Journal of Chromatography A, 1190 (2008) 333–341

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Journal of Chromatography A

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Comparative evaluation of liquid chromatography–mass spectrometry versusgas chromatography–mass spectrometry for the determination ofhexabromocyclododecanes and their degradation products in indoor dust

Mohamed Abou-Elwafa Abdallaha, Catalina Ibarraa, Hugo Neelsb, Stuart Harrada, Adrian Covacib,∗

a Division of Environmental Health and Risk Management, School of Geography, Earth and Environmental Sciences, University of Birmingham, Birmingham B15 2TT, UKb Toxicological Centre, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium

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Article history:Received 21 January 2008Received in revised form 2 March 2008Accepted 4 March 2008Available online 7 March 2008

Keywords:Liquid chromatographyGas chromatographyMass spectrometryHexabromocyclododecanesHBCDsDegradation productsIndoor dust

a b s t r a c t

Domestic and office dustgas chromatography–elecuid chromatography–elecmethod to quantify HBCDwhile 13C-labeled �-HBCDand �-HBCD diastereomerdiastereomers and using acentrations obtained via Gusing LC–ESI/MS/MS. Theas I.S. and the average RFr > 0.9) were obtained bet0.76 to 1.36. Pentabromocwere detected in the studaration from the parent csuggests that the eliminat

1. Introduction

Hexabromocyclododecane (HBCD) is a high-volume bromi-nated flame retardant (BFR) with a total world market demandof 16.7 × 106 kg in 2001, making it the second most widely usedBFR in Europe [1]. It is used as an additive to expanded andextruded polystyrene foams for thermal insulation and to a lesserextent for back-coating of fabrics for furniture [1]. HBCD is analiphatic, brominated cyclic alkane produced by bromination ofcyclododecatri-1,5,9-ene, a process which leads theoretically tothe formation of a mixture of 16 stereoisomers. The commercialmixtures consist mainly of the �-, �-, and �-HBCD diastereomers,with the latter being predominant [2]. HBCD’s low vapor pressure(6.27 × 10−5 Pa), very low water solubility (3.4 �g/L), fairly highlog Kow value of 5.6 combined with its persistence, indicate that itmay bioaccumulate in fatty tissues [3]. This is compounded by thefact that oral exposure to HBCDs may induce hepatic cytochromeP450 enzymes in rats [4] and can alter the normal uptake of neu-

∗ Corresponding author. Tel.: +32 3 820 2704; fax: +32 3 820 2722.E-mail address: adrian.covaci@ua.ac.be (A. Covaci).

0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.doi:10.1016/j.chroma.2008.03.006

les (n = 37) were analyzed for hexabromocyclododecanes (HBCDs) usingcapture negative ionization–mass spectrometry (GC–ECNI/MS) and liq-ray tandem mass spectrometry (LC–ESI/MS/MS). To determine the bestg GC–ECNI/MS, BDE 128 was used as internal standard (I.S.) in all samples,used as I.S. in some samples. Total HBCD concentrations (sum of �-, �-,re calculated using response factors (RFs) for �- and �-HBCD as individualrage RF for both diastereomers. Statistical comparison showed that con-NI/MS were statistically indistinguishable (p > 0.05) from those obtainedst match between the two techniques was obtained using [13C]�-HBCD- and �-HBCDs. Excellent linear correlations (Pearson coefficient valuesthe GC–ECNI/MS and LC–ESI/MS/MS results, with slopes ranging from

odecenes (four isomers) and tetrabromocyclododecadienes (two isomers)amples and were identified as degradation products of HBCDs after sep-und on the basis of both retention time and mass spectrum. This findingf HBr is the major degradation pathway for HBCDs in dust.

© 2008 Elsevier B.V. All rights reserved.

rotransmitters in rat brain [5]. There are also indications that it can

disrupt the thyroid hormone system [6] and induce cancer througha non-mutagenic mechanism in humans [7].

Following its first detection in fish and sediment samples fromthe River Viskan in Sweden [8], HBCD has attracted increasingattention, with its levels being reported in a wide variety of bioticand abiotic samples [9]. Traditionally, HBCD has been analyzedby gas chromatography–mass spectrometry (GC–MS) operated inelectron-capture negative ionization (ECNI) mode. Bromide iondetection (m/z = 79 and 81) is used commonly because of its highsensitivity [3]. However, the GC technique has a number of seriousdrawbacks, specifically the impossibility of separating the HBCDdiastereomers, the thermal rearrangement of HBCD diastereomersabove 160 ◦C, the decomposition of HBCDs at temperatures above240 ◦C, and the partial or complete degradation of HBCDs in dirty GCsystems [10]. Furthermore, isotopically-labeled (e.g. [13C]-) HBCDstandards cannot be used when only the bromide ion is monitored.In addition, other brominated compounds of potential use as inter-nal standards, such as polybrominated diphenyl ethers (PBDEs),have a better thermal stability than HBCDs and therefore cannotcompensate for the decomposition that occurs in the GC system[3].

