Evaluation of BDE-47 Hydroxylation Metabolic Pathways Based on

Article pubs.acs.org/est

Evaluation of BDE-47 Hydroxylation Metabolic Pathways Based on a Strong Electron-Withdrawing Pentafluorobenzoyl Derivatization Gas Chromatography/Electron Capture Negative Ionization Quadrupole Mass Spectrometry Chao Zhai,† Shunv Peng,† Limin Yang,† and Qiuquan Wang*,†,‡ †

Department of Chemistry and the MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, Fujian 361005, China ‡ State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen, Fujian 361005, China S Supporting Information *

ABSTRACT: Understanding the metabolic pathways of polybrominated diphenyl ethers (PBDEs) is a key issue in the evaluation of their cytotoxicity after they enter the biota. In order to obtain more information concerning the metabolic pathways of PBDEs, we developed a strong electronwithdrawing pentafluorobenzoyl (PFBoyl) derivatization capillary gas chromatography/electron capture negative ionization quadrupole mass spectrometry (GC/ECNI-qMS). PFBoyl esterification greatly improves separation of the metabolites of PBDEs such as hydroxylated PBDEs (OHPBDEs) and bromophenols (BPs) metabolites in rat liver microsomes (RLMs). On the other hand, the strong electronwithdrawing property of PFBoyl derivatized on OH-PBDEs and/or BPs makes cleavage of the ester bond on ECNI easier resulting in higher abundance of the structure-informative characteristic fragment ions at a high m/z region, which facilitate the identification of OH-PBDEs metabolites. Subsequent quantification can be performed by monitoring not only 79Br− (or 81Br−) but also their characteristic fragment ions, achieving more accurate isotope dilution quantification using GC/ECNI-qMS. These merits allow us to identify totally 12 metabolites of BDE-47, a typical example of PBDEs, in the RLMs in vitro incubation systems. In addition to the already known metabolites of BDE-47, one dihydroxylated 3,6-di-OH-BDE-47 and one dihydroxylated 3,5-di-OH-tetrabrominated dioxin were found. Moreover, the second hydroxylation took place on the same bromophenyl ring, where the first hydroxyl group was located, and was further confirmed via the identification of the dihydroxylated 2′,6′-di-OH-BDE-28 of an asymmetric 2′-OH-BDE-28. This methodological development and its subsequent findings of the metabolic pathways of BDE-47 provided experimental evidence for understanding its dioxin-like behavior and endocrine disrupting risk.

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INTRODUCTION After the application for decades of polybrominated diphenyl ethers (PBDEs) as typical flame retardants, they have been detected in human breast milk and blood,1−3 in addition to their wide distribution in the environment and biota.4−7 The resulting internal exposures lead to the fact that PBDEs can be further metabolized to hydroxylated PBDEs (OH-PBDEs) and methoxylated PBDEs (MeO-PBDEs) under the catalysis of a superfamily of P450 enzymes.8,9 The more water-soluble metabolites used to be considered favorable for excretion, but they present a greater toxicity than their parent compounds because they are more similar in structure to triiodothyronine and tetraiodothyronine, being more competitive in binding with thyroxin transporter transthyretin.10 These findings provide a clue to understand the toxicological mechanism of PBDEs in the potential disruption of thyroxin homeostasis.11 The accumulating evidence indicates that PBDEs and their © 2014 American Chemical Society

hydroxylated metabolites have dioxin-like properties and endocrine disrupting potential and thus are threats to mammals and plants.12 Up to now, studies on metabolism of different PBDE congeners in not only various incubation systems like hepatocytes and liver microsomes but also living bodies have already been carried out.9,13−17 Several dominant metabolites such as 5-OH-BDE-47 and 3-OH-BDE-47 were confirmed, and other novel findings like dihydroxylated metabolites are in search for more evidence to support.8 A full understanding of the metabolism and toxicity of PBDEs depends significantly on accurate and simultaneous determination of PBDEs and their metabolites in a biological Received: Revised: Accepted: Published: 8117

December 20, 2013 June 10, 2014 June 13, 2014 June 13, 2014 dx.doi.org/10.1021/es405446y | Environ. Sci. Technol. 2014, 48, 8117−8126

Environmental Science & Technology

Article

Figure 1. Pentafluorobenzoylation of OH-PBDEs.

