Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2016 Sep 10.
Published in final edited form as: Anal Chim Acta. 2015 Aug 25;892:140–147. doi: 10.1016/j.aca.2015.08.026

“One-shot” analysis of polybrominated diphenyl ethers and their hydroxylated and methoxylated analogs in human breast milk and serum using gas chromatography-tandem mass spectrometry

Deena Butryn a, Michael Gross a, Lai-Har Chi b, Arnold Schecter c, James Olson b, Diana Aga a,*
PMCID: PMC4589300  NIHMSID: NIHMS724032  PMID: 26388484

Abstract

The presence of polybrominated diphenyl ethers (PBDEs) and their hydroxylated (OH-BDE) and methoxylated (MeO-BDE) analogs in humans is an area of high interest to public health due to their neurotoxic and endocrine disrupting effects. Consequently, there is a rise involving the investigation of these three classes of compounds together in order to understand their bioaccumulation patterns in environmental matrices and in humans. Analysis of PBDEs, OH-BDEs, and MeO-BDEs using liquid chromatography-mass spectrometry (LC-MS) can be accomplished simultaneously, but detection limits for PBDEs and MeO-BDEs in LC-MS is insufficient for trace level quantification. Therefore, fractionation steps of the phenolic (OH-BDEs) and neutral (PBDEs and MeO-BDEs) compounds during sample preparation are typically performed so different detection techniques can be used to achieve the needed sensitivities. However, this approach involves multiple injections, ultimately increasing analysis time. In this study, an analytical method was developed for a “one-shot” analysis of 12 PBDEs, 12 OH-BDEs, and 13 MeO-BDEs using gas chromatography tandem mass spectrometry (GC-MS/MS). This overall method includes simultaneous extraction of all analytes via pressurized liquid extraction followed by lipid removal steps to reduce matrix interferences. The OH-BDEs were derivatized using N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide, producing OH-TMDS derivatives that can be analyzed with PBDEs and MeO-BDEs by GC-MS/MS in “one shot” within a 25-min run. The overall recoveries were generally greater than 65%, and the limits of detection ranged from 2–14 pg in both breast milk and serum. The applicability of the method was successfully validated on four paired human breast milk and serum samples. The mean concentrations of total PBDEs, OH-BDEs, and MeO-BDEs in breast milk were 59, 2.2, and 0.57 ng g−1 lipid, respectively. In serum, the mean total concentrations were 79, 38, and 0.96 ng g−1 lipid, respectively, exhibiting different distribution profiles from levels detected in the breast milk. This GC-MS/MS method will prove useful and cost-effective in large-scale studies needed to further understand the partitioning behavior and ultimately the adverse health effects of these important classes of brominated diphenyl ethers in humans.

Keywords: Polybrominated diphenyl ethers, Hydroxylated polybrominated diphenyl ethers, Methoxylated polybrominated diphenyl ethers, Breast Milk, Serum, Gas chromatography tandem mass spectrometry

1. Introduction

Polybrominated diphenyl ethers (PBDEs) are additive flame retardants that are mixed with, or coated onto consumer products to prolong flame dispersion. Because these compounds are not chemically bound to the polymer and have low vapor pressures, they are leached into the surroundings more readily [1]. Consequently, PBDEs are ubiquitous in the environment and have been found to bioaccumulate in humans [2]. Furthermore, the hydroxylated (OH-BDE) and methoxylated (MeO-BDE) analogs of PBDEs have become a concern to environmental chemists and toxicologists because of their retention in higher trophic level organisms such as in fish, birds, seals, polar bears, sharks, and humans [3–8]. The OH-BDEs and MeO-BDEs are not commercially produced, but are biotransformation products of PBDEs and are also known to be naturally occurring in the environment [9]. These three classes of brominated diphenyl ethers (BDEs) have also been linked to the development of neurological disorders, and are considered endocrine disrupting chemicals with effects related to developmental delays, disruptions of neurotransmitter release, and cytotoxicity [10–12]. Due to their lipophilic nature, BDEs have all been detected in human blood (maternal and fetal), breast milk, and umbilical cord tissue, and their levels vary depending on age, diet, occupation, and geographical location [13–17]. Therefore, understanding their accumulation patterns can ultimately provide information on their sources and metabolism in humans.

Previously established methods for analyzing PBDEs in human samples often include extraction with a non-polar solvent, sample clean-up, and separation by gas chromatography (GC) or liquid chromatography (LC) [1]. However, inclusion of OH-BDEs and MeO-BDEs necessitates that the sample treatment and instrumental analysis be modified in order to account for these classes of compounds. While simultaneous determination of PBDEs, OH-BDEs, and MeO-BDEs is theoretically possible using liquid chromatography-tandem mass spectrometry (LC-MS/MS), limits of detection (LOD) for PBDEs and MeO-BDEs are much higher than OHBDEs due to their poor ionization efficiencies using APCI [8]. Therefore, to achieve the necessary detection limits, PBDEs, OH-BDEs, and MeO-BDEs have been analyzed separately through fractionation using an acidified silica column and then analyzing the OH-BDEs by LC-MS/MS, and the PBDEs and MeO-BDEs by GC/MS [18]. Another common technique used to separate the neutral (PBDEs and MeO-BDEs) and phenolic (OH-BDEs) analytes involves partitioning with potassium hydroxide [13, 14, 17, 19–22]. However, an added concern of this method is that small amounts of PBDEs can be detected in the phenolic fractions resulting from incomplete separation, which may lead to erroneous results [22]. Gel-permeation chromatography is also used to isolate target analytes from interfering lipids and as well as be able to fractionate compounds based on polarity and size; however this approach is laborious and not environmentally friendly because of the high organic solvent requirement [6, 23].

In the past, PBDEs have been analyzed by GC with an electron capture detector (ECD) because of its inherently high sensitivity for halogenated compounds [24]. However, GC-ECD is less selective than GC-MS because the former relies solely on retention times, and co-eluting compounds cannot be distinguished from each other. Therefore, GC with electron capture negative ionization mass spectrometry (ECNI-MS), high resolution mass spectrometry (HRMS), and triple quadrupole mass spectrometry (QqQ-MS) have gained popularity because of their enhanced selectivity. While ECNI-MS is very sensitive, it generally monitors for the signal of the negative ions of the bromine isotopes m/z 79 and 81. Consequently, structural isomers of BDEs are difficult to differentiate, and isotopically-labeled standards (13C-BDEs) are not useful for quantification. On the other hand, the use of electron ionization (EI) coupled to HRMS or QqQ can take advantage of isotopically-labeled compounds for accurate quantification based on isotope dilution. The latter technique also has the additional advantage of identifying co-eluting BDEs based on the characteristic mass spectral fragmentation obtained from the collision cell [25].