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334 M.A.-E. Abdallah et al. / J. Ch

In 2003, Budakowski and Tomy [11] introduced liquid chro-matography coupled tandem mass spectrometry (LC–MS/MS) forthe chromatographic resolution and determination of the majorHBCD diastereomers. Since then, several refinements and mod-ifications to enhance resolution and/or sensitivity have beenreported for the LC–MS/MS analysis of HBCDs [10,12,13]. Presently,LC–MS/MS is considered the method of choice for the analysis ofHBCDs, because, in addition to its ability to chromatographicallyresolve the HBCD diastereomers, no thermal degradation or analytecomposition transmutation occurs during analysis and it is com-patible with the use of isotopically-labeled HBCD diastereomersas internal standards. These labeled standards can compensate forboth instrumental fluctuations of the MS system and for matrix-related ion suppression or enhancement effects that can occur inthe ion source [10,12].

Despite these clear advantages of LC–MS/MS, the higher capi-tal cost of such instrumentation, means that it is possible that theuse of GC–MS, at least for screening purposes, has been too earlydiscounted. Currently, very little is known about the comparativeperformance of GC–MS and LC–MS/MS techniques for the deter-mination of �HBCDs. Recently, an interlaboratory comparison forHBCDs in fish and standard solutions [14] has shown good agree-ment between results obtained by GC–MS or LC–MS. However, noneof the reporting laboratories presented results using both tech-niques. If such a comparison revealed GC–MS to offer acceptableperformance, then it would open up a simple, more widely avail-able and sensitive technique for the determination of HBCDs, thatcould for example be used to screen samples for HBCDs and PBDEs,in the same GC–ECNI/MS run.

The principal aim of this study is to compare the performance ofGC–ECNI/MS and LC–ESI/MS/MS for the determination of �HBCDsin indoor dust. In addition, strategies to enhance the selectivityand accuracy of the GC–ECNI/MS method for �HBCD determina-tion are discussed. The present study also reports for the first timethe identification by LC–MS/MS of degradation products of HBCDs,and their detection in indoor dust. Elucidation of such productsoffers potential insights into the pathways of HBCD degradation.

2. Experimental

2.1. Chemicals

All solvents used in the extraction and analysis procedures forLC–MS/MS were of HPLC-grade quality (Fisher Scientific, Loughbor-

ough, UK). Pesticide-grade Florisil (60–100 mesh) and anhydroussodium sulfate were obtained from Acros Organics (Geel, Bel-gium). Native �-, �-, and �-HBCD standards were obtained fromCambridge Isotope Labs. (Andover, MA, USA), while isotopically-labeled [13C]�-, �-, and �-HBCDs and [2H18]�-HBCD (�-HBCD-d18)were purchased from Wellington Labs. (Guelph, Canada). Forthe preparation of samples subjected to GC–MS analysis, silica(60–100 mesh), concentrated sulfuric acid and anhydrous sodiumsulfate were obtained from Merck (Darmstadt, Germany). All sol-vents were of GC grade quality (Merck). BDE 128 was purchasedfrom Accustandard (New Haven, CT, USA), while the polychlo-rinated biphenyl (PCB) 206 was obtained from Dr. Ehrenstorfer(Augsburg, Germany).

2.2. Sample collection

Dust samples (n = 37) were collected from randomly chosenhouses and offices in Birmingham, UK using a Nilfisk Sprint Plus1600 W vacuum cleaner. Sampling was conducted in March–June2007 according to a standard protocol, where 1 m2 of carpet wasvacuumed for 2 min in each location and in case of bare floors 4 m2

ogr. A 1190 (2008) 333–341

for 4 min [15,16]. Samples were collected using nylon sample socks(25 �m pore size) that were mounted in the furniture attachmenttube of the vacuum cleaner. After sampling, socks were closed witha twist tie, sealed in an air-tight plastic bag and stored at −20 ◦C.Before and after sampling, the furniture attachment was cleanedthoroughly using an isopropanol-impregnated disposable wipe.

2.3. Analytical protocols

2.3.1. LC–MS/MS analysisThe analytical procedure used for the sample preparation and

analysis of HBCDs in dust via LC–MS/MS has been described previ-ously [16]. A brief summary follows.

2.3.1.1. Sample preparation and extraction. Samples were sievedthrough a 500 �m mesh size sieve, weighed accurately and thenextracted using pressurized liquid extraction (ASE 300 system;Dionex, Oakville, Canada). Dust samples (typically between 100and 250 mg) were loaded into pre-cleaned 66 mL cells containing1.5 g Florisil and Hydromatrix (Varian, Palo Alto, CA, USA) to fill thevoid volume of the cells, spiked with 25 ng of each of 13C-labeled�-, �, and �-HBCD as internal standards and extracted with hex-ane:dichloromethane (1:1, v/v) at 90 ◦C and 1500 psi. The heatingtime was 6 min, static time 5 min, purge time 100 s, flush volume50%, and the number of static cycles was 3.