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EXPERIMENTAL SECTION Instrumental Analysis. All GC/qMS analysis was performed on a GC-MS QP2010 (Shimadzu, Kyoto, Japan) equipped with a quadrupole mass analyzer together with EI and/or an ECNI source. A DB-1 MS (100% methylpolysiloxane) fused silica capillary column (J&W Scientific, Folsom, CA) of 30 m × 0.25 mm i.d. × 0.25 μm film thickness was used with helium as the carrier gas (1.2 mL/min, 99.999%, Linde China). The sample (1 μL) was injected under splitless mode using an AOC-20i auto sampler at 280 °C. The oven temperature was programmed from 80 °C (held for 2 min) ramped at 10 °C/ min to 280 °C (held for 6 min), and the interface temperature was maintained at 280 °C. The optimized conditions were then set: an electron energy of 70 eV, emission current of 60 μA, and ion source temperature of 200 °C for both EI and ECNI, as well as a moderating gas (CH4) pressure of 2.5 × 10−3 Pa when using ECNI. Full scan and selected ion monitoring (SIM) spectra were recorded for each of the PFBoylated products. All the monitored characteristic fragment ions together with 79Br− are listed in Table S1, Supporting Information. Derivatization, Extraction, and Clean-up. An aqueous tetrabutylammonium carbonate buffer solution (0.25 M, pH 11.6), prepared by mixing appropriate amounts of TBAOH and sodium bicarbonate (Sinopharm Chemical Reagent Ltd., China), was used during the PFBCl (J&K Scientific Ltd., China) derivatization. Bromophenols (BPs) (100 μg/mL) and OH-PBDEs (50 μg/mL) together with the surrogate standard 13 C-6-OH-BDE-47 (5 μg/mL) were converted to their PFBoyl esters by adding 500 μL of buffer solution and 10 μL of 10% PFBCl toluene solution (Figure 1). After shaking for 10 min, additional n-hexane was used to extract the PFBoyl products in the n-hexane phase three times (1 mL each time). The separated n-hexane phase was then cleaned up using 400 μL of concentrated H2SO4 and inverting for 5 min (in the cases of RLMs and CYP isoforms incubation). After phase separation, the n-hexane phase was washed using ultrapure water and dried with anhydrous sodium sulfate. Finally, the n-hexane phase obtained was concentrated to 1 mL under a gentle stream of nitrogen at room temperature, and BDE-66 (10 ng) was added as a volumetric standard before further GC/ECNI-qMS analysis. In order to evaluate the derivatization efficiency using HPLC with a UV detector (214 nm) (see Supporting Information, Table S2), considering the detection power of the UV detector, higher concentration BPs (500 μg/mL) and OHPBDEs (500 μg/mL) together with the surrogate standard 13C6-OH-BDE-47 (500 μg/mL) and the corresponding 10% PFBCl toluene solution were employed. OH-PBDEs were also derivatized using TMS-DM (SigmaAldrich Company Ltd., US), acetic anhydride, isobutyl chloroformate, isopropyl bromide, acetyl chloride, and trichloroacetic chloride (J&K Scientific Ltd., China) based on the methods reported elsewhere,22−24,27 for comparative studies.

system. Gas chromatography (GC) based techniques are routinely employed for the determination of PBDEs and MeO-PBDEs,18,19 but they are not suitable for direct analysis of OH-PBDEs under the same instrumental conditions because of the relatively high polarity of OH-PBDEs.20 Chemical derivatization provides a way to lower the polarity and thus to obtain a better chromatographic behavior. In the literature, diazomethane (DM) and trimethylsilyl diazomethane (TMSDM) are most frequently used for the methylation of OHPBDEs.21,22 However, it is hard to distinguish the methylated derivatives of OH-PBDEs and the MeO-PBDEs that may already exist in the system owing to their totally identical structure. Acetic anhydride, methylchloroformate (MCF), and N,O-bis(trimethylsilyl)-trifluoroacetamide were then used to avoid this possible overlap.15,23,24 In all these studies, electron impact ionization (EI) and electron capture negative ionization (ECNI) mass spectrometry (MS) have been employed together. EI-MS offers structural information, while ECNI-MS permits sensitive quantification of the trace OH-PBDE derivatives in complex biological samples mainly via the monitoring of highly abundant but structure-unspecific bromine ions (79Br− and/or 81Br−). In the case of ECNI-MS, however, the resolution of different OH-PBDEs merely depends on GC separation, and isotope dilution quantification (IDQ) using a 13C-surrogated standard is not possible. The acyl chloride moiety in pentafluorobenzoyl chloride (PFBCl) can react with the hydroxyl group of organic molecules via an SN 2 nucleophilic attack, forming a pentafluorobenzoyl (PFBoyl) ester.25,26 We hypothesized that PFBCl esterification would not only lower the polarity of OHPBDEs resulting in better chromatographic separation but also stabilize the rich-structure informative fragment and even molecular ions of OH-PBDEs during their ionization under the ECNI source due to the fact that the five-fluorine-containing pentafluorobenzene moiety has a strong ability to capture more electrons and cause the ester bond to cleave more easily, benefiting structural identification of OH-PBDEs rather than only monitoring the 79Br− and/or 81Br−. More importantly, the more abundant fragment and/or molecular ions would make it possible to use isotopic surrogates performing an accurate IDQ without losing detection sensitivity when compared with only monitoring 79Br− and/or 81Br−. All these characteristics will benefit a more comprehensive evaluation of PBDE metabolic pathways where many unknown OH-PBDEs like metabolites need identification and quantification. We therefore proposed a systematic study of gas chromatography/electron capture negative ionization quadrupole mass spectrometry (GC/ ECNI-qMS) together with the PFBCl derivatization for the metabolic study of BDE-47 in the rat liver microsomes (RLMs) and the recombinant rat individual cytochrome P450 (CYP) isoform in vitro incubation systems. 8118