The objective of this study is to develop an efficient extraction, clean-up, and detection method for the simultaneous analysis of 12 PBDEs, 12 OH-BDEs, and 13 MeO-BDEs using gas chromatography with tandem mass spectrometry (GC-MS/MS). Derivatization via silylation with N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) was chosen based on the high analytical responses and distinct fragmentation patterns observed in the derivatives formed [26]. A programmable temperature vaporizer (PTV) injector was optimized to avoid thermal degradation and analyte discrimination of BDEs due to their range of vapor pressures, molecular weights, and degrees of bromination [27]. The GC-MS/MS was operated under selected reaction monitoring (SRM) mode to achieve high selectivity and signal-to-noise ratios. Four paired breast milk and serum samples were used for method validation to demonstrate the applicability of the method in analyzing human samples for trace levels of PBDEs, OH-BDEs, and MeO-BDEs. The novelty of this “one-shot” GC-MS/MS method lies in its ability to simultaneously quantify the three classes of BDEs, with high specificity and sensitivity, without the need for the tedious fractionation that lends to analyte losses. The method can prove useful in epidemiological studies requiring large number of samples for analysis, such as in determining the partitioning behavior of the three classes of BDEs in complex biological samples.

2. Materials and methods

2.1. Chemicals and reagents

Chromatographic silica gel (60 Å, 40–63 um) was purchased from Sorbent Technologies (Norcross, GA). The derivatization agent, MTBSTFA, was obtained from Sigma Aldrich (St. Louis, MO) and analytical standards including all individual PBDEs, OH-BDEs, and MeO-BDEs were purchased from Accustandard (New Haven, CT). A surrogate mix containing nine stable isotope-labeled 13C- PBDEs (13C- PBDE-3, -7, -15, -28, -47, -99, -153, -154, -183) and the internal standard solution of 13C-PBDE-77 were obtained from Wellington Laboratories, Inc. (Guelph, ON, Canada). For method development, the sheep serum was obtained from Quad Five (Ryegate, MT), and human breast milk was donated from lactating mothers in Buffalo, NY. For the analysis of paired breast milk and serum specimens analyzed, volunteers from the Mothers’ Milk Bank in Austin, Texas each donated breast milk and blood serum which were collected in chemically clean containers. More detail on these paired samples have been previously published [28].

2.2. Sample preparation

2.2.1. Extraction

Sheep serum and donated human breast milk were used for all method development and samples were stored at −40° C until extraction. Five grams of homogenized breast milk and one gram of serum were each spiked with a 13C- labeled surrogate mixture as shown in the schematic diagram of the method in Fig. 1. Isopropanol was added to the milk (5 mL) and serum (1 mL) to denature proteins, and then samples were mixed with 7 g of pre-washed Hydromatrix™ (Agilent Technologies, Santa Clara, CA). The extraction procedure was briefly modified based off a previously published method using pressurized liquid extraction with an accelerated solvent extractor (Dionex, Sunnyvale, CA) with 50/50 hexanes:dichloromethane (hex:DCM) at a temperature of 100° C, pressure of 1500 psi, 5 min cycle time performed 3 times, flush volume of 150%, and purge time of 100 seconds, resulting in an extraction volume of 120 mL [29]. Then, lipid content for breast milk was determined gravimetrically by evaporating the extracts down to dryness. Due to the small sample volume of serum, lipid content was determined by calculating total triglyceride and cholesterol levels enzymatically, presented previously by Schecter et al. [28].

Fig. 1.

Fig. 1

Open in a new tab

Schematic diagram showing each step of the sample preparation procedure for the simultaneous detection of PBDEs, OH-BDEs, and MeO-BDEs in breast milk and serum.

2.2.2. Lipid clean-up

The extracts were cleaned-up with a liquid-liquid extraction (2×) using 2 mL concentrated sulfuric acid and 10 mL hexane. The organic layers were combined and evaporated down to 0.75 mL. This extract was then passed through an activated silica column consisting of a self-packed glass cartridge with 1.2 g pre-baked anhydrous sodium sulfate, and 3.2 g acidified silica (44% sulfuric acid), held by a layer of glass wool at the bottom of the cartridge. The PBDEs, OH-BDEs, and MeO-BDEs were eluted with 10 mL 50/50 hex:DCM. Samples were quantitatively transferred to 2 mL amber vials and derivatized by adding 500 µL of acetonitrile and 50 µL of MTBSTFA, followed by a 30-min incubation in an oven at 80° C. The derivatized extracts were then evaporated to dryness and reconstituted with isooctane in 50 uL inserts. Finally, 20 ng mL−1 of 13C-PBDE-77 was spiked into each sample as an internal standard for GC-MS/MS analysis.

2.3. GC-MS/MS analysis

Sample analysis was performed on a Trace GC Ultra coupled to a TSQ Quantum XLS triple quadrupole mass spectrometer (Thermo Fisher Scientific, West Palm Beach, FL). Separation was achieved on a 30-m DB-5HT capillary column, with a 0.25-mm inner diameter, and a 0.10-µm film thickness (Agilent Technologies, Santa Clara, CA.). The oven temperature program was as follow: initial temperature of 120° C was held for 2 min, ramped at 23° C min−1 to 250° C, followed by a second ramp at 3° C min−1 to 275° C, and then finally increased at a rate of 33° C min−1 to a final temperature of 330° C which was held for 5 min. The flow rate was held at 1.2 mL min−1 with helium (99.99% purity) as the carrier gas. For the inlet, the PTV was set at an initial temperature of 89° C and was increased at a rate of 7.5° C min−1 for 0.2 min. The analytes were then transferred to the column at a temperature of 330° C for 0.1 min. The split flow was 60 mL min−1 with a splitless time of 1.13 min.

The GC-MS/MS was initially operated under the full scan mode at 60 eV, scanning from m/z of 100–1000 to determine the retention times and the m/z values of the most abundant ions for each analyte. To optimize for fragmentation ions, product scans were then conducted from 10–50 eV to obtain the collision energies that provided the most abundant signal of the characteristic fragment ion. Collision energies were chosen based on a 5–10% relative intensity remaining for the base peak. Once the m/z values for the transitions were identified, a GC-MS/MS method based on SRM mode was created for the analysis of all PBDEs, derivatized OH-BDEs (OH-TMDS-BDEs), and MeO-BDEs within a 25-min run time. The observed retention times, m/z values of ions monitored, collision energies, recoveries, and method LODs (mLOD) for all analytes in the breast milk and serum matrices are presented in Table 1.

Table 1.