2.3.1.2. Clean up. The crude extracts were concentrated to 0.5 mLusing a Zymark Turbovap II (Hopkinton, MA, USA), then washedwith 98% sulfuric acid. After phase separation, the hexane layer wastransferred onto a Florisil column topped with sodium sulfate andeluted with 30 mL of hexane:dichloromethane (1:1, v/v). The eluatewas evaporated to dryness under a gentle stream of N2 and thedried extract was reconstituted in 100 �L of �-HBCD-d18 (25 pg/�Lin methanol) used as recovery determination (or syringe) standard,used to determine the recoveries of internal standards for qualityassurance/quality control (QA/QC) purposes.

2.3.1.3. Analysis. Separation of �-, �-, and �-HBCDs was achievedusing a dual pump Shimadzu LC-20AB Prominence liquid chro-matograph (Kyoto, Japan) equipped with SIL-20A autosampler, aDGU-20A3 vacuum degasser and a Varian Pursuit XRS3 C18 ana-lytical column (150 mm × 2 mm I.D., 3 �m particle size). A mobilephase of (a) 1:1 methanol/water with 2 mM ammonium acetate and(b) methanol at a flow rate of 120 �L/min was applied for elution ofthe target compounds; starting at 50% (b) then increased linearly to

100% (b) over 3 min, held for 5 min, followed by a linear decrease to65% (b) over 2.5 min and held for 5.5 min. The three diastereomerswere baseline separated on the C18 column with retention times of12.3, 12.9 and 13.3 min for �-, �- and �-HBCD, respectively.

Mass spectrometric analysis was performed using a Sciex API2000 triple quadrupole mass spectrometer (Applied Biosystems,Foster City, CA, USA) operated in electrospray negative ionization(ESI) mode. Direct infusion of the target compounds (�-, �- and�-HBCDs, native and 13C-labeled isomers, 2 ng/�L each in MeOH)into the MS/MS system was performed with a built-in Harvardsyringe pump at a flow rate of 10 �L/min. The infusion exper-iments served for the tuning and adjusting the source and thecompound-specific parameters during the method development.MS/MS detection operated in the MRM mode was used for quantita-tive determination of the HBCD isomers based on m/z 640.6 → m/z79, m/z 652.4 → m/z 79 and m/z 657.7 → m/z 79 for the native,13C-labeled and 2H18-labeled diastereomers, respectively. The opti-mized MS/MS parameters for the selected MRM transitions aregiven in Table 1. The optimum collision energy, declustering andcollision cell potentials were found to be the same for all the targetcompounds.

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M.A.-E. Abdallah et al. / J. Ch

Table 1Optimized MS/MS parameters for the analysis of HBCDs

Parameter (units) Value

Curtain gas (a.u.) 35Turbo gas temperature (◦C) 500Ionspray voltage (V) −4500Declustering potential (V) −5Focusing potential (V) −365Collision gas (a.u.) 5Collision energy (eV) 40Cell entrance potential (V) −6Collision cell exit potential (V) −10

These parameters were obtained through infusion experiments using native and13C-labeled �-, �- and �-HBCDs (2 ng/�L each in MeOH). a.u., arbitrary units

2.3.2. GC–MS analysis2.3.2.1. Sample preparation and extraction. Dust samples (typicallybetween 150 and 250 mg) were spiked with internal standards(50 ng BDE 128 and 100 ng [13C]�-HBCD) and were then extractedwith 100 mL n-hexane:acetone (3:1, v/v) in hot Soxhlet extractionmode for 2 h as described previously [17].

2.3.2.2. Clean up. The crude extract was cleaned up by 8 gof acidified silica (44%, w/w, concentrated sulfuric acid).HBCDs were eluted with 20 mL of hexane, followed by 15 mLof dichloromethane. The eluate was concentrated by rotary-evaporation and evaporated to dryness by a gentle nitrogen stream.The extract was reconstituted in 200 �L of isooctane (containing100 pg/�L PCB 206) used as recovery determination standard.