dx.doi.org/10.1021/es405446y | Environ. Sci. Technol. 2014, 48, 8117−8126


Article

In Vitro Incubation of BDE-47. BDE-47 was chosen as the substrate, which was incubated in an RLMs in vitro incubation system (containing 80 mM Tris-HCl buffer, pH 7.4, and 5 mM of MgCl2) initiated by the addition of NADPH.28,29 The RLMs were purchased from Sigma-Aldrich and stored in the temperature of −70 °C before use. NADPH was formed by an instant reaction of NADPH regenerating system (BD Biosciences, Mississauga, Ontario, Canada) containing Solution A (NADP+) and Solution B (glucose-6-monophosphate and glucose 6 phosphate dehydrogenase). Just before use, the incubation system was prepared in an ice bath to keep a robust life vitality of CYP enzymes. Briefly, 5 μL of 2 mM BDE-47 hexane solution was added to 1 mL of the RLMs system, and the incubation mixture was prewarmed for 5 min at 37 °C in a thermal vortex mixer (Aosheng, China); then, the premixed NADPH was added to initiate the reactions. The final concentrations of microsomal proteins and NADPH were 1.0 mg mL−1 and 1 mM. After incubation for 2 h, 400 μL of a 0.25 M solution of tetrabutylammonium carbonate was added to stop the reaction and to provide an alkaline derivatization environment for later phase-translocation and PFBCl esterification. To determine whether the biotransformation of BDE47 was enzyme-mediated, experiments with heat-denatured RLMs were conducted. Moreover, recombinant rat individual CYP isoforms (Baculovirus-insect cell microsomes coexpressing rat CYP enzymes) including CYP1A1, 2A2, 2B1, 2C1, 2C6, 3A1, and 3A2 (30 pmol each corresponding to 30 nM) were used instead of the RLMs as the CYP donors. Incubation reactions were allowed to proceed for 30 min. Baculovirusinsect cell control microsomes with rat CYP oxidoreductase and cytochrome b5 but no CYP enzyme were used as control groups. The above experiments were repeated at least three times. Formation rate of hydroxylated metabolites of BDE-47 was evaluated using the recombinant CYP isoform (30 pmol/ 30 nM) under a 30 min incubation. Rate of metabolite formation was plotted as a function of metabolite concentration following the 30 min incubation of BDE-47 (10 μM) with the recombinant CYP isoforms (30 pmol/30 nM), and tangent lines of the curve modeled by nonlinear regression analysis represented rates of metabolite formation. The average rate during 30 min was calculated, and the data represent mean values. In parallel, a typical asymmetric 2′-OH-BDE-28 (0.1 μM) was used to study the possible second hydroxylation position of monohydroxylated OH-BDE under the same conditions.

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RESULTS AND DISCUSSION Esterification of OH-PBDEs and BPs with PFBCl. When evaluating the metabolic pathway of a compound, one may expect to obtain information on the types and contents of possible metabolites produced together with the depletion of their parent compound. A nonpolar capillary column commonly used for separating PBDEs was selected to analyze an artificial sample containing seven typical PBDEs, 12 OHPBDEs, and 5-MeO-BDE-47 as well as two BPs of 25 ng mL−1 each, using GC/ECNI-qMS without any chemical derivatization, in which 79Br− was monitored at first (Figure 2A). The results indicated that the PBDEs and 5-MeO-BDE-47 could be quantitatively separated and sensitively detected. However, both the resolution and sensitivity for the OH-PBDEs and BPs were remarkably low, even coeluted and/or not detectable. Polar OH-PBDEs with Zerewitinoff active hydrogen atoms have a weak interaction with the nonpolar stationary phase

Figure 2. GC/ECNI-qMS chromatograms of typical PBDEs, OHPBDEs, MeO-BDE-47, and BPs before and after PFBCl derivatization. (A) PBDEs, 5-MeO-BDE-47, and OH-PBDEs as well as BPs without PFBCl derivatization when monitoring bromine ion at m/z 79.0 with SIM mode; (B) PBDEs and MeO-BDE-47 and PFBCl-derivatized OH-PBDEs and BPs monitoring bromine ion at m/z 79.0 with SIM mode; (C) monitoring 79Br− for PBDEs and MeO-BDE-47 and the corresponding characteristic fragment ions of PFBCl-derivatized OHPBDEs and BPs through different time windows with SIM mode. The peak number, compound name, and monitoring ions as well as their monitoring time windows are listed in Table S3, Supporting Information.