GC-MS/MS parameters of PBDEs, OH-BDEs, and MeO-BDEs and their recoveries (n=2) and method limits of detection in breast milk and serum

Analytes Retention
Time (min)		 Base Peak
(m/z)		 Q1, Q2
(m/z)		 Collision Energy
(eV)		 Recovery (%) mLODs (pg)
PBDEs Breast Milk Serum Breast Milk Serum
PBDE-10 6.98 328 168, 147 18 8 ± 2 2 ± 3 n.d n.d
PBDE-7 7.23 328 168, 147 15 103 ± 4 100 ± 9 5.3 4.2
PBDE-15 7.53 328 168, 142 18 82 ± 2 75 ± 15 3.3 3.1
13C-PBDE-15 7.53 340 180, 151 25 n/a n/a n/a n/a
PBDE-28 8.26 406 248, 246 18 103 ± 1 102 ± 1 7.5 3.2
13C-PBDE-28 8.26 258 150, 178 25 n/a n/a n/a n/a
PBDE-49 9.90 486 326, 328 15 86 ± 3 82 ± 5 3.8 4.9
PBDE-47 10.21 486 326, 328 18 84 ± 2 85 ± 0 6.0 4.0
13C-PBDE-47 10.21 338 230, 228 28 n/a n/a n/a n/a
13C-PBDE-77 (IS) 10.89 498 338, 388 25 n/a n/a n/a n/a
PBDE-100 11.93 564 404, 406 20 86 ± 3 91 ± 2 8.2 7.0
PBDE-99 12.52 564 404, 406 18 91 ± 3 91 ± 1 10.0 11.8
13C-PBDE-99 12.52 416 308, 306 30 n/a n/a n/a n/a
PBDE-85 13.81 564 404, 406 20 92 ± 2 72 ± 6 6.6 9.0
PBDE-154 14.59 484 375, 324 33 82 ± 4 82 ± 4 4.1 10.9
13C-PBDE-154 14.59 496 335, 388 38 n/a n/a n/a n/a
PBDE-153 15.82 484 326, 324 38 77 ± 3 86 ± 4 8.4 12.2
13C-PBDE-153 15.82 496 387, 386 33 n/a n/a n/a n/a
PBDE-138 17.78 484 377, 324 35 88 ± 9 96 ± 6 5.9 8.0
PBDE-183 20.51 564 457, 484 30 77 ± 2 85 ± 5 13.6 9.6
13C-PBDE-183 20.51 576 468, 418 38 n/a n/a n/a n/a
OH-BDEs
2'-OH-BDE-3 8.22 321 166, 227 15 0 ± 0 1 ± 0 n.d n.d
2'-OH-BDE-7 9.37 401 166, 320 20 1 ± 0 8 ± 2 n.d n.d
3'-OH-BDE-7 9.90 401 225, 320 15 0 ± 0 0 ± 0 n.d n.d
3'-OH-BDE-28 12.07 479 148, 399 23 28 ± 0 86 ± 2 12.7 3.8
4'-OH-BDE-17 12.98 479 400, 228 13 3 ± 0 6 ± 1 n.d n.d
6-OH-BDE-47 13.25 559 324, 478 25 89 ± 3 87 ± 5 2.1 6.2
2'-OH-BDE-68 14.28 559 324, 326 23 90 ± 5 79 ± 2 8.5 6.7
5-OH-BDE-47 14.67 559 318, 138 23 104 ± 2 84 ± 1 4.5 3.4
4'-OH-BDE-49 15.94 559 318, 227 23 83 ± 3 84 ± 6 7.6 6.3
6'-OH-BDE-99 16.01 639 324, 326 25 86 ± 1 82 ± 3 3.8 7.9
3-OH-BDE-47 17.24 559 318, 399 25 87 ± 5 77 ± 8 6.1 6.8
4-OH-BDE-42 18.06 559 318, 226 20 80 ± 5 86 ± 4 7.3 2.9
6-OH-BDE-85 18.56 639 404, 402 25 81 ± 3 80 ± 7 4.0 3.8
5'-OH-BDE-99 18.56 639 398, 139 25 94 ± 1 87 ± 2 9.6 6.2
6-OH-BDE-82 18.72 639 404, 402 25 84 ± 2 79 ± 1 6.8 5.7
4-OH-BDE-90 22.60 639 398, 227 25 89 ± 2 84 ± 6 5.5 8.0
MeO-BDEs
2'-MeO-BDE-07 8.08 358 264, 262 20 2 ± 0 18 ± 4 n.d n.d
3'-MeO-BDE-07 8.30 358 198, 183 15 1 ± 0 4 ± 0 n.d n.d
2'-MeO-BDE-28 9.66 436 342, 340 20 74 ± 4 77 ± 0 5.7 5.7
4'-MeO-BDE-17 9.98 436 261, 233 25 71 ± 4 81 ± 1 5.0 5.4
3'-MeO-BDE-28 9.98 436 276, 278 13 48 ± 9 64 ± 3 5.0 5.4
2'-MeO-BDE-68 10.99 516 422, 420 20 87 ± 2 102 ± 3 2.7 10.3
6-MeO-BDE-47 11.36 516 422, 420 23 89 ± 1 98 ± 9 8.8 7.3
3-MeO-BDE-47 11.79 516 356, 358 15 91 ± 4 103 ± 3 2.5 6.3
5-MeO-BDE-47 11.94 516 356, 341 15 85 ± 4 100 ± 5 2.0 4.5
4'-MeO-BDE-49 12.05 516 341, 501 23 90 ± 2 97 ± 4 5.8 5.9
4-MeO-BDE-42 13.23 516 501, 356 20 84 ± 3 90 ± 4 8.0 7.6
5'-MeO-BDE-100 13.63 436 393, 421 25 82 ± 1 74 ± 3 10.3 5.1
6'-MeO-BDE-99 13.87 596 436, 500 20 91 ± 1 84 ± 2 11.6 12.3
3-MeO-BDE-100 14.00 596 421, 393 15 87 ± 5 86 ± 8 8.2 6.7
4-MeO-BDE-90 14.89 596 581,421 15 85 ± 1 85 ± 3 10.1 6.7
Open in a new tab

IS: internal standard

n/a: not applicable

n.d.: not determined due to loss of analytes in sulfuric acid treatment during clean-up step (Figure S-1)

2.4. Quality control

Isotope dilution was used for the quantification of PBDEs using 13C-PBDEs, and a 7-point external calibration curve from 0.75–50 ng mL−1 was used to quantify OH-TMDS-BDEs and MeO-BDEs. Method detection limits ranged from 2–14 pg and were determined based on the standard deviation of the lowest spiked concentration (2.5 ng mL−1) in both serum and milk matrices, along with a signal to noise ratio greater than 3. For positive identification, the retention time of an analyte was set to match that of the standard within ±0.1 min; and isotopic ratios of the monitored m/z had to be within ±15% of the expected ratios of the standard. Intra-day precision of this method proved reproducible with relative standard deviations (RSD) of <8% for all PBDEs, OH-BDEs, and MeO-BDEs in both breast milk and serum. A calibration curve using a concentration range of 0.625 to 360 ng mL−1 showed good linearity resulting in R2 >0.995 for all analytes. The extraction and clean-up steps were performed in a positive pressure laboratory to limit atmospheric contamination. All glassware were pre-washed with Alconox®, rinsed with deionized water, and then submerged in a 2% nitric acid bath overnight for removal of potential contaminants adsorbed on the glass. Reported concentrations were background-subtracted by subtracting the endogenous levels in unspiked samples.

