at 4 degrees C for up to four weeks until the entire set of 10 samples was received.

Sample preparation. Cord blood samples were transferred from the blood collection bags into measuring cylinders to determine the total sample volume. About 5 mL of the sample was then transferred into a polypropylene tube for perfluorochemical analysis. The remainder was transferred to a glass container. Both portions were stored at -20 degrees C. Concentrations of chemicals in blood samples were computed based on the total sample weight less the weight of the anticoagulant.

Analysis of PCDD/PCDFs, PBDD/PBDFs, PCBs, PBDEs, PCNs. Analyses for the following groups of compounds were achieved on a single 50 gram portion of the blood-anticoagulant mixture: polychlorinated dioxins and furans (PCDD/PCDFs), polybrominated dibenzodioxins and dibenzofurans (PBDD/PBDFs), polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and polychlorinated naphthalenes (PCNs). The sample was first spiked with an extensive suite of 13C labeled surrogate standards including compounds from all of the target analyte groups. The sample was extracted by shaking with 40 mL of ethanol and 40 mL of saturated ammonium sulfate solution followed by liquid-liquid extraction into 150 mL of hexane. The extract was cleaned up and fractionated by a series of adsorption chromatographic columns (silica, alumina, florisil and carbon) and analyzed by gas chromatography with high resolution mass spectrometric detection (GC/HRMS). Five separate analyses were conducted: PCBs following the protocols of EPA Method 1668A; PBDEs following the protocols for EPA Method 1614; PCDD/PCDFs following the protocols for EPA Method 1613B; and, separately, PCNs and PBDD/PBDFs following in-house methods patterned after the EPA 1600 series methods.

GC/HRMS analyses were performed using a Micromass Autospec Ultima magnetic sector high resolution mass spectrometer equipped with a Hewlett-Packard 6890 gas chromatograph. Quantification of target analytes was achieved by isotope dilution quantification using the 13C labeled surrogate standards.

Analysis of organochlorine pesticides. Another 40 gram aliquot of blood-anticoagulant mixture was used for analysis of organochlorine pesticides. The sample was first spiked with multiple 13C labeled surrogate pesticide standards. The sample was extracted by the procedure described above, cleaned up by gel permeation chromatography on BioBeads SX-3 and split into 2 fractions on Florisil. Each fraction (non-polar compounds and polar compounds) was analyzed by GC/HRMS for pesticides. Organochlorine pesticides were analyzed on a Micromass VG70 magnetic sector high resolution mass spectrometer equipped with a Hewlett-Packard 5890 gas chromatograph.

Analysis of Polyaromatic Hydrocarbons (PAHs). PAH analysis was conducted on a 20-gram aliquot of blood anticoagulant mixture. A suite of 2D labeled surrogate standards was added to the sample which was then extracted as described above. The extract was cleaned up by gel permeation chromatography on BioBeads SX-3 and adsorption chromatography on silica. It was then analyzed by gas chromatography with a mass selective detector (GC/MSD) using an Agilent 6890 gas chromatograph coupled to an Agilent 5973N mass selective detector operated at unit resolution in the electron impact ionization mode using multiple ion detection. Quantification of target analytes was achieved by isotope dilution quantification using the 2D labeled surrogate standards.

Analysis of Perfluorochemicals (PFCs). Analysis for perfluorochemicals was conducted on a 2 gram aliquot of sample. The sample was first spiked with two 13C labeled perfluorochemical surrogate standards, extracted with acetonitrile and cleaned up on a C-18 solid phase extraction cartridge. The extract was analyzed by LC/MS/MS using a Micromass Quattro Ultima MS/MS coupled with a Waters 2690 liquid chromatographic system. Quantification of target analytes was achieved by isotope dilution quantification using the 13C labeled surrogate standards.

Analysis for Methylmercury. Analysis for methylmercury was conducted by Flett Research Ltd. (Winnipeg, MB). Approximately 0.3 g of blood-anticoagulant mixture was analyzed by KBr extraction, followed by ethylation, purge and trap and cold vapor atomic fluorescence spectroscopy (CVAFS). The analysis batch included a procedural blank and a reference sample (DORM-2, National Research Council of Canada).

Procedures for quality assurance and quality control (QA/QC). All organic analyses were conducted in accordance with AXYS' accredited QA/QC program. Regular participation in international inter-laboratory calibration programs is a component of this program. Each analysis batch also included a procedural blank, a laboratory blank, blood bag blanks (extractions from ethanol and from q water-corn oil water mixture added to the standardized cord blood container), and an analysis duplicate. 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 data quality. A positive finding was determined as three times the minimum quantifiable area. Subsequently, to correct for possible procedural or equipment contamination, the blank sample analyses were subtracted from the respective determined analyte values.