based on the like-dissolves-like rule, resulting in a short retention time and therefore a poor resolution.30 On the other hand, the existence of the hydroxyl group in OH-PBDEs makes OHPBDEs less volatile compared to the corresponding PBDEs because of the formation of an intermolecular hydrogen bond, 8119



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Figure 3. ECNI-qMS of 3-OH-BDE-47 and 3-PFBoyl-O-BDE-47 and EI-qMS of 3-PFBoyl-O-BDE-47. (A) 3-OH-BDE-47; (B) 3-PFBoyl-O-BDE47; (C) Br-isotopic distribution in the fragment ion [O-BDE-47]− under ECNI; (D) 3-PFBoyl-O-BDE-47 under EI.

partly responsible for the at least 1 order of magnitude lower sensitivity of OH-PBDEs than those of PBDEs and MeOPBDEs on the ECNI-qMS. In order to improve the chromatographic behavior and sensitivity of OH-PBDEs and BPs to ensure a simultaneous analysis together with their parent PBDEs under GC/ECNIqMS, esterification of the hydroxyl group in OH-PBDEs and BPs was performed using PFBCl (Figure 1). The formation of [O-PBDE]− and/or [O-C6H5−xBrx]− from OH-PBDEs and BPs was in a water-based alkaline medium containing 0.25 M tetrabutylammonium carbonate (pH 11.6), and then, the [OPBDE]− and/or [O-C6H5−xBrx]− was carried by TBAOH, a phase-transfer catalyst, into a 10% PFBCl toluene phase for pentafluorobenzoylation. Only 10 min of hand-shaking was required to achieve derivatization efficiencies ranging from 87.4% to 99.8% evaluated using HPLC (Table S2, Supporting Information). The derivatization efficiency depends on the position of the hydroxyl group on the brominated benzene ring with a different number of bromine substituents. Bromine has a strong electron-withdrawing orientation and is a typical orthopara directing group. 2,4,6-TBP and 3-OH-BDE-47 have displayed relatively high derivatization efficiency of 99.8% and 98.4% due to the similar structure with the two bromines on the ortho-position to the hydroxyl group. The high uniformity of esterification efficiency of 6-OH-BDE-82, 6-OH-BDE-85, and 6-OH-BDE-87 indicated a relative weak influence of the ether oxygen bond linked brominated benzene ring on pentafluorobenzoylation. Moreover, the PFBoylated derivatives

of OH-PBDEs (for example, 3-OH-BDE-47 and 5-OH-BDE47) were stable when concentrated H2SO4 was used to remove possible phospholipids extracted from the RLMs incubation system (Figure S1, Supporting Information). After PFBCl derivatization of the artificial sample, it was analyzed using GC/ECNI-qMS with monitoring of 79Br− (Figure 2B) and the characteristic fragment ions (Figure 2C). Except for the overlap of the PFBoylated TBP (2) and BDE-17 (3) at a retention time of 20.45 min (Figure 2B), which could be distinguished by monitoring the characteristic fragment ion of [O-TBP]− at m/z 338.0 (Figure 2C), all the others were baseline separated with sharp peaks. These improvements in separation degree and detection sensitivity were ascribed to the effective pentafluorobenzoylation of the hydroxyl group in OHPBDEs and BPs, and the easier cleavage of the ester bond (PFB-C(O)−O-BDE) on ECNI owing to the very strong electron-withdrawing feature of the five fluorine in PFBoyl, which led to more abundant fragment ions of [O-BDE]− being detected. ECNI-qMS Spectra of the PFBoylates of OH-PBDEs and BPs. ECNI-qMS proved to be very sensitive for the determination of an electronegative atom-containing PBDEs via monitoring 79Br−/81Br− ions. However, in most cases, extremely low abundance structure-informative fragment ions at higher m/z range were hardly observed. One had to increase the sample volume, for example, using a pulsed large-volume injection technique, to see the structure-informative fragment ions of PBDEs during a GC/ECNI-qMS.31 In the case of OH8120



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Figure 4. ECNI mass spectra for (A1) phenol; (A2) 2,4-DBP; (A3) 2,4,6-TBP; (B1) 2′-OH-BDE-28; (B2) 3′-OH-BDE-28; (B3) 4′-OH-BDE-17; (C1) 5-OH-BDE-47; (C2) 3-OH-BDE-47, and (C3) 13C-6-OH-BDE-47 after PFBCl derivatization. The inset panels illustrate the isotope distribution of the characteristic ions.