3. Results and discussion

3.1. Sample clean-up

To develop a robust analytical method to detect trace levels of PBDEs, OH-BDEs, and MeO-BDEs in breast milk and serum, appropriate clean-up steps were investigated. Concentrated sulfuric acid and an additional activated silica adsorption column were used to denature proteins and effectively remove interfering lipids that could cause ion suppression in GC-MS/MS analysis. Percent recoveries were generally higher than 65% for tri- to hexa-BDEs, but were much lower (<15%) for mono- and di-BDEs in breast milk and serum (Table 1). To determine the cause of analyte loss, hexane was spiked with 25 ng mL−1 of PBDE-10, 2’-OH-BDE-03, 2’-OH-BDE-07, 3’-OH-BDE-07, 2’-MeO-BDE-07, and 3’-MeO-BDE-07, and individual recoveries were determined either after treatment with concentrated sulfuric acid (18M) or after passing through an activated silica column. Data presented in Fig. S1 indicates that the poor recoveries of lower brominated analytes can be attributed mainly to the analyte losses during treatment with 18M sulfuric acid, even though other factors such as evaporation and elution steps could contribute to analyte loss as well. However, treatment with 18M sulfuric acid is necessary to remove proteins in the samples and reduce the high fat content in breast milk causing matrix effects. Samples that were not properly cleaned-up caused significant decrease in GC-MS/MS signals, as observed from a series of injections that showed loss of sensitivity over time. This is the first study that provides an explanation for the loss of lower brominated BDE congeners that is commonly observed. Since treatment with 18M sulfuric acid in combination with the activated silica column was essential for an effective clean-up, the lower BDE congeners were not quantified in this study. Many studies have not been able to detect lower BDE congeners in biological samples, most likely because of their degradation during sulfuric acid clean-up.

3.2. Instrumental performance

The derivatization step converting OH-BDEs into OH-TDMS-BDEs did not affect the amounts and composition of MeO-BDEs and PBDEs, as depicted in Fig. 1. The selective derivatization of OH-BDEs in the presence of PBDEs and MeO-BDEs is key to the successful development of a “one-shot” GC-MS/MS method that can quantify all PBDEs, OH-TMDS-BDEs, and MeO-BDEs in one run without the need to separate the phenolic BDEs from the neutrals prior to derivatization, which was done in previous methods [13, 14, 17, 20, 30–32].

3.2.1. PTV optimization

The main advantage of using a PTV in splitless mode is the ability to have controlled transfer of analytes from the inlet to the column to achieve lower detection limits and avoid analyte discrimination [27]. In this study, five parameters were investigated: transfer temperature, rate of increase in transfer temperature, evaporation rate, split flow, and splitless time. A transfer temperature of 330° C was chosen because temperatures higher than 330° C resulted in signal reduction due to thermal degradation of analytes in the inlet liner. This coincided with maximum responses at a transfer temperature rate of 7.5° C min−1. No apparent difference was observed with changes in evaporation rate. However, the rate of solvent evaporation should be equivalent to the rate of analyte transfer to prevent flooding onto the column, that may lead to poorly resolved peaks [33]. Therefore, an evaporation rate of 7.5° C sec−1 was chosen along with a split flow of 60 mL min−1 to efficiently trap analytes and to release the solvent through the solvent vent. A splitless time of 1.13 min, defined as the duration of the whole injection step, was long enough to ensure quantitative transfer of all analytes onto the column. All PTV parameters were optimized on a 3-uL injection volume and are presented in Fig. S2.

3.2.2. Mass spectra fragmentation

The mass spectral patterns of BDEs were characterized using the isotopic bromine signature of 79Br and 81Br. The base peak monitored for di- through penta-substituted PBDEs was the molecular ion [M]+, while for any hexa- and hepta-substituted PBDEs the base peak was [M-2Br]+. The fragmentation of PBDEs occurred with a common loss of two bromines except PBDE-138 and PBDE-154, which resulted in the loss of [M-COBr]+. For MeO-BDEs, the base peak was [M]+ for the para- and ortho- MeO-BDE position, and [M-2Br]+ for the meta-MeO-BDE position. The fragmentation patterns of MeO-BDEs in the samples were consistent with the generalized fragmentation patterns reported by Athanasiadou et al [25] for the different positional isomers of MeO-BDEs in electron ionization mode.

For the OH-BDE derivatives using MTBSTFA, the base peak was the loss of the t-butyl moiety [M-C(CH3)3]+. Mass spectra of the OH-BDE derivatives can be seen in Fig. S3. Fragmentation of the ortho-substituted OH-TMDS-BDEs was monitored via cleavage of the ether bond, corresponding to the loss of the opposing phenyl ring and its subsequent bromines from the attached silyl group (Fig. S3a). Furthermore, fragmentation of both meta- and para-substituted OH-TMDS-BDEs shared common losses of an odd number of bromines, as depicted in Figs. S3b and S3c for 5-OH-TMDS-BDE-47, and 4’-OH-TMDS-BDE-49, respectively.

3.3. Analysis of BDEs in human samples

3.3.1. Method validation through PBDE comparison

To demonstrate the applicability of the “one-shot” method in biological samples, four paired human breast milk and serum samples were analyzed. The PBDE concentrations in these paired samples have been previously determined using GC-HRMS, and results have been published [28]. Therefore, these samples were useful in validating the accuracy of this developed GC-MS/MS method. A comparison of breast milk and serum concentrations for the 6 PBDE congeners (PBDE-28, -47, -99, -100, -153, and -154) most commonly detected in humans determined by GC-HRMS and GC-MS/MS shows good correlation (R2=0.75; slope=0.91) in Fig. 2. However, the previous study did not analyze for OH-BDEs and MeO-BDEs in these breast milk and serum samples, and therefore no comparison can be made for these compounds. Nevertheless, it is reasonable to infer based on the results of the spiked samples, and the high correlation between PBDE concentrations determined by the two methods, that the determination of OH-BDEs and MeO-BDEs using this “one-shot” GC-MS/MS method is reliable.

