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Novel Methoxylated Polybrominated Diphenoxybenzene Congeners and Possible Sources in Herring Gull Eggs from the Laurentian Great Lakes of North America Da Chen,*,†,‡ Robert J. Letcher,*,†,‡ Lewis T. Gauthier,† Shaogang Chu,† Robert McCrindle,§ and Dave Potter|| †
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Wildlife and Landscape Directorate, Science and Technology Branch, Environment Canada, National Wildlife Research Centre, Carleton University, Ottawa, ON, K1A 0H3, Canada ‡ Department of Chemistry, Carleton University, Ottawa, ON, K1S 5B6, Canada § Chemistry Department, University of Guelph, Guelph, ON, N1G 2W1, Canada Wellington Laboratories, Research Division, Guelph, ON, N1G 3M5, Canada
bS Supporting Information ABSTRACT: An increasing number of brominated flame retardants and other brominated substances are being reported in herring gull eggs from the Laurentian Great Lakes basin. Yet, in extracts from gulls’ eggs, numerous bromide anion response peaks in electron capture negative ion (ECNI) mass chromatograms remain unidentified. Using archived herring gull egg homogenates, we characterize the structures of three major and three minor, new and unique brominated substances. After extensive cleanup and separation to isolate these substances from the extracts, high-quality ECNI and electron impact (EI) mass spectra revealed fragmentation patterns consistent with congeners of methoxylated polybrominated diphenoxybenzene (MeO-PBDPB), where four congeners contained five bromines and the other two contain four and six bromines, respectively. Optimized, semiquantitative analysis revealed sum concentrations of the MeOPBDBP congeners ranged from 12 h) prior to use. Bakerbond SPE silica gel (SiOH) disposable, solid phase extraction (SPE) columns (6 mL, 500 mg, 4760 μm) were purchased from VWR (Mississauga, ON, Canada). Solvents used were HPLC grade (Caledon Laboratories, Georgetown, ON, Canada). The 6-MeOBDE-137 and 40 -MeO-BDE-201 standards were purchased from AccuStandards Inc. (New Haven, CT). Samples. For qualitative identification of target unknown analytes, herring gull eggs were collected annually during the 1999 2001 period from the Channel-Shelter Island (Lake Huron) colony. These eggs (n = 1013 each year) were pooled and archived at 40 °C at Environment Canada’s National Wildlife Specimen Bank. Herring gull eggs collected in late-April to early-May of 2009, from 14 colony sites in the Laurentian Great Lakes (Figure 1), were used to quantify and examine the spatial distribution of target analytes. For each colony site, n = 1013 individual eggs were pooled on an equal wet weight basis. The Great Lakes egg samples were collected as part of Environment Canada’s Great Lakes Herring Gull Monitoring Program.16 Egg Sample Preparation. Approximately 40 g of pooled egg homogenate from Channel-Shelter Island (19992001) were extracted in ten replicates (4 g each). Each replicate was ground with DE and then subjected to accelerated solvent extraction (ASE) (Dionex ASE 200, Sunnyvale, CA) with 50:50 dichloromethane:hexane (DCM:HEX). A method blank (DE only) was also extracted along with the egg samples. The extract was purified by gel permeation chromatography (GPC) (OI Analytical, TX) followed by cleanup on a Bakerbond SPE cartridge. After prewashing with 6 mL HEX, the target analytes were eluted from the cartridge with 8 mL 20:80 DCM:HEX. The generated fraction was further cleaned and separated on a column (250 11 mm i.d.) packed with 7-g silica gel (Grade 62, 60200 mesh, Sigma-Aldrich). The first fraction containing PBDEs was eluted with 100 mL HEX, followed by 30 mL 20:80 DCM:HEX. The second fraction eluted with 16 mL 80:20 DCM:HEX contained the target analytes and were concentrated for various gas chromatographymass spectrometry analyses. Gas ChromatographyLow Resolution Mass Spectrometry (GCLRMS). The target compounds were analyzed using an Agilent 6890 GC (Agilent Tech., Palo Alto, CA) coupled to a low resolution, single quadrupole mass analyzer (Agilent 5973 MS), and in both ECNI and electron impact (EI) ionization modes. The column used was a 15-m DB-5 HT column (0.25 mm i.d., 0.1 μm, J&W Scientific, Agilent Tech.). The injector was operated in pulsed-splitless mode, held at 240 °C. The initial oven temperature was held at 100 °C for 2 min, increased to 250 at 25 °C/min, then to 260 at 1.5 °C/min, and finally to 325 at 25 °C/min (held for 7 min). For EI (ion source 230 °C) and ECNI (ion source 250 °C), full scan (scan range 30800 m/z) mass spectra were generated for the target analytes. The heated transfer line temperature was 280 °C and the quadrupole temperature was 150 °C. Methane was used as reagent gas for ECNI-MS. Figure 2 shows the GC full scan ECNI mass chromatograms of a partial time range of 14 to 18 min for the Channel-Shelter herring gull egg fraction (showing three major (U1, U2, and U3) and three minor (U4, U5, and U6) analytes) and PBDE standard mixture (containing BDE-194, -205, and -206). Gas ChromatographyHigh Resolution Mass Spectrometry (GCHRMS). Accurate mass determination was performed using an Agilent 6890 GC coupled to a Waters Autospec Ultima MS. 9524
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Figure 2. Gas chromatographylow resolution mass spectrometry (electron-capture negative ionization mode; full scan m/z 30 800 amu) mass chromatograms of methoxylated polybrominated diphenoxybenzene (MeO-PBDPB) congeners (U1U6) in a fraction from a representative egg pool from Channel-Shelter herring gulls (top) and a PBDE standard mixture (bottom). The mass chromatograms are for a partial GC elution time range of 1418 min.
The fraction containing target analytes was injected onto the GCHRMS system and analyzed in full-scan mode (501000 amu). Voltage scan experiments were created to scan narrow mass ranges that encompassed the molecular fragment of interest. These narrow mass ranges also included 1 to 3 reference peaks from the mass-calibrant, perfluorokerosene (PFK). The instrument was tuned to a resolution of at least 10 000. A calibration curve was constructed using the PFK masses in a portion of the TIC where no peaks eluted. A secondary reference correction (Masslynx 4.1) was applied to the acquired data file to autocorrect each mass in the spectrum using the PFK reference peaks. The corrected data file was then opened and the background was subtracted from the region of interest to generate the final mass spectrum. The Masslynx 4.1 elemental composition program was utilized to determine the best fitting formula for each isotope signal in the mass spectrum of interest. All injections
were in the splitless mode at a temperature of 280 °C. The oven temperature program was the same as that for GCLRMS analysis. Quantitative Analysis in Herring Gull Eggs. The target analytes (U1U6) (Figure 2) are identified as methoxylated polybrominated diphenoxybenzene (MeO-PBDPB) congeners, and the characterization is described in detail in the Results and Discussion section. To semiquantify the six MeO-PBDPBs, approximately 1 g egg pool homogenate from each of fourteen gull colonies and two method blanks were spiked with 20 ng of 6-MeO-BDE-137 as an internal standard and subjected to ASE extraction. After gravimetric determination of lipid content using 10% of the ASE extract, the remaining extract was subjected to GPC, followed by SPE cleanup. The fraction eluted with 8 mL 20:80 DCM:HEX was evaporated and reconstituted to 250 μL in isooctane, and analyzed on the Agilent 6890 GC-5973 MS using 9525
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Figure 3. Electron-capture negative ionization (scan range m/z 30 800 amu) and electron impact (scan range m/z 30 800 amu) mass spectra (GCLRMS) of the methoxylated polybrominated diphenoxybenzene (MeO-PBDPB) congener U1 in the extract of Channel-Shelter Island herring gull egg. The base structure of this congener is also shown (hydrogen atoms are omitted for clarity).
ECNI mode. The analytical column and temperature program, as well as GC parameters, were the same as those described in the previous section. Semiquantification of target analytes U1, U2, U5, and U6 was achieved via selected ion monitoring (SIM) for 79 Br and 81Br, and based on the calibration curve for the 40 MeO-BDE-201. This could be accomplished due to the comparable molecular structures and equitable cleanup and isolation from egg homogenate, as well as their close retention times on GC (i.e., 16.25 min for 40 -MeO-BDE-201 versus 16.17 min for U1). Due to the lack of suitable methoxylated PBDE standards
that elute on GC at similar retention times as U3, BDE-206 was used instead as the quantification standard for U3 (Figure 2). This could be accomplished as the quantification based on the calibration curves for 40 -MeO-BDE-201 and BDE-194 produced comparable results for U1 or U2 (Supporting Information (SI) Table S1). Although the peaks of BDE-206 and U3 were slightly overlapped (Figure 2), the quantification results for U3 via selected ion monitoring (SIM) for 79Br and 81Br were deemed credible, as our previous report indicated that the BDE-206 concentrations in herring gull eggs from the same locations were 9526
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Figure 4. An electron impact (positive) ionization fragmentation pathway scheme of one representative, methoxylated polybrominated diphenoxybenzene (MeO-PBDPB) congener (Br5). Hydrogen atoms are omitted for clarity.
mostly below 0.1 ng/g ww.5 For the same reason, U4 was semiquantified based on the calibration curve for BDE-170. Quality Control and Assurance. The precision, accuracy and recovery efficiency of the MeO-PBDPB analytical method was assessed using commercial chicken eggs (n = 6) spiked with 20 ng each of BDE-194, BDE-206 and 6-MeO-BDE-137. The mean ((standard deviation) recoveries were 91((8.6)% and 93((7.5)% for BDE-194 and -206, respectively. The recoveries of internal standard 6-MeO-BDE-137 were 95((14)%. Based on 6-MeO-BDE-137, the method limits of quantification (MLOQs) of the MeO-PBDPBs were estimated as an analyte response six times the standard deviation of the noise and they were 0.2 ng/g ww, assuming that the MeO-PBDPBs have the same ECNI responses as 6-MeO-BDE-137. MeO-PBDPBs were not detectable (nd: signal/noise