two Br at ortho-position and one at para-position in their molecules, only [PFBoyl-O-C6H5]− at m/z 288.1 could be observed as the base peak in the case of phenol after PFBClesterification (Figure 4 A1); in the case of 2,4-DBP, [OC6H3Br2]− with its isotopic distribution at m/z 249.0/251.0/ 253.0, formed through the cleavage of the ester bond, and [PFB-O-C6H3Br]− at m/z 336.9/338.9, losing a neutral CO (which has a relatively low molar enthalpy change of −111 kJ mol−1 and is able to depart during the cleavage reaction of carbonyl-containing PFBoylates) and one Br, became noticeable but [PFBoyl-O-C6H3Br2]− disappeared (Figure 4 A2). In the case of 2,4,6-TBP, one more Br at the ortho-position resulted in [O-C6H2Br3]− at m/z 328.8 (the most abundant one in the isotope clusters of 324.8/326.8/328.8/330.8) as the base peak, and also [PFB-O-C6H2Br2]− at m/z 414.9/416.9/ 418.9 remarkably increased (Figure 4 A3). Clearly, the increase in the number of Br, especially in the ortho-position, led to the competition between Br and the PFBoyl for electron capturing and weakened the ester bond strength, making the departure of PFBoyl easier and resulting in higher abundance of the characteristic ions. This became more complicated in the case of the PFBoylates of OH-PBDEs, since an additional inductive effect from the bromophenoxyl moiety in the PFBoylates had to be taken into account. For example, 2′-PFBoyl-O-BDE-28 contains an ortho-position dibrominated phenoxyl moiety and a meta-position Br, the peak at m/z 420.7 of the most abundant

PBDEs, for example, 3-OH-BDE-47 without PFBCl-derivatization, only 79Br−/81Br− was observed while no fragment ions at higher m/z presented in noticeable abundance (Figure 3A). After pentafluorobenzoylation, its structure-informative fragment ions could be observed (Figure 3B), such as [-O-BDE47]− at m/z 496.7/498.7/500.7/502.7/504.7 with a fair Brisotopic distribution (Figure 3C). Even though the derivatized PFBoyl group containing a carbonyl carbon cation ([PFBoylH]+) was not detected on the ECNI-qMS, [PFBoyl-H]+ leaving at m/z 195.0 could be observed on the EI-qMS operating in positive mode (Figure 3D), confirming the effective pentafluorobenzoylation. The full-scan ECNI spectra of pentafluorobenzoylated OH-PBDEs and BPs recorded when monitoring their characteristic fragment ions (Table S1, Supporting Information) can be used for identification. The results listed in Table S1, Supporting Information, suggested that the abundance of the higher m/z fragment ions of the OH-PBDEs and/or BPs PFBoylates significantly depended on the position of the hydroxyl group on their molecular structures, where PFBCl-derivatization took place; on the other hand, the number and position of Br substitutes on OH-PBDEs and/or BPs also influenced the cleavage of the ester bond, resulting in different abundances of the higher m/z fragment ions (Figure 4). Taking as an example phenol with no Br, 2,4-DBP with one Br located at ortho-position and one at para-position related to the hydroxyl group, and 2,4,6-TBP with 8121


a

2,4-DBP 2,4,6-TBP 2′-OH-BDE-3

3′-OH-BDE-7 2′-OH-BDE28 3′-OH-BDE28 4′-OH-BDE17 5-OH-BDE-47 3-OH-BDE-47 6-OH-BDE-87 6-OH-BDE-82 6-OH-BDE-85 6-OH-BDE137 13 C-6-OHBDE-47

1 2 5

9 11

8122

4.9 8.0 8.5 6.6 14.2 17.3

3.6

30.987

17.0

4.0

2.3 4.1

2.5 2.0 1.5

IDL (fg)

31.980 32.210 33.553 34.053 34.447 34.677

30.360

30.283

26.713 29.100

17.890 20.450 22.093

R.T. (min)

9.2

11.3 13.0 28.5 26.6 27.1 22.3

17.0

14.0

8.3 14.1

1.5 2.0 3.5

MDL (ng L−1)

27.3

36.7 43.1 88.7 96.6 91.2 62.7

45.4

36.8

27.8 46.9

5.0 6.7 11.7

LOQ (ng L−1)

Measurement precision for the PFBoylated OH-PBDEs standards.

23

15 16 19 20 21 22

13

12

name

peak no.

79

0.996

0.993 0.998 0.993 0.993 0.991 0.999

0.999

0.982

0.9529 0.995

0.994 0.998 0.999

R2

Br−

1.2

0.4 0.5 0.6 2.1 2.2 3.5

2.4

0.8

0.4 1.1

0.4 0.3 0.8

RSD % n=6

a

90.0

83.5 78.9 88.5 91.4 106.3 84.2

111.4

78.5

88.6 83.3

95.0 96.3 89.6

recovery % (50 ng)

500.7 500.7 580.6 580.6 580.6 656.6 512.7

[13C-O-tetra-BDE]−

515.7

420.8

[O-tri-BDE]− [PFBoyl-O-tri-BDEBrF]− [O-tetra-BDE]− [O-tetra-BDE]− [O-penta-BDE]− [O-penta-BDE]− [O-penta-BDE]− [O-hexa-BDE]−