2.3.2.3. Analysis. The determination of HBCDs was performedwith an Agilent 6890GC-5973MS system equipped with a15 m × 0.25 mm I.D., 0.10 �m DB-5 capillary column (J&W Scien-tific) and operated in the ECNI mode. The ion source, quadrupoleand interface temperatures were 230, 150 and 300 ◦C, respectively.Helium was used as carrier gas at constant flow (1.5 mL/min) and

Fig. 1. LC–MS/MS chromatogram of standard solution (200 pg/�L) of [12C]- and [13C]HBCand the absence of degradation products (MRM 480.4 to 79 for TBCDs and MRM 560.8 to

ogr. A 1190 (2008) 333–341 335

methane as reagent gas. The MS was operated in the selected ionmonitoring (SIM) mode and the electron multiplier voltage was setat 2100 V. A 1-�L volume of the extract was injected in solventvent mode (injector temperature at 90 ◦C, kept for 0.05 min, thenincreased with 700 ◦C/min to 295 ◦C, vent time 0.03 min, vent flow75 mL/min). The splitless time was 1.25 min. The oven temperatureswas programmed from 90 ◦C, kept for 1.25 min, then increased with10 ◦C/min to 200 ◦C, then increased with 20 ◦C/min to 300 ◦C, keptfor 10 min. Dwell times were 35 ms. Ions m/z 79 and 81, togetherwith ions m/z = 561 and 573 deriving from the fragment [M − Br]−

for HBCD and [13C]HBCD, respectively, were monitored for theentire run, while the recovery standard PCB 206 was measuredusing ions m/z = 464 and 466.

2.4. Quality assurance/quality control

Analyte identification was based on its retention time relative tothe internal standard used for quantification, ion chromatogramsand intensity ratios of the monitored ions. A deviation of the ionintensity ratios within 20% of the mean values obtained for thecalibration standards was considered acceptable.

For LC–MS/MS, individual five-point calibration plots (peak arearatios − analyte/internal standard − plotted against concentrationratios − analyte/internal standard) were created for the �-, �-,and �-HBCD diastereomers using the corresponding 13C-labeleddiastereomer as internal standard. The response factors (RFs) foreach diastereomer were calculated at each of the five concentra-tions used for the calibration plot and averaged. With each batch ofsamples, a calibration standard containing the three HBCD diastere-omers and their 13C-labeled analogues was analyzed. For data fromthat batch to be deemed acceptable, the RFs obtained from thisstandard had to fall within 25% of the average of the RFs obtainedfor the five concentration levels of the calibration plot.

For GC–MS, the RFs for �- and �-HBCDs were calculated rel-ative to [13C]�-HBCD. This approach most closely parallels the

Ds and �-HBCD-d18 showing baseline separation of the three HBCD diastereomers79 for PBCDs).

romatogr. A 1190 (2008) 333–341

336 M.A.-E. Abdallah et al. / J. Ch

degradation pattern of the native compounds during GC analysis.Interestingly, the two diastereomers had different RF values of 1.02and 0.74, respectively. This finding is in agreement with the fact thatHBCD diastereomers exhibit different thermal stability and thus,different thermal degradation during GC analysis [18,19]. The RFsfor �- and �-HBCDs (monitored ions m/z 561 or 79) were also cal-culated based on BDE 128 as internal standard. The �- and �-HBCDdiastereomers again generated two different RF values of 0.006 and0.004 for m/z 561 and of 0.606 and 0.430 for m/z 79. Despite thesediastereomer-specific RFs, the inability of GC to quantify the relativeabundance of the different diastereomers in samples emphasizedthat an RF based on the average of those derived for �- and �-HBCDshould also be derived. Concentrations of �HBCDs were calculatedbased on each of these diastereomer-specific and averaged RFs, inan effort to identify the most appropriate computation method.

For every five samples, one method blank was conducted. Thisconsisted of 1 g anhydrous sodium sulfate treated in identical fash-ion to samples. Concentrations of HBCDs in blanks never exceeded1% of the concentration in a sample from the same batch and datawere thus not blank-corrected. Field blanks (n = 6) were also con-ducted. These comprised of sodium sulfate (1 g) “sampled” usingthe vacuum cleaner and sock according to the standard protocoland treated as a sample. For LC–MS/MS, method detection limitsfor individual diastereomers were governed by field blanks andwere typically 0.2 ng/g. In comparison, method detection limitsfor �HBCDs via GC–MS were 5 and 50 ng/g calculated based onm/z = 79 and 561, respectively.

In the absence of an appropriate standard reference materialfor which certified concentrations of HBCDs exist, method perfor-mance was assessed through the analysis of five replicates of SRM2585 (house dust) which has indicative values for HBCDs [20]. On-going monitoring of method accuracy was performed through theanalysis of one SRM sample for every 20 dust samples. For sampleswhere particularly elevated concentrations of �HBCDs were found(i.e. such that the internal standard “spiking” levels were inappro-priately low), a fresh aliquot of the dust sample in question wasanalyzed using a smaller quantity of dust and a higher amount ofinternal standards.

3. Results and discussion

3.1. Chromatographic properties of HBCDs

The major three diastereomers of HBCD were baseline separated

via the LC–ESI/MS/MS upon monitoring the ion transition m/z 640.6[M − H]− → 79 in the negative ion mode (Fig. 1). In contrast to GC,the absence of high temperatures in the LC system means that nodegradation was observed for the native, 2H18- or 13C-labeled HBCDstandards (Fig. 1). Within the tandem MS, the turbo gas tempera-ture of 500 ◦C is used to maintain a clean ion source, but it hasno direct effect because of the “uniform cushioning” of the analytewith the solvent in the ES source which, together with the veryshort transit time, allows the HBCDs to be ionized and enter the MSsystem without being thermally decomposed.