Fig. 2.

Fig. 2

Open in a new tab

PBDE concentrations in paired milk and serum subjects determined using the “one-shot” method developed in this study compared with the values obtained for the same samples using a previously reported method employing GC-HRMS [28]. The PBDE congeners represented here are PBDE -28, -47, -99, -100, -153, and -154. The equation of the line is y = 0.91x + 2.67 with an R2 of 0.75 showing good agreement between the two methods.

3.3.2. Evaluation of PBDEs, OH-BDEs and MeO-BDEs in breast milk and serum

A total of 9 PBDEs were detected in the breast milk and serum samples; and the concentrations are represented in Table 2. PBDE-47 and PBDE-99 were the most abundant congeners detected in both matrices, with concentrations that are 44% (±13%) and 25% (±9%) relative to the other PBDE congeners, respectively. This accumulation reflects the high presence of PBDE-47 and PBDE-99 in the commercial penta- and octa-PBDE mixtures that were used in consumer goods [34]. Total levels of PBDEs in the paired breast milk and serum samples ranged from 16.6–112 and 16.5–146 ng g−1 lipid, respectively.

Table 2.

Concentrations in ng g−1 lipid of PBDEs, OH-BDEs, and MeO-BDEs detected in the four paired breast milk and serum subjects.

Sample #1 Sample #2 Sample #3 Sample #4
Breast Milk Serum Breast Milk Serum Breast Milk Serum Breast Milk Serum
PBDE-28 2.16 ND 1.31 ND 0.554 ND 2.20 1.95
PBDE-49 0.520 <LOD 0.190 <LOD 0.367 1.67 0.415 1.85
PBDE-47 57.2 43.1 19.6 22.2 9.42 2.22 32.6 58.4
PBDE-85 1.84 1.91 0.444 1.30 0.201 0.990 0.564 1.30
PBDE-99 23.5 34.4 6.61 19.2 2.89 6.72 7.53 25.3
PBDE-100 13.1 16.8 3.12 2.06 1.34 2.46 4.93 19.1
PBDE-153 12.0 10.2 2.71 <LOD 1.77 2.42 23.0 38.5
PBDE-154 1.32 1.37 0.234 ND 0.091 ND 0.249 ND
PBDE-183 0.186 ND 0.082 ND ND ND 0.202 ND
ΣPBDEs 112 108 34.3 44.8 16.6 16.5 71.7 146
3’-OH-BDE-28 ND ND ND ND ND ND <LOD ND
3-OH-BDE-47 3.01 ND ND 7.46 ND ND 0.393 11.7
5-OH-BDE-47 0.171 7.53 0.181 0.562 ND 7.42 0.481 9.15
6-OH-BDE-47 0.090 4.68 0.084 4.16 ND 4.37 0.408 5.72
4-OH-BDE-42 ND ND 0.186 10.2 ND ND 0.442 12.6
4’-OH-BDE-49 ND ND ND 5.15 ND 0.668 0.285 7.93
2’-OH-BDE-68 ND ND 0.011 <LOD ND ND 0.180 <LOD
6-OH-BDE-82 0.124 ND 0.14 ND ND ND 0.300 9.14
6-OH-BDE-85 0.116 ND 0.141 ND 0.821 7.63 ND ND
5’-OH-BDE-99 0.311 ND ND 17.2 ND 20.7 0.718 ND
6’-OH-BDE-99 ND ND ND ND ND ND 0.201 ND
ΣOH-BDEs 3.81 12.2 0.743 44.7 0.821 40.8 3.41 56.2
2’-MeO-BDE-28 ND ND 0.059 0.214 ND 0.224 0.360 0.397
3’-MeO-BDE-28 ND ND ND <LOD ND ND 0.344 <LOD
3-MeO-BDE-47 ND ND ND ND ND ND 0.163 0.325
5-MeO-BDE-47 ND ND ND 0.230 ND ND 0.310 0.415
6-MeO-BDE-47 ND <LOD ND 0.174 ND <LOD 0.148 0.176
4-MeO-BDE-42 ND ND ND 0.162 ND ND 0.214 0.416
4’-MeO-BDE-49 0.062 ND ND 0.301 ND ND 0.166 ND
2’-MeO-BDE-68 0.048 ND 0.066 0.240 ND ND 0.284 0.560
ΣMeO-BDEs 0.110 0 0.125 1.32 0 0.224 1.99 2.29
Open in a new tab

ND: not detected

<LOD: Below method limits of detection

Results from these analyses were consistent with previous studies that indicated a dominance of tetra- and penta-brominated OH-BDEs and MeO-BDEs in biological samples [17, 19, 35]. The relative abundances of OH-BDEs were highly variable within individual serum subjects and were detected at 10–100 fold greater levels in serum than in breast milk (Table 2). This pattern of OH-BDE accumulation is very similar to the observed behavior of hydroxylated polychlorinated biphenyls [14]. Note that 5-OH-BDE-47 and 6-OH-BDE-47 had the highest detection frequencies in all samples (88%), and 5’-OH-BDE-99 was detected at the highest concentration (20.7 ng/g lipid). Interestingly, these findings mirror the results from in vitro metabolism studies on PBDE-47 and PBDE-99 in humans [36, 37].

Total MeO-BDE concentrations were much lower than total OH-BDEs, ranging from none detected to 1.99 ng g−1 lipid in breast milk, and none detected to 2.29 ng g−1 lipid in serum. Notably, 2’-MeO-BDE-68, one of the MeO-BDE congeners that has been postulated to be naturally occurring in the environment [5, 7], was the most frequently detected (63%) in the four paired samples, along with 2’-MeO-BDE-28. Sample chromatograms of OH-BDEs and MeO-BDEs detected in the breast milk and serum subject #2 are presented in Figure S-4a and S-4b, respectively.

We summarized the levels of BDEs reported in literature (Table 3), which do not appear to vary amongst men and women, but vary widely based on geographical location. In paired samples, PBDE levels were either similar to or lower than OH-BDEs, specifically in paired blood/cord blood samples (No MeO-BDEs were analyzed) [15, 38]. Since prenatal exposure to BDEs is of major concern, the concentrations of these compounds in blood/cord blood samples can provide insight on the transfer of BDEs from the mother to the infant during pregnancy [15, 17]. One study from Hong Kong, China, reported elevated levels of MeO-BDEs in blood plasma (ng g−1 lipid), which suggested that a seafood-rich diet was the main source of these compounds [19]. All other studies examining MeO-BDEs in either breast milk or serum reported lower levels (pg g−1 lipid) [13, 35, 39]. However, further studies with a larger number of human subjects are necessary to investigate cytochrome P-450 mediated oxidative metabolism and dietary factors which may contribute to the large inter-individual variability in the retention of PBDEs, OH-BDEs, and MeO-BDEs in serum, and the partitioning of these agents into breast milk [36, 37, 40].