263.0 420.8

250.8 330.8 460.0

m/z

[C6H3Br2]− [C6H2Br3]− [PFBoyl-O-monoBDE]− [O-mono-BDE]− [O-tri-BDE]−

characteristic fragment ion

3.2

5.5 6.8 8.3 11.3 13.5 16.6

3.3

34.8

2.4 5.4

12.0 15.0 1.2

IDL (fg)

10.1

12.5 13.8 12.1 14.3 13.5 16.6

3.3

11.8

2.4 5.4

1.2 1.5 1.2

MDL (ng L−1)

32.1

44.0 44.3 33.3 41.0 11.7 55.3

11.0

43.5

8.0 18.0

4.0 5.0 4.0

LOQ (ng L−1)

characteristic fragment ions

Table 1. Method Performance of OH-PBDEs and BPs on the GC/ECNI-qMS When Monitoring Bromine and Characteristic Ions

0.998

0.996 0.994 0.994 0.996 0.995 0.994

0.992

0.997

0.991 0.992

0.992 0.998 0.990

R2

0.9

0.5 0.5 1.5 3.0 4.6 6.6

2.5

0.8

0.4 1.2

0.5 0.3 0.8

RSD %a n=6

90.0

83.0 80.6 88.5 107.0 85.4 83.6

80.8

89.7

89.6 105.0

91.0 96.3 89.9

recovery % (50 ng)

Environmental Science & Technology Article



Article

Figure 5. Chromatogram of BDE-47 metabolites biotransformed in the RLMs in vitro incubation system (A); summary of metabolic pathways of BDE-47 in the RLM in vitro incubation system (B); ECNI-MS spectra and their deduced structure of di-OH-BDE-47 (C) and di-OHtetrabrominated dioxin of BDE-47 (D). Monitoring ions and corresponding time-window for the metabolites of BDE-47 were listed in Table S6, Supporting Information.

phenomena observed might be helpful to judge the hydroxylated position of PBDEs in later studies of PBDE metabolism in the RLMs in vitro incubation system. Method Performance. Pentafluorobenzoylation of OHPBDEs and BPs not only improved their chromatographic separation on a nonpolar capillary gas chromatographic column but also allowed us to extract the structure-related fragment ions for identification, making it possible to identify and selectively quantify OH-PBDEs and BPs together with their parent PBDEs using GC/ECNI-qMS for the metabolic study. The obtained IDLs (3S/N) of the OH-PBDEs and BPs studied here ranged from 1.2 to 12.6 fg when monitoring their corresponding signature fragment ions, comparable to those (1.5 to 28.5 fg) when monitoring 79Br− (Table 1), resulting in at least 2 orders of magnitude improvement when compared with those without PFBCl derivatization. This method displayed linearity in the concentration range from 1 to 100 ng mL−1 (R2 > 0.990) for all the OH-PBDEs and BPs by monitoring both of the signature ions and/or 79Br−. The relative standard deviations (RSDs) at a concentration of 25 ng mL−1 (n = 6) of the OH-PBDE standards were in the range of 0.4% to 6.6% when monitoring their signature ions, comparable to those of 0.3% to 3.5% when monitoring 79Br− (Table 1). Because no standard reference materials are currently available, the present method was validated by the RLMs samples (purchased from Sigma-Aldrich) spiked with two concentrations (5 and 50 ng mL−1) of the OH-PBDEs and BPs

mass in the isotope clusters of 418.7/420.7/422.7/424.7 ([2′O-BDE-28]−) became the base peak (Figure 4 B1); while 3′PFBoyl-O-BDE-28 contained a meta-position dibrominated phenoxyl moiety and an ortho-position Br, the peak at m/z 420.7 of the most abundant mass in the isotope clusters of 418.7/420.7/422.7/424.7 ([3′-O-BDE-28]−) decreased to 30% (Figure 4 B2). 4′-PFBoyl-O-BDE-17, also an OH-tri-BDE PFBoylate, has a para-position dibrominated phenoxyl moiety and meta-position Br; the peak at m/z 420.7 (the most abundant mass in the isotope clusters of 418.7/420.7/422.7/ 424.7 of [4′-O-BDE-17]−) decreased to 10% (Figure 4 B3), but [tetra-FBoyl-O-di-BDE]− formed by losing one F and one Br at m/z 515.7 became obvious. 5-PFBoyl-O-BDE-47 and 3PFBoyl-O-BDE-47 have the same meta-dibrominated phenoxyl moiety in their molecules; the difference is that there is one ortho-position Br in 5-PFBoyl-O-BDE-47 and two orthoposition Br atoms in 3-PFBoyl-O-BDE-47. As discussed in the case of BPs, the ortho-position Br played an important role in the cleavage of the ester bond. [O-BDE-47]− fragment ion was the base peak in the mass spectra (Figure 4 C1 and C2) of both 5-PFBoyl-O-BDE-47 and 3-PFBoyl-O-BDE-47, but [3PFB-O-tri-BDE]− increased more remarkably in the case of 3PFBoyl-O-BDE-47 than 5-PFBoyl-O-BDE-47. As for the 13Csurrogate 6-PFBoyl-O-BDE-47, which has an ortho-dibrominated phenoxyl moiety and two meta-position Br atoms, its [OBDE-47]− was again the base peak, demonstrating the effect of the ortho-position Br once more (Figure 4 C3). These 8123