In GC–ECNI/MS, only one peak is obtained corresponding to thetotal HBCDs due to the co-elution of the three isomers. Several ionswere monitored and evaluated for their ability to quantify HBCDs.BDE 128 was used as internal standard in all analyzed samples;however, BDE 128 is more thermally stable compared to HBCDand hence cannot compensate for the thermal decomposition ofHBCDs during GC elution. In an effort to minimize underestima-tion of �HBCDs in real samples caused by thermal decompositionat temperatures >240 ◦C, 13C-�-HBCD was used as internal stan-dard in some samples, on the premise that 13C-labeled and nativeHBCDs will behave identically in the GC system. Hence, quantifying

Fig. 2. GC–ECNI/MS total ion chromatogram and full scan mass spectra of 13C-�-HBCD standard (concentration 1 ng/�L in isooctane) (A) and �-HBCD standard(concentration 1 ng/�L in isooctane) (B). The retention time of HBCD peak was16.6 min. The standard solutions were analyzed using conditions described in Sec-tion 2.

relative to [13C]HBCD should compensate for within-GC degrada-

tion, as well as for instrumental variations in sensitivity during andbetween sample runs.

In GC–ECNI/MS, [13C]�-HBCD was monitored using m/z 573(Fig. 2A), while the native HBCDs were monitored at m/z 561(Fig. 2B) corresponding to the ion [M − Br]¯. This enhances methodselectivity and allows better structural confirmation of HBCDs. Thepresent paper reports for the first time the use of [13C]�-HBCD forGC–ECNI/MS using specific ions such as [M − Br]−. However, thisenhancement in method selectivity is accompanied by a decreasein sensitivity as the peak at m/z 561 has much less intensity thanthe “traditionally” monitored peak at m/z 79 (Fig. 2A and B). There-fore, in samples with low concentrations of HBCDs, where the peakcorresponding to m/z 561 cannot clearly be measured, only m/z 79was used for quantitation.

Concentrations of �HBCD in dust samples were calculatedbased on RFs obtained using [13C]�-HBCD and BDE 128 as internalstandards. The obtained GC–ECNI/MS results were classified intothree sets according to the internal standard and ions used for quan-titation (Table 2). The data in each set were compared statisticallyto the LC–MS/MS results obtained for the corresponding samplesusing a paired t-test for means and F-test for variances (Table 3). No

M.A.-E. Abdallah et al. / J. Chromatogr. A 1190 (2008) 333–341 337

Table 2Total HBCD concentrations (ng/g) in dust samples using LC–MS/MS and GC–ECNI/MS

Sample LC–ESI/MS/MS GC–ECNI/MS

[13C]HBCDs internal standard BDE 128 internal standard

m/z 561 m/z 79

RF of �-HBCD RF of �-HBCD Average RF of �-HBCD RF of �-HBCD Average RF of �-HBCD RF of �-HBCD Average

S1 12,200 12,900 17,700 15,300 11,700 17,600 14,600S2 2,500 1,500 2,100 1,800 5,300 7,900 6,600S3 2,400 1,500 2,000 1,800 1,600 2,300 1,900S4 880 640 880 750 770 1,200 960S5 3,500 4,200 5,800 5,000 7,000 10,500 8,700S6 780 1,100 1,500 1,300 1,200 1,800 1,500S7 4,000 3,900 5,300 4,600 5,600 8,400 7,000S8 1,500 1,300 1,800 1,300 1,500 2,200 1,800S9 1,050 1,200 1,200 1,200 1,200 1,800 1,500S10 1,600 1,400 1,900 1,600 1,300 1,900 1,600S11 8,500 9,700 13,400 11,600 11,300 16,900 14,100S12 890 610 830 720 650 970 810S13 390 80 110 90 80 120 100S14 6,400 4,800 6,600 5,700 4,570 6,900 5,700S15 15,700 9,400 13,000 11,200 11,320 17,000 14,200S16 4,700 3,900 5,300 4,600 4,200 6,300 5,300

S17 2,100 1,200S18 111,000 96,000S19 960 690S20 1,300 860S21 13,700 14,000S22 2,000 1,900S23 1,700 2,800S24 1,400 1,400S25 11,900 8,100S26 16,100 13,200S27 14,000 7,200S28 14,400 8,800S29 6,800 6,200S30 7,400 4,900S31 18,500 15,500

S32 50 –a

S33 170 –a

S34 730 –a

S35 320 –a

S36 180 –a

S37 290 –a

a Concentrations based on m/z could not be calculated due to low ion intensities.

statistically significant differences (p > 0.05) were found betweenthe three sets of GC/MS data and the concentrations obtained viaLC–MS/MS. The closest match between the two techniques wasobtained when [13C]�-HBCD was used as internal standard andthe RF deployed was the average of those obtained for �- and �-HBCD. An important point is that the superior performance of thisaveraged RF may be due to the roughly equal abundance of the�- and �-HBCD diastereomers in most dust samples revealed bythe LC–MS/MS results. For matrices such as biota, where the �-HBCD diastereomer predominates, then a RF derived solely for the�-HBCD may be more appropriate.