Table 3.

Summary showing the ranges of concentrations in ng g−1 lipid of PBDEs, OH-BDEs, and MeO-BDEs in humans reported in literature.

PBDEs OH-BDEs MeO-BDEs Matrix Cohort Location Reference
0.17–29 0.07–11 ND Blood Maternal Indiana, USA Qiu et al. (2009)
0.10–550 0.03–250 ND Blood Fetal Indiana, USA
0.14–91 2.5–140a NA Blood Maternal Ohio, USA Chen et al. (2013)b
0.41–96 2.5–230a NA Cord blood Maternal Ohio, USA
0.99–58.4 0.56–20.7 0.16–0.56 Blood Maternal Texas, USA This study (2015)b
0.082–57.2 0.011–3.01 0.048–0.36 Breast Milk Maternal Texas, USA
0.16–290 0.11–32 NA Pooled blood Children Managua, Nicaragua Athanasiadou et al. (2008)
0.023–1,100 0.021–0.89 0.012–15 Breast milk Maternal Barcelona, Spain Lacorte et al. (2009)
NA NA 1.1–3.0 Breast milk Maternal Bizerte, Tunisia Hassine et al. (2015)
0.09–2.0 NDc 0.25d Breast milk Maternal Okinawa, Japan Fujii et al. (2014)
<LOQ-0.003 0.17c NDd Blood Maternal Okinawa, Japan
11–95e 2.5–51e NA Blood Maternal Kashiwa, Japan Kawashiro et al. (2008)b
17–670e NA NA Breast milk Maternal Kashiwa, Japan
0.57–15e 0.83–11e NA Cord blood Maternal Kashiwa, Japan
0.47–11e 1.6–19e NA Umbilical cord Maternal Kashiwa, Japan
2.0–2,800e 1.2–60e 0.40–6.1e Blood E-waste workers Bangalore, India Eguchi et al. (2012)
1.4–2,700e 0.67–120e 0.54–18e Blood Coastal area residents Chidambaram, India
0.11–18 0.012–0.22 0.50–47 Blood Female Hong Kong, China Wang et al. (2012)
0.40–16 0.062–0.30 0.40–16 Blood Male Hong Kong, China
0.19–230 7.5–360 ND Blood E-waste workers Shantou, China Ren et al. (2010)
Open in a new tab

ND: not detected, NA: not analyzed

a

Units reported in pg mL−1

b

Study involves paired samples

c

Only 6-OH-BDE-47 was targeted

d

Only 6-MeO-BDE-47 was targeted

e

Units reported in pg g−1 wet weight

4. Conclusion

In this study, we developed an efficient method that can simultaneously analyze PBDEs, OH-BDEs, and MeO-BDEs in a “one-shot” GC-MS/MS run. This “one-shot” GC-MS/MS method offers several advantages over existing methods for PBDEs, OH-BDEs and MeO-BDEs such as: (1) simultaneous extraction and clean-up steps that effectively minimize matrix effects, (2) ability to detect PBDEs, OH-BDEs and MeO-BDEs simultaneously in one GC-MS/MS run without the need for multiple injections, ultimately decreasing analysis time, and (3) high efficiency separation by GC coupled with the selectivity offered by MS/MS detection allowing improved LOD and specificity. While the four paired breast milk and serum subjects are limited in sample number, this preliminary data provided insight on the partitioning of PBDEs, OH-BDEs, and MeO-BDEs in humans, which is still not fully understood. The mean concentrations of total PBDEs, OH-BDEs, and MeO-BDEs in breast milk were 59, 2.2, and 0.57 ng g−1 lipid, respectively. In serum, the mean total concentrations were 79, 38, and 0.96 ng g−1 lipid, respectively, exhibiting different distribution profiles from levels detected in the breast milk. The developed “one-shot” GC-MS/MS method described in this paper provides an efficient and cost-effective analytical approach that will be used for further investigations with in large-scale studies to understand the fate, accumulation patterns, and potential health effects of these compounds in humans.

Supplementary Material

NIHMS724032-supplement.docx (270.8KB, docx)

  1. A simultaneous sample preparation method was developed for the “one-shot” analysis of PBDEs, OH-BDEs, and MeO-BDEs in human breast milk and serum.

  2. Method positively correlated with previously published literature on PBDE concentrations in the same paired samples, with r2 = 0.75.

  3. Findings provide insight on the different partitioning behavior of PBDEs, OH-BDEs, and MeO-BDEs in humans.

Acknowledgements

The work described in this paper was supported by the National Institute of Environmental Health Sciences (grant #ES021554). We would like to thank the Austin Milk Bank in Austin, Texas, especially Kim Updegrove and Gretchen Flatau for the donation of the breast milk and serum.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