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standards as well as 13C-6-OH-BDE-47. On the basis of the recovery of 13C-6-OH-BDE-47 (90.0%), their recoveries were in the range of 75% to 112%, and the RSDs (50 ng mL−1, n = 6) were below 10%, suggesting that this method could be applied to the study of PBDE metabolism in the RLMs incubation system. The MDLs (3 S/N) obtained in the case of the spiked RLMs for the OH-PBDEs and BPs studied were 1.2 to 34.8 pg mL−1 and 1.5 to 28.5 pg mL−1 when monitoring their signature ions and 79Br−, and the corresponding LOQs (10 S/N) of 4.0 to 55.3 pg mL−1 and 5.0 to 88.7 pg mL−1 were accordingly obtained with the quality assurance and quality control criteria (see Supporting Information). In the early stage of our research, we tried several other derivatization reagents, including TMS-DM, acetic anhydride, iso-butyl chloroformate (an analogue of MCF), and isopropyl bromide, to derivatize the OH-PBDEs and BPs. The results indicated that the IDLs of 5-OH-BDE-47, for example, were 2.7 to 6.9 times higher than that using PFBCl when monitoring its signature ion [O-BDE-47] − at m/z 500.7 (Table S4, Supporting Information). When compared with the methods reported using LC/MS, the MDLs of the present method are at least 2 orders of magnitude lower than the direct determination of OH-PBDEs without any derivatization32,33 and comparable to those with dansyl chloride derivatization (Table S5, Supporting Information).34 BDE-47 Biotransformation under Cytochrome P450 Catalysis. BDE-47, the most abundant low-brominated congener of PBDEs in the environment and biota, was taken as an example of PBDEs to investigate the metabolic pathways under P450 enzyme catalysis in the RLMs in vitro incubation system. Although six monohydroxylated tetrabrominated metabolites were theoretically expected to be formed from BDE-47 by direct hydroxylation of Br-free positions or by an NIH shift via an arene oxide,35 12 metabolites were detected using the highly selective and sensitive PFBCl-derivatization GC/ECNI-qMS developed in this study (Figure 5A,B). After incubation for 2 h, the parent BDE-47 depletion rate in the RLMs in vitro incubation system was determined to be 2.1%, which is in agreement with those of 0.1−10% reported previously.34 On the basis of their corresponding retention time and ECNI mass spectra as well as peak area (Figures 5A and 4C), three monohydroxylated products were determined as major metabolites including 6-OH-BDE-47 of 4.5 ± 0.6 pg (No. 10 peak), 5-OH-BDE-47 of 16.5 ± 1.2 pg (11), and 3OH-BDE-47 of 3.8 ± 0.4 pg (12), as well as 2,4-DBP of 46.2 ± 6.8 pg (2). The observation of one OH-di-BDE of 7.1 ± 0.5 pg (7) and 3′-OH-BDE-7 3.1 ± 0.2 pg (8) and 2′-OH-BDE-28 5.5 ± 0.6 pg (9) indicated that there was a debromination and/or hydroxylation metabolic pathway of BDE-47 in the RLMs incubation system. In addition, the debrominated products, BDE-17 of 4.1 ± 0.2 pg (3) and BDE-28 of 10.3 ± 0.9 pg (4), could be also identified. It should be noted that a trace amount of dihydroxylated BDE-47 (13) was detected and identified (Figure 5C), which provides experimental evidence to prove the further possible biotransformation of monohydroxylated BDE-47.36 The highly abundant fragment ions [(PFBoyl-O)2BDE-47]− at m/z 902.7/904.7/906.7/908.7/910.7, [PFBoylO2-BDE-47]− at m/z 707.7/709.7/711.7/713.7/715.7, and [HO2-BDE-47]− at m/z 512.7/514.7/516.7/518.7/520.7 validated the di-OH-BDE-47 formation in the system. The observed characteristic fragment ion [C6H3Br2O]− at m/z 249.0/251.0/253.0, a half symmetrical structure with an ether bond of BDE-47 (Figure 5C), suggested that the two hydroxyl