The analyzed dust samples were subjected to a thorough cleanup process prior to analysis which reduces substantially the occur-

Table 3Statistical comparison (t-test, F-test and Pearson correlation coefficients) between values

[13C]HBCDs internal standard (n = 16)* BDE 128 in

m/z 561 (n

RF of �-HBCD RF of �-HBCD Average RF of �-HB

t-test paired for means 1.39 1.51 0.20 2.15F-test for variances 1.34 1.42 1.06 1.34Pearson correlation (r) 0.92 0.92 0.92 0.99Slope GC versus LC 1.05 0.76 0.89 1.15

The confidence level was set at 95%. * t-critical = 2.13, F-critical = 2.40; ** t-critical = 2.73, F

1,800 1,540 1,200 1,700 1,500144,000 120,000 81,000 114,000 98,000

1,000 860 700 970 8401,300 1,100 870 1,200 1,000

20,900 17,400 13,200 18,400 15,8002,800 2,400 1,900 2,700 2,3004,200 3,500 2,600 3,600 3,1002,100 1,800 1,400 2,000 1,700

12,200 10,200 8,100 12,200 10,20019,800 16,500 13,000 18,100 15,50010,800 9,000 7,100 10,000 8,60013,200 11,000 8,700 12,200 10,4009,300 7,800 6,200 8,600 7,4007,300 6,100 4,800 6,800 5,800

23,200 19,300 14,500 20,300 17,400

–a –a 30 30 30–a –a 90 120 100–a –a 540 750 640–a –a 100 140 120–a –a 110 150 130–a –a 220 300 260

rence of matrix effects during MS analysis. This, together withthe deployment of an RF based on the average of the RFs forthe �- and �-HBCDs, are likely to account for the good cor-relations obtained between the GC–MS and LC–MS/MS results(Table 3). The R-values obtained here exceed those reported byvan Leeuwen and de Boer [21] for the correlation between GC–MSand LC–MS results of HBCD analysis in fish samples. In our study,the slopes of the linear correlations between the GC–MS andLC–MS/MS results ranged from 0.76 to 1.36 indicating a closematch between the results obtained by the two techniques. Thisalso differs from the results of van Leeuwen et al. who reportedGC–MS results to be five times higher than LC–MS results ineels [22] and a slope of 0.23 for the linear correlation between

of total HBCDs issued by LC–MS/MS and GC–ECNI/MS

ternal standard

= 31)** m/z 79 (n = 21)***

CD RF of �-HBCD Average RF of �-HBCD RF of �-HBCD Average

2.43 1.46 1.95 1.15 1.650.60 0.86 1.09 0.94 1.290.99 0.99 0.99 0.99 0.990.77 0.92 1.36 0.97 1.13

-critical = 1.84; *** t-critical = 2.08, F-critical = 2.12.

338 M.A.-E. Abdallah et al. / J. Chromatogr. A 1190 (2008) 333–341

Table 4

Concentrations of HBCDs (ng/g dust) in SRM 2585 compared to indicative valuesreported by Keller et al. [20]

Average concentrations ± SD

�-HBCD �-HBCD �-HBCD �HBCDs

Indicative values [19] 19 ± 3.7 4.3 ± 1.1 120 ± 22 143 ± 27LC–MS/MS (n = 5) 19 ± 3.6 4.4 ± 0.4 125 ± 20 150 ± 20

[13C]HBCDs internal standard (n = 1)RF of �-HBCD 105RF of �-HBCD 145Average 125

BDE 128 internal standardm/z 561 (n = 5)

RF of �-HBCD 95 ± 9RF of �-HBCD 140 ± 15Average 120 ± 10

m/z 79 (n = 4)RF of �-HBCD 105 ± 10RF of �-HBCD 150 ± 15Average 130 ± 10

LC–ESI/MS/MS and GC–ECNI/MS results of HBCDs analysis in fish

[21].

3.2. Method performance

The accuracy of the applied analytical methods was checked viareplicate analysis (n = 5) of SRM 2585 (house dust) which has indica-tive values for HBCDs ([20], Table 4). There was a good agreementbetween the LC–MS/MS results and the indicative values, while forthe GC–ECNI/MS results, only the concentrations estimated usingthe RF of �-HBCD are closely matching the indicative values. Themost probable explanation for this is that the �-HBCD isomer is thepredominant isomer in the SRM 2585 material (>83% of total HBCDsas determined by LC–MS/MS). Furthermore, using the RF of thethermally stable �-HBCD isomer may underestimate substantiallythe total HBCD concentration as it decomposes in the GC systemto a lesser extent than the �-HBCD isomer does. Method precisionwas calculated as the relative standard deviation of concentrationsobtained from five replicates of SRM 2585. Method precision (RSD)was <15% for both LC–MS/MS and GC–ECNI/MS methods, indicatinggood precision for both methods.

Fig. 4. GC–EI/MS chromatogram of a dust sample (S18) showing the target compounds (Hsimilar to those described in Section 2 except for the analytical column (HT-8, 25 m × 0.22

Fig. 3. GC–ECNI/MS chromatograms (selected ion monitoring mode) of ions m/z79 (A) and m/z 561 (B) for a dust sample (S26) showing enhanced selectivity anddecreased sensitivity (see absolute value of the abundance) obtained when monitor-ing the ion m/z 561 ion. Degradation products, PBCDs and TBCDs, are also indicated.The sample was analyzed using conditions described in Section 2.

Average recoveries ± standard deviations for the [13C]HBCDdiastereomers as determined by LC–MS/MS were: �-HBCD = 82 ± 8,�-HBCD 88 ± 5, and �-HBCD 83 ± 8%. No HBCDs could be detectedin method blanks (n = 6) for the dust analysis procedures consist-ing of sodium sulfate (1 g). Detectable, but very low concentrations(typically 0.1–0.5 ng/g �HBCDs) were obtained for field blanks(n = 6). These consisted of sodium sulfate (1 g) “sampled” usingthe vacuum cleaner and sock according to the standard proto-col and treated as a sample. Concentrations in samples were thuscorrected for the contamination detected in the associated fieldblank.

BCDs) and the degradation products (PBCDs and TBCDs). Analytical conditions aremm I.D., 0.25 �m) and temperature of the ion source (230 ◦C).

M.A.-E. Abdallah et al. / J. Chromat

3.3. Degradation products of HBCD in dust

The thermal isomerization and degradation of HBCDs at thehigh temperatures used during the GC/MS analysis have beenreported previously [18]. These degradation products are observed

Fig. 5. Full scan EI–MS spectra of HBCDs (A), PBC

ogr. A 1190 (2008) 333–341 339

in the GC–ECNI/MS chromatograms for both samples and standards(Fig. 3), which makes it very difficult to determine if these degrada-tion products are present originally in the samples or produced as aresult of thermal degradation in the GC system. The identity of thedegradation products was investigated by GC–MS which separated

Ds (B) and TBCDs (C) for dust sample S18.

romatogr. A 1190 (2008) 333–341

340 M.A.-E. Abdallah et al. / J. Ch

the degradation products from HBCDs (Fig. 4) and provided theirfull scan mass spectra (Fig. 5). The issue of whether these productsare merely formed in the GC system may be resolved by the use ofLC–MS/MS, during which no thermal degradation occurs and there-fore, no degradation products are observed in the chromatogramsof standards (Fig. 1). Two different degradation products wereobserved in almost all dust samples and in SRM 2585. The prod-ucts thus identified are pentabromocyclododecenes (PBCDs) andtetrabromocyclododecadienes (TBCDs), which were monitored attransitions m/z 560.8 → 79 and m/z 480.4 → 79, respectively.

The mass spectra of HBCDs, PBCDs and TBCDs are very similarsuggesting that these compounds have a similar structure (Fig. 5).Furthermore, the low-mass fragment ions at m/z 53, 65/67, 79, 91,105, and 117 deepens the assumption of an aliphatic backbone forthe degradation products. At higher masses, the fragment ions atm/z 317 (2 Br) and m/z 399 (3 Br) are also in agreement with themass spectrum of HBCDs. Unfortunately, GC–MS does not allowthe correct identification of the number of PBCD or TBCD isomers,since, similar to HBCDs, these compounds also co-elute. No peakswere detected for lower brominated (1–3 Br atoms) degradationproducts of HBCDs in the dust samples.

The LC–MS/MS chromatograms of PBCDs (Fig. 6) showed fourwell-resolved peaks indicating the presence of at least four major

isomers. The PBCDs were well separated from the parent HBCDson retention time basis (Figs. 1 and 6), with the PBCDs elutingbetween 10.13 and 11.90 min and the HBCDs between 12.32 and13.32 min. The MS/MS technique enabled further structure confir-mation through the monitoring of the PBCDs at m/z 560.8 → 79.0,which differs from that of the parent HBCDs monitored at m/z640.9 → 79.0. These peaks were assigned the names of PBCD1 toPBCD4, with the first eluting isomer being PBCD1. PBCDs werereported previously by Barontini et al. [23,24] as thermal degra-dation products of HBCDs having a molecular weight of 561 g/moland a total number of seven isomers detected and identified viaGC–MS. Hiebl and Vetter [25] also detected PBCD (one isomer) asa metabolite/degradation product of HBCDs in chicken egg and infish via GC–EI/MS. However, in the present study, only four PBCDisomers were detected and identified via LC–MS/MS indicating thatthe degradation pathways of HBCDs under ambient environmentalconditions are different from those induced under the harsher ther-mal conditions (300–400 ◦C) employed by Barontini et al. [23,24].This can also explain the difference in the profiles of degradationproducts obtained by GC–MS and LC–MS/MS analysis of dust sam-ples. During the GC run, the sample is exposed to high temperatures

Fig. 6. LC–MS/MS chromatogram of a dust sample (S26) showing PBCD isomers(MRM 560.8 to 79).

