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Methodology

Overview

The mothers and daughters tested in this study were part of a cohort of 22 U.S. adolescents and adults ranging in age from 17 to 71, whose blood and urine were tested for 70 common consumer product chemicals. Samples were collected in November and December of 2005, and analyzed at AXYS Analytical in Sydney, British Columbia and BrooksRand in Seattle, Washington. Sampling and analytical protocol are described below.

Sample acquisition and storage

Urine acquisition and storage. Study participants were instructed to collect their first morning urine in a 120 mL amber glass jar. Samples were placed into two zip-lock plastic bags, and then placed on ice and/or refrigerated until shipping.

Blood acquisition and storage. Approximately 100 ml of blood were drawn each study participant into a series of glass and plastic vacutainer tubes. The blood used for lead and mercury analysis was drawn into a 7 mL plastic tube containing EDTA anti-coagulant, inverted 20 times, and placed on ice until shipping. The blood used for perfluorinated chemical analysis was drawn into a 10 mL plastic tube, and left to rest at room temperature for an hour before being centrifuged; the blood serum was then decanted into another plastic tube and placed on ice until shipping. The blood used for bisphenol A and polybrominated diphenyl ether analysis was drawn into eight, 10 mL glass tubes, and left to rest at room temperature for an hour before being centrifuged; the blood serum was then decanted into two amber glass tubes and placed on ice until shipping.

Analysis of samples

Analysis of phthalate monoesters in urine samples by LC/MS/MS. Urine samples were spiked with a suite of 13C-labelled surrogate standards prior to enzymatic hydrolysis with b-glucuronidase. The deconjugated samples were cleaned up by solid phase extraction (SPE) and spiked with 13C-labelled recovery (internal) standards prior to analysis by liquid chromatography-mass spectrometry (LC/MS/MS).

Analysis of sample extracts for phthalate monoesters was performed on a high performance liquid chromatograph with a Micromass Quattro Ultima triple quadrupole mass spectrometer and a data system running MassLynx 4.0 software for targeting and quantification. The MS was run at unit mass resolution with negative ion electrospray ionization. Data were acquired in the Multiple Reaction Monitoring (MRM) mode. A Reverse Phase C18 column (10.0 cm, 2.1 mm i.d., 3.5 µm particle) was used for separation of the targets.

Concentrations of target analytes were determined using isotope dilution or internal standard quantification. Concentrations were determined using weighted linear regression calibration procedures. Target analytes determined were: Monomethyl phthalate (mMP); Monoethyl phthalate (mEP); Mono-n-butyl phthalate (MBP); Monobenzyl phthalate (mBzP); Mono-2-ethylhexyl phthalate (mEHP); Mono-(2-ethyl-5-oxohexyl) phthalate (DEHP Metabolite VI) (mEOHP); and Mono-(2-ethyl-5-hydroxyhexyl) phthalate (DEHP (Metabolite IX) (mEHHP).

Analysis of perfluorinated organic compounds (PFCs) in blood serum by LC/MS/MS. Blood serum samples were spiked with a suite of 13C-labelled surrogate standards prior to extraction and cleanup. Samples were extracted in formic acid and cleaned up using an SPE cartridge. Final extracts were spiked with 13C-labelled recovery (internal) standards prior to analysis by liquid chromatography-mass spectrometry (LC/MS/MS).

Analysis of sample extracts for perfluorinated organics by LC/MS/MS was performed on a high performance liquid chromatograph coupled with a Micromass Quattro Ultima triple quadrupole mass spectrometer and a data system running manufacturers MassLynx v.4.0 software for targeting and quantification. The MS was run at unit mass resolution with negative ion electrospray ionization. Data are acquired in the Multiple Reaction Monitoring (MRM) mode. Chromatographic separation was achieved using a Reverse Phase C18 column (10.0 cm, 2.1 mm i.d., 3.5 µm particle size).

Target compounds were quantified using the isotope dilution or internal standard quantification against matrix matched calibration standards carried through the analysis procedure alongside the samples. Concentrations were determined using weighted linear regression calibration procedures. Target analytes determined were: Perfluorobutanoate (PFBA); Perfluorobutanesulfonate (PFBS); Perfluoropentanoate (PFPeA); Perfluorohexanesulfonate (PFHxS); Perfluorohexanoate (PFHxA); Perfluorooctanesulfonate (PFOS); Perfluoroheptanoate (PFHpA); Perfluorooctane sulfonamide (PFOSA); Perfluorooctanoate (PFOA); Perfluorononanoate (PFNA); Perfluorodecanoate (PFDA); Perfluoroundecanoate (PFUnA); and Perfluorododecanoate (PFDoA).

Analysis of lead in whole blood by EPA Draft Method 1638, Modified. Blood samples were prepared with nitric acid (HNO3) and heated in an oven overnight prior to analysis. This method incorporates ionization of the sample in inductively coupled RF plasma, with detection of the resulting ions by mass spectrometer on the basis of their mass-to-charge ratio.

Analysis of total mercury in whole blood by EPA Method 1631, Rev E. 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 total monomethyl mercury in whole blood by EPA Draft Method 1630 and modifications. 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 total bisphenol A and bisphenol a diglycidyl ether in blood serum by LC/MS/MS. Blood serum samples were spiked with perdeuterated surrogate standard prior to enzymatic hydrolysis with b-glucuronidase. The deconjugated samples were cleaned up by solid phase extraction (SPE) and spiked with a 13C-labelled recovery (internal) standard prior to analysis by liquid chromatography-mass spectrometry (LC/MS/MS) for bisphenol A (BPA) and bisphenol A diglycidyl ether (BADGE).

Analysis of sample extracts for BPA and BADGE by LC/MS/MS was performed on a high performance liquid chromatograph coupled with a Micromass Quattro Ultima triple quadrupole mass spectrometer and a data system running MassLynx 4.0 software for targeting and quantification. The MS was run at unit mass resolution with negative ion electrospray ionization. Data were acquired in the Multiple Reaction Monitoring (MRM) mode. A Reverse Phase-C18 column (10.0 cm, 2.1 mm i.d., 3.5 µm particle size) was used for separation of the targets.

BPA and BADGE were quantified using the isotope dilution method of quantification. Concentrations were determined using weighted linear regression calibration procedures.

Analysis of brominated diphenylethers (BDEs) in blood serum by HRGC/HRMS. Blood serum samples were spiked with a suite of 13C12-labelled surrogate standards prior to extraction. The samples were extracted by shaking with a solution of ethanol, hexane and ammonium sulphate and then backwashed with reagent water to remove residual ethanol.

The final extracts were cleaned up on an automated (Fluid Management Systems, Inc 'Power-PrepTM') system using acidic silica, layered acid base silica, Florisil and alumina chromatographic clean up columns. The resulting extract was reduced in volume and spiked with labelled recovery (internal) standard prior to instrumental analysis.

Instrumental analysis was performed on a Micromass Ultima high resolution MS equipped with a Hewlett Packard 6890 GC and a CTC autosampler. Chromatographic separation is achieved using a DB-5 HT (30 m, 0.25 mm I.D., 0.1 µm film); a split/splitless injection sequence is used. The MS was operated at a mass resolution of 5000 (static) in the electron impact mode using multiple ion detection, acquiring at least two ions for each target and surrogate compound.

Concentrations of target analytes were calculated using the isotope dilution (internal standard) method of quantification in accordance with EPA Method 1614B. Compounds were quantified by comparing the area of the quantification ion to that of the corresponding 13C-labelled standard and correcting for response factors.