groups metabolized were on the same bromophenyl ring of BDE-47. As is discussed above in the section related to the ECNI-qMS spectra of the PFBoylates of OH-PBDEs and BPs, the hydroxyl group at the ortho-position related to the etherbond led to a higher abundance of high-mass characteristic fragment ions, and taking the steric hindrance effect into consideration, it was deduced that the two hydroxyl groups are located at the ortho-position and meta-position related to the ether bond (3,6-di-OH-BDE-47). In order to further confirm that the dihydroxylation of BDE-47 happened on the same bromophenyl ring, an asymmetric brominated monohydroxylated congener, 2′-OH-BDE-28, was incubated in the same RLMs incubation system. A dihydroxylated metabolite of 2′OH-BDE-28 was identified (Figure S2, Supporting Information) with the bared [C6H3Br2O]− at m/z 249.0/251.0/253.0 in addition to the observation of [(PFBoyl-O)2-BDE-28]− at m/z 824.0/826.0/828.0/830.0 and [PFBoyl-O2-BDE-28]− at m/z 629.0/631.0/633.0/635.0 as well as [HO2-tri-BDE]− at m/ z 434.7/436.7/438.7/440.7, confirming again that the further hydroxylation was on the monohydroxylated bromophenyl ring but not the other nonhydroxylated one. This result not only suggested that the second hydroxyl group metabolized was on the bromophenyl ring where the first hydroxyl group was located but also complied with the discovery that most of the hydroxylated metabolites of PBDEs detected in the biota share the common character of an ortho-hydroxylation related to the ether bond.37 Besides, a di-OH-tetrabrominated dioxin metabolite of BDE-47 was identified in the RLMs incubation system based on its signature ions at m/z 528.7/530.7/532.7/ 534.7/536.7 and 913.7/915.7/917.7/919.7/921.7 (Figure 5D and No. 6 peak in Figure 5A), providing experimental evidence regarding its dioxin-like cytotoxic property. Moreover, the recombinant individual CYP isoforms including CYP1A1, 2A2, 2B1, 2C1, 2C6, 3A1, and 3A2 were used to evaluate their contributions to the metabolism of BDE47. The results obtained indicated that CYP2B1, CYP2A2, and CYP1A1 played a greater role in the metabolism of BDE-47. The parent BDE-47 depletion rates were 16.5%, 12.7%, and 8.4% when catalyzed by CYP2B1, CYP2A2, and CYP1A1 at the 30 nM level, respectively. The metabolic products were dominated with monohydroxylated BDE-47 like 3-OH-BDE47 (126.3 ± 0.3 pg), 5-OH-BDE-47 (156.4 ± 0.5 pg), and trace amounts of 6-OH-BDE-47 (12.2 ± 0.2 pg) in the case of CYP2B1 with the formation rates of 141.0 ± 0.5, 173.3 ± 0.2, and 13.3 ± 0.1 mg min−1 mol−1, respectively. CYP2A2 was also involved in the formation of 3,6-di-OH-BDE-47 (48.0 ± 0.2 pg; 53.3 ± 0.1 mg min−1 mol−1) and di-OH-tetrabrominated dioxin (36.2 ± 0.2 pg; 41.2 ± 0.5 mg min−1 mol−1). CYP1A1 contributed to the debromination and hydroxylation processes resulting in the formation of 2′-OH-BDE-7 (40.2 ± 0.2 pg; 40.3 ± 0.2 mg min−1 mol−1), 3′-OH-BDE-7 (33.0 ± 0.2 pg; 33.4 ± 0.3 mg min−1 mol−1), and 2′-OH-BDE-28 (26.2 ± 0.2 pg; 33.2 ± 0.2 mg min−1 mol−1); CYP2B1 and CYP2A2 played a significant role in the cleavage of the ether bond and were thus responsible for the formation of 2,4-DBP and 4-BP. It is worth noting that, not limited to the CYP isoforms studied here, more isoforms from the P450 superfamily, which has over 50 CYP isoforms, are expected to be involved to come to a more comprehensive conclusion indicating which isoform(s) is/are significantly responsible for the biotransformation pathways of BDE-47 using the developed PFBCl esterification GC/ECNIqMS methodology in the near future. 8124



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ASSOCIATED CONTENT

S Supporting Information *

Additional information including chemicals and reagents, the derivatization efficiency of the OH-PBDEs, the peak number, compound name, and monitoring ions of OH-PBDEs as well as their time windows, quality assurance, and quality control, stability of PFBoyl-O-BDE-47 and comparison of detection limits of different derivatization methods based on GC/MS and HPLC/MS as well as the ECNI-qMS spectrum of the dihydroxylated metabolite of 2′-OH-BDE-28. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86 592 2181796; fax: +86 592 2187400; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This study was supported financially by the National Basic Research 973 Program of China (2014CB932004 and 2009CB421605) and the National Natural Science Foundation of China (21035006 and 21275120). We thank Professor John Hodgkiss for his help with the English in this article.

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Evaluation of BDE-47 Hydroxylation Metabolic Pathways Based on

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