References

  • 1.Covaci A, Voorspoels S, Boer Jd. Determination of Brominated Flame Retardants, with Emphasis on Polybroinated Diphenyl Ethers (PBDEs) in Environmental and Human Samples- A Review. Environmental International. 2003;29:735–756. doi: 10.1016/S0160-4120(03)00114-4. [DOI] [PubMed] [Google Scholar]
  • 2.Hites RA. Polybrominated Diphenyl Ethers in the Environment and in People: A Meta-Analysis of Concentrations. Environmental Science and Technology. 2004;38:945–956. doi: 10.1021/es035082g. [DOI] [PubMed] [Google Scholar]
  • 3.Verreault J, Gabrielsen GW, Chu S, Muir DCG, Andersen M, Hamaed A, Letcher RJ. Flame Retardants and Methoxylated and Hydroxylated Polybrominated Diphenyl Ethers in Two Norwegian Arctic Top Predators: Glaucous Gulls and Polar Bears. Environmental Science & Technology. 2005;39:6021–6028. doi: 10.1021/es050738m. [DOI] [PubMed] [Google Scholar]
  • 4.Liu J, Luo X-J, Yu L-H, He M-J, Chen S-J, Mai B-X. Polybrominated diphenyl ethers (PBDEs), polychlorinated biphenyles (PCBs), hydroxylated and methoxylated-PBDEs, and methylsulfonyl-PCBs in bird serum from South China. Archives Of Environmental Contamination And Toxicology. 2010;59:492–501. doi: 10.1007/s00244-010-9487-4. [DOI] [PubMed] [Google Scholar]
  • 5.Marsh G, Athanasiadou M, Bergman A, Asplund L. Identification of Hydroxylated and Methoxylated Polybrominated Diphenyl Ethers in Baltic Sea Salmon (Salmo Salar) Blood. Environmental Science and Technology. 2004;38 doi: 10.1021/es034671j. [DOI] [PubMed] [Google Scholar]
  • 6.Berghe MV, Weijs L, Habran S, Das K, Bugli C, Rees J-F, Pomeroy P, Covaci A, Debier C. Selective Transfer of Persistent Organic Pollutants and their Metabolites in Grey Seals During Lactation. Environmental International. 2012;46:6–15. doi: 10.1016/j.envint.2012.04.011. [DOI] [PubMed] [Google Scholar]
  • 7.Wan Y, Wiseman SB, Chang H, Zhang X, Jones PD, Hecker M, Kannan K, Tanabe S, Hu J-Y, Lam MH-W, Giesy JP. Origin of Hydroxylated Brominated Diphenyl Ethers: Natural Compounds or Man-Made Flame Retardants? Environmental Science and Technology. 2009;43:7536–7542. doi: 10.1021/es901357u. [DOI] [PubMed] [Google Scholar]
  • 8.Kato Y, Okada S, Atobe K, Endo T, Matsubara F, Oguma T, Haraguchi K. Simultaneous determination by APCI-LC/MS/MS of hydroxylated and methoxylated polybrominated diphenyl ethers found in marine biota. Analytical Chemistry. 2009;81:5942–5980. doi: 10.1021/ac9009885. [DOI] [PubMed] [Google Scholar]
  • 9.Wiseman SB, Wan Y, Chang H, Zhang X, Hecker M, Jones PD, Giesy JP. Polybrominated Diphenyl Ethers and their Hydroxylated/Methoxylated Analogs: Environmental Sources, Metabolic Relationships, and Relative Toxicities. Marine Pollution Bulletin. 2011;63:179–188. doi: 10.1016/j.marpolbul.2011.02.008. [DOI] [PubMed] [Google Scholar]
  • 10.Meerts I, Letcher RJ, Hoving S, Marsh G, Bergman A, Lemmen JG, Van den Berg B, Brouwer A. In Vitro Estrogenicity of Polybrominated Diphenyl Ethers, Hydroxylated PBDEs, and Polybrominated Bisphenol A Compounds. Environmental Health Perspectives. 2001;109:399–407. doi: 10.1289/ehp.01109399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Dingemans MML, van den Berg M, Bergman A, Westerink RHS. Calcium-Related Processes Involved in the Inhibition of Depolarization-Evoked Calcium Increase by Hydroxylated PBDEs in PC12 Cells. Toxicological Sciences. 2009;114:302–309. doi: 10.1093/toxsci/kfp310. [DOI] [PubMed] [Google Scholar]
  • 12.Su G, Zhang X, Liu H, Giesy JP, Lam MH-W, Lam PKS, Siddiqui MA, Musarrat J, Al-Khedhairy A, Yu H. Toxicogenomic Mechanisms of 6-OH-BDE-47, 6-MeO-BDE-47, and BDE-47 in E. Coli. Environmental Science and Technology. 2012;46:1185–1191. doi: 10.1021/es203212w. [DOI] [PubMed] [Google Scholar]
  • 13.Fujii Y, Nishimura E, Kato Y, Harada KH, Koizumi A, Haraguchi K. Dietary exposure to phenolic and methoxylated organohalogen contaminants in relation to their concentrations in breast milk and serum in Japan. Environmental International. 2014;63:19–25. doi: 10.1016/j.envint.2013.10.016. [DOI] [PubMed] [Google Scholar]
  • 14.Athanasiadou M, Cuadra SN, Marsh G, Bergman A, Jakobsson K. Polybrominated Diphenyl Ethers (PBDEs) and Bioaccumulative Hydroxylated PBDE Metabolites in Young Humans from Managua, Nicaragua. Environmental Health Perspectives. 2008;116 doi: 10.1289/ehp.10713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Chen A, Park J-S, Linderholm L, Rhee A, Petreas M, DeFranco EA, Dietrich KN, Ho S-m. Hydroxylated Polybrominted Diphenyl Ethers in Paired Maternal and Cord Sera. Environmental Science and Technology. 2013;47:3902–3908. doi: 10.1021/es3046839. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Mazdai A, Dodder NG, Abernathy MP, Hites R, Bigsby RM. Polybrominated Diphenyl Ethers in Maternal and Fetal Blood Samples. Environmental Health Perspectives. 2003;111:1249–1252. doi: 10.1289/ehp.6146. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Qiu X, Bigsby RM, Hites R. Hydroxylated Metabolites of Polybrominated Diphenyl Ethers in Human Blood Samples from the United States. Environmental Health Perspectives. 2009;117 doi: 10.1289/ehp.11660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Sun J, Liu J, Liu Q, Qu G, Ruan T, Jiang G. Sample Preparation Method for the Speciation of Polybrominated Diphenyl Ethers and their Methoxylated and Hydroxylaed Analogues in Diverse Environmental Matrices. Talanta. 2012;88:669–676. doi: 10.1016/j.talanta.2011.11.059. [DOI] [PubMed] [Google Scholar]
  • 19.Wang H-S, Chen Z-J, Ho K-L, Ge L-C, Du J, Lam MH-W, Giesy JP. Hydroxylated and Methoxylated Polybrominated Diphenyl Ethers in Blood Plasma of Humans in Hong Kong. Environmental International. 2012;47:66–72. doi: 10.1016/j.envint.2012.06.004. [DOI] [PubMed] [Google Scholar]
  • 20.Eguchi A, Nomiyama K, Subramanian A, Parthasarathy P, Bulbule KA, Takahashi S, Tanabe S. Organohalogen and Metabolite Contaminants in Human Serum Samples from Indian E-Waste Recycling Workers. Interdisciplinary Studies on Environmental Chemistry. 2010:167–174. [Google Scholar]
  • 21.Ren G, Yu Z, Ma S, Zheng K, Wang Y, Wu M, Sheng G, Fu J. Determination of polybrominated diphenyl ethers and their methoxylated and hydroxylated metabolites in human serum from electronic waste dismantling workers. Analytical Methods. 2011;3:408–413. doi: 10.1039/c0ay00571a. [DOI] [PubMed] [Google Scholar]
  • 22.Stapelton HM, Kelly SM, Pei R, Letcher RJ, Gunsch C. Metabolism of Polybrominated Diphenyl Ethers (PBDEs) in Human Hepatocytes in Vitro. Environmental Health Perspectives. 2009;117:197–202. doi: 10.1289/ehp.11807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Sun J, Liu J, Jiang G. Hydroxylated and Methoxylated Polybrominated Diphenyl Ethers in Mollusks from Chinese Coastal Areas. Chemosphere. 2013;92:322–328. doi: 10.1016/j.chemosphere.2013.03.042. [DOI] [PubMed] [Google Scholar]
  • 24.Hovander L, Athanasiadou M, Asplund L, Jensen S, Wehler EK. Extraction and cleanup methods for analysis of phenolic and neutral organohalogens in plasma. Journal of analytical toxicology. 2000;24:696–703. doi: 10.1093/jat/24.8.696. [DOI] [PubMed] [Google Scholar]
  • 25.Athanasiadou M, Marsh G, Athanassiadis I, Asplund L, Bergman A. Gas Chromatography and Mass Spectrometry of Methoxylated Polybrominated Diphenyl Ethers (MeO-PBDEs) Journal of Mass Spectrometry. 2006;41:790–801. doi: 10.1002/jms.1035. [DOI] [PubMed] [Google Scholar]
  • 26.Schummer C, Delhomme O, Appenzeller BMR, Wennig R, Millet M. Comparison of MTBSTFA and BSTFA in Derivatization Reactions of Polar Compounds Prior to GC/MS Analysis. Talanta. 2009;77:1473–1482. doi: 10.1016/j.talanta.2008.09.043. [DOI] [PubMed] [Google Scholar]
  • 27.Stapelton HM. Instrumental Methods and Challenges in Quantifying Polybrominated Diphenyl Ethers in Environmental Extracts: A Review. Analytical Bioanalytical Chemistry. 2006;386:807–817. doi: 10.1007/s00216-006-0400-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Schecter A, Colacino J, Sjödin A, Needham L, Birnbaum L. Partitioning of polybrominated diphenyl ethers (PBDEs) in serum and milk from the same mothers. Chemosphere. 2010;78:1279–1284. doi: 10.1016/j.chemosphere.2009.12.016. [DOI] [PubMed] [Google Scholar]
  • 29.Woods R, Vallero RO, Golub MS, Suarez JK, Ta TA, Yasui DH, Chi LH, Kostyniak PJ, Pessah IN, Berman RF, LaSalle JM. Long-lived epigenetic interactions between perinatal PBDE exposure and Mecp2308 mutation. Hum Mol Genet. 2012;21:2399–2411. doi: 10.1093/hmg/dds046. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Weijs L, Shaw SD, Berger ML, Neels H, Blust R, Covaci A. Methoxylated PBDEs (MeO-PBDEs), hydroxylated PBDEs (OH-PBDEs) and hydroxylated PCBs (HO-PCBs) in the liver of harbor seals from the northwest Atlantic. Science of the Total Environment. 2014;493:606–614. doi: 10.1016/j.scitotenv.2014.06.028. [DOI] [PubMed] [Google Scholar]
  • 31.Malmvarn A, Marsh G, Kautsky L, Athanasiadou M, Bergman A, Asplund L. Hydroxylated and Methoxylated Brominated Diphenyl Ethers in the Red Algea Ceramium tenuicorne and Blue Mussels from the Baltic Sea. Environmental Science and Technology. 2005;39:2990–2997. doi: 10.1021/es0482886. [DOI] [PubMed] [Google Scholar]
  • 32.Zota AR, Park J-S, Wang Y, Petreas M, Zoeller RT, Woodruff TJ. Polybrominated diphenyl ethers (PBDEs), hydroxylated PBDEs (OH-PBDEs), and measures of thyroid function in second trimester pregnant women in California. Environmental Science and Technology. 2012;45:7896–7905. doi: 10.1021/es200422b. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Tollback P, Bjorklund J, Ostman C. Large-volume programmed-temperature vaporiser injection for fast gas chromatography with electron capture and mass spectrometric detection of polybrominated diphenyl ethers. Journal of Chromatography A. 2003;991:241–253. doi: 10.1016/s0021-9673(03)00174-2. [DOI] [PubMed] [Google Scholar]
  • 34.La Guardia MJ, Hale RC, Harvey E. Detailed Polybrominated Diphenyl Ether (PBDE) Congener Composition of the Widely Used Penta-, Octa-, and Deca-PBDE Technical Flame-retardant Mixtures. Environmental Science & Technology. 2006;40:6247–6254. doi: 10.1021/es060630m. [DOI] [PubMed] [Google Scholar]
  • 35.Lacorte S, Ikonomou MG. Occurrence and Congener Specific Profiles of Polybrominated Diphenyl Ethers and their Hydroxylated and Methoxylated Derivatives in Breast Milk from Catalonia. Chemosphere. 2009;74:412–420. doi: 10.1016/j.chemosphere.2008.09.050. [DOI] [PubMed] [Google Scholar]
  • 36.Erratico CA, Szeitz A, Bandiera SM. Oxidative Metabolism of BDE-99 by Human Liver Microsomes: Predominant Role of CYP2B6. Toxicological Sciences. 2012;129:280–292. doi: 10.1093/toxsci/kfs215. [DOI] [PubMed] [Google Scholar]
  • 37.Feo ML, Gross MS, McGarrigle BP, Eljarrat E, Barcelo D, Aga DS. Biotransformation of BDE-47 to Potentially Toxic Metabolites is Predominantly Mediated by Human CYP2B6. Environmental Health Perspectives. 2013;121 doi: 10.1289/ehp.1205446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Kawashiro Y, Fukata H, Omori-Inoue M, Kubonoya K, Jotaki T, Takigami H, Sakai S-I, Mori C. Perinatal Exposure to Brominated Flame Retardants and Polychlorinated Biphenyls in Japan. Endocrine Journal. 2008;55:1071–1084. doi: 10.1507/endocrj.k08e-155. [DOI] [PubMed] [Google Scholar]
  • 39.Hassine SB, Ameur WB, Eljarrat E, Barcelo D, Touil S, Driss MR. Methoxylated polybromianted diphenyl ethers (MeO-PBDE) in human milk from Bizerte, Tunisia. Environmental Research. 2015;138:32–37. doi: 10.1016/j.envres.2015.01.016. [DOI] [PubMed] [Google Scholar]
  • 40.Gross MS, Butryn DM, McGarrigle BP, Aga DS, Olson JR. Primary Role of Cytochrome P450 2B6 in the Oxidative Metabolism of 2,2',4,4',6-Pentabromodiphenyl Ether (BDE-100) to Hydroxylated BDEs. Chemical research in toxicology. 2015 doi: 10.1021/tx500446c. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

NIHMS724032-supplement.docx (270.8KB, docx)

RESOURCES