Fig. 7. LC–MS/MS chromatogram of SRM 2585 (house dust) showing TBCD isomers(MRM 480.4 to 79).

and the degradation products may be different from those origi-nally present in the sample and detected as such by the LC–MS/MS.

Efforts were made to baseline resolve PBCD3 from PBCD4 on theLC column by slightly changing the solvent gradients and flow rate.However, since this was not possible, no further trials were madeto avoid alterations of the method parameters that might affect theseparation and retention times of the main target compounds, inthis case the �-, �-, and �-HBCD isomers.

Only two peaks were observed for TBCDs in chromatogramsindicating the presence of two congeners (Fig. 7). However, the firsteluted peak (TBCD1) always was less intense than the second peak(TBCD2), which is also observed much more frequently in the dustsamples. The detection of these two TBCD isomers in dust samplesindicates that the degradation pathways of HBCDs in indoor dustare different from those in waste water sludge and freshwater sed-iments in which Davis et al. [26] reported only one TBCD isomer asa degradation product of HBCDs (via LC–APPI/MS). They concludedthat HBCDs degrade via a dibromoelimination reaction resulting inthe loss of two bromines from vicinal carbons with the subsequentformation of a double bond between the adjacent carbon atoms.

However, the detection and identification of the four PBCDstogether with the two TBCDs in the present study rules out vic-inal debromination as the main degradation pathway of HBCDsin indoor dust. The detection of degradation products, PBCDs and

TBCDs, and their relationship to the parent HBCDs in the analyzeddust samples, favors the elimination of HBr. This results in theformation of a double bond (adjacent to the initial carbon atomscontaining Br) and it is suggested to be a significant degradationpathway of HBCDs in indoor dust. This hypothesis is in agreementwith the degradation pathway suggested by Hiebl and Vetter [25]for HBCDs in chicken egg and white fish.

Accurate quantitation of the detected degradation products wasnot possible due to the lack of either native or labeled standards forthese compounds which are (to the authors’ knowledge) reportedhere in indoor dust for the first time. Furthermore, similar to HBCDs,the LC–MS/MS method allows the detection of different PBCD andTBCD diastereomers.

Although LC–MS/MS remains the technique of choice for theisomer-specific analysis of HBCDs, the present study indicates thatGC–ECNI/MS can be used as a screening tool to estimate the totalHBCD concentrations with acceptable accuracy. This is facilitated bythe use of [13C]HBCDs as internal standards and by deployment ofan RF based on the average of those obtained for the �- and �-HBCDdiastereomers. However, this enhancement in method accuracyand specificity using [13C]HBCDs as internal standards (monitoring

romat

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[[

C.H. Marvin, Rapid Commun. Mass Spectrom. 19 (2005) 2819.[13] N.G. Dodder, A.M. Peck, J.R. Kucklick, L.C. Sander, J. Chromatogr. A 1135 (2006)

M.A.-E. Abdallah et al. / J. Ch

of ion m/z = 573) is accompanied by a decrease in the method sensi-tivity. As an alternative, BDE 128 (or another PBDE congener whichis not present in the PBDE technical mixture) can be used as internalstandard (monitoring of ion m/z = 79) with lower method detectionlimits, but less selectivity. Nevertheless, the need for monitoring oftotal HBCDs in food and feed has recently been underlined by theEuropean Food Safety Agency, which has included HBCDs (as sum ofisomers) on the list with priority BFRs [27]. PBCDs and TBCDs, iden-tified in the present study as the degradation products of HBCDs,were observed in the GC–MS chromatograms of dust samples. UsingLC–MS/MS, it could be demonstrated that they are not only the

result of thermal degradation in the GC, but are originally presentin the dust samples.

Acknowledgements

The authors gratefully acknowledge the provision of stu-dentships from the Egyptian government and Egyptian Ministryof Higher Education (M.A.A.), the National Council of Science andTechnology – Mexico (CONACYT) (C.I.), and of a postdoctoral fel-lowship from the Research Scientific Foundation of Flanders (FWO)(A.C.). The support of the UK POPs network training fund for thevisit of M.A.A. and C.I. to the University of Antwerp is also gratefullyacknowledged.

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