Isotope Dilution Method for Determination of Polybrominated Diphenyl

Aug 4, 2009 - SRM 2585 Organic Contaminants in House Dust was purchased from ..... analytical techniques is given in Table 3, while the full set of re...
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Anal. Chem. 2009, 81, 7460–7467

Isotope Dilution Method for Determination of Polybrominated Diphenyl Ethers Using Liquid Chromatography Coupled to Negative Ionization Atmospheric Pressure Photoionization Tandem Mass Spectrometry: Validation and Application to House Dust Mohamed Abou-Elwafa Abdallah,*,†,‡ Stuart Harrad,† and Adrian Covaci§ Division of Environmental Health and Risk Management, School of Geography, Earth and Environmental Sciences, University of Birmingham, Birmingham, B15 2TT, United Kingdom, Department of Analytical Chemistry, Faculty of Pharmacy, Assiut University, 71526 Assiut, Egypt, and Toxicological Center, University of Antwerp, Universiteitsplein 1, 2610 Wilrijk, Belgium A total of 14 tetra- to deca- PBDE congeners were separated on a C18 reversed phase liquid chromatographic column. PBDEs 47, 85, 99, 100, 153, 154, 183, 196, 197, 203, 206, 207, 208, and 209 were eluted using a gradient methanol/water/toluene mobile phase system at a flow rate of 0.5 mL min-1. 13 C-BDE-47, 13C-BDE-99, 13C-BDE-153, BDE-128, and 13C-BDE-209 were used as internal standards, while 13C-BDE-100 was used as a syringe standard. Separated analytes were ionized using an atmospheric pressure photoionization (APPI) source equipped with a 10 eV krypton lamp and operated in negative ion mode. [M-Br + O]- ions were monitored as precursor ions for all studied PBDEs, except for BDE-208 and BDE-209 which produced higher intensity at the [C6Br5O]- ion cluster. [Br]ions were monitored as fragment ions for all target compounds. Method detection limits ranged from 12 to 30 pg. The method was applied to determination of PBDEs in standarad reference material (SRM 2585), and favorable results were obtained. Unlike GC methods, no thermal degradation was encountered in the analysis of higher brominated PBDEs. This rendered the method useful for quantification of BDE-209 debromination products. The method also allows the use of 13C-labeled internal standards, which compensate for instrumental fluctuations and matrix-related ion suppression or enhancement. Polybrominated diphenyl ethers (PBDEs) are a group of brominated flame retardants which are or have been produced commercially in three main formulations: Penta (mainly BDE* Corresponding author. E-mail [email protected]; phone +44 121 414 5431; fax +44 121 414 3078. † University of Birmingham. ‡ Assiut University. § University of Antwerp.

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47 and BDE-99, 38-49% each, alongside other tri- to heptaBDEs), Octa (a mixture of hexa- to deca-BDEs, the exact congener composition varies between the two principal formulations marketed), and Deca (92-97% decabromodiphenyl ether, plus various nona-(principally) and octa-BDEs).1 The main uses for these commercial formulations are the Pentaproduct to flame retard polyurethane foams in carpets, vehicle interiors, furniture and bedding, as well as printed circuit boards and microprocessor packaging in computers; the Octaformulation to treat thermoplastics, such as high-impact polystyrene (HIPs) and acrylonitrile-butadiene-styrene copolymers (ABS); and the Deca-product used in HIPs applied mainly in plastic housings for electrical goods, like TVs and computers, as well as textiles.2 Several studies have discussed the levels of PBDEs in both biotic and abiotic matrixes.3-5 Because of their persistent, bioaccumulative and toxic (PBT) characteristics, tetra-to-hepta- BDEs were listed recently as persistent organic pollutants (POPs) under the Stockholm Convention.6 Meanwhile, the European Court of Justice ruled against the exemption of Deca-BDE from the RoHS directive and decided that its use must be phased out by July 1, 2008.7

(1) La Guardia, M. J.; Hale, R. C.; Harvey, E. Environ. Sci. Technol. 2006, 40 (20), 6247–6254. (2) Harrad, S.; Ibarra, C.; Diamond, M.; Melymuk, L.; Robson, M.; Douwes, J.; Roosens, L.; Dirtu, A. C.; Covaci, A. Environ. Int. 2008, 34 (2), 232– 238. (3) Law, R. J.; Herzke, D.; Harrad, S.; Morris, S.; Bersuder, P.; Allchin, C. R. Chemosphere 2008, 73 (2), 223–241. (4) Hites, R. A. Environ. Sci. Technol. 2004, 38 (4), 945–956. (5) Covaci, A.; Voorspoels, S.; de Boer, J. Environ Int. 2003, 29 (6), 735–756. (6) Stockholm convention on POPs. Governments unite to step-up reduction on global DDT reliance and add nine new chemicals under international treaty. http://chm.pops.int/Convention/Pressrelease/COP4Geneva8May 2009/tabid/542/language/en-US/Default.aspx, 2009 (accessed June 5, 2009). (7) Judgement of the European Court of Justice on Joint Cases C-14/06 and C-295/06. http://curia.europa.eu/en/actu/communiques/index.htm, 2008 (accessed December 12, 2008). 10.1021/ac901305n CCC: $40.75  2009 American Chemical Society Published on Web 08/04/2009

The extraction, chromatographic separation, and mass spectrometric detection of PBDEs have been reviewed recently.8,9 However, a relative lack of sensitivity is observed with GC-EI/ MS analysis of higher brominated PBDEs (defined as those containing more than six Br). Therefore, electron capture negative ionization (ECNI), based on monitoring m/z 79 and 81 bromide ions, is the method most widely used for the analysis of higher PBDEs (hepta to deca-BDE congeners).8 This method does not provide mass discrimination between native and 13C-labeled isomers and hence prevents the use of the latter as internal standards, except for 13C-BDE-209 which produces abundant [C6Br5O]- ions.10 The GC-ECNI/MS analysis of BDE-209 is hampered by thermal degradation problems, which necessitates shorter columns with higher temperature limits and higher phase ratios to minimize oncolumn degradation. Additionally, optimized injection techniques (i.e., on-column injection, pressure pulses, or split vent timing) are required to minimize the time spent in the heated injector zone to prevent thermal decomposition.8,9 The thermal degradation problems encountered with GC analysis may eventually be avoided using LC-based methods. Recently, atmospheric pressure photoionization (APPI) has appeared as a very soft ionization technique, allowing the analysis of hydrophobic compounds using LC-MS/MS. APPI is a natural evolution of APCI (atmospheric pressure chemical ionization) in which a UV source initiates the ionization process, as opposed to a corona discharge as in APCI. Briefly, the energy supplied by a UV source is used to ionize the molecules of a doping agent (substance of favorable ionization energy lower than the energy of the supplied photons). The produced dopant radical cations may then ionize the analyte through charge exchange. Alternatively, the dopant cations can ionize solvent molecules by proton transfer; then the protonated solvent molecules proceed to ionize the analyte. In negative ion mode, the ionization can occur via deprotonation, electron capture, or charge exchange. The formation of a radical cation makes it possible to ionize nonpolar molecules that cannot be efficiently analyzed by either ESI (electrospray ionization) or APCI.11 Riu et al.12 reported the use of an APPI ion source in negative ion (NI) mode to give “promising” results in the analysis of tetra-to-deca- BDEs using LC-MS/MS (quadrupole ion trap). No such promising results were obtained using ESI or APCI sources, but unfortunately, no quantitative data were provided. This was rectified recently by Lagalante and Oswald13 who reported on the use of LC-APPI-MS/MS for determination of the concentrations of eight PBDEs (octa and nona-BDEs not included) in dust. However, concentrations were calculated using an external standard method (i.e., no internal standard used). (8) Covaci, A.; Voorspoels, S.; Ramos, L.; Neels, H.; Blust, R. J. Chromatogr., A 2007, 1153 (1-2), 145–171. (9) Stapleton, H. M. Anal. Bioanal. Chem. 2006, 386 (4), 807–817. (10) Bjo ¨rklund, J.; Tollback, P.; Ostman, C. J. Mass Spectrom. 2003, 38 (4), 394–400. (11) Kauppila, T. J.; Kuuranne, T.; Meurer, E. C.; Eberlin, M. N.; Kotiaho, T.; Kostiainen, R. Anal. Chem. 2002, 74 (21), 5470–5479. (12) Riu, A.; Zalko, D.; Debrauwer, L. Rapid Commun. Mass Spectrom. 2006, 20 (14), 2133–2142. (13) Lagalante, A. F.; Oswald, T. D. Anal. Bioanal. Chem. 2008, 391 (6), 2249– 2256.

In light of the above, the aims of this study are (1) to develop and validate a new analytical method based on LC-NI-APPIMS/MS for determination of 14 PBDEs in one run using 13Clabeled isotopes as internal standards; (2) to apply the developed method for determination of PBDEs in indoor dust samples; (3) to compare the results of the developed method to those obtained using well established GC-ECNI-MS and GC-EI-MS methods;14,15 and (4) to investigate the potential applicability of the method to evaluating debromination of BDE209 in dust samples. EXPERIMENTAL SECTION Chemicals. All solvents used in the extraction and analysis procedures were of HPLC grade quality (Fisher Scientific, Loughborough, U.K.). Individual standards of native BDEs 47, 85, 99, 100, 128, 153, 154, 183, 196, 197, 201, 202, 206, 207, 208, and 209 and 13C-BDEs 47, 99, 100, 153, and 209 were purchased from Wellington Laboratories (Guelph, ON, Canada). Standards of BDEs 198, 203, 204, and 205 were bought from Accustandard (New Haven, CT). For cleanup of dust samples, silica (70-130 mesh), concentrated sulfuric acid, and anhydrous sodium sulfate were obtained from Sigma-Aldrich (St. Louis, MO). SRM 2585 Organic Contaminants in House Dust was purchased from the National Institute of Standards and Technology (NIST, Gaithersburg, MD). Dust Sampling. Dust samples were collected between September 2006 and June 2007 from 25 microenvironments within the West Midlands conurbation in the U.K. All microenvironments comprised a convenience sample of acquaintances of the authors. The following microenvironment categories were sampled: homes (living rooms, n ) 9), offices (n ) 8), and cars (n ) 8). Dust samples were collected using a Nilfisk Sprint Plus 1600 W vacuum cleaner. Sampling was conducted according to a clearly defined standard protocol2 by one of the research team. In offices and homes, 1 m2 of carpet was vacuumed for 2 min, and in the case of bare floors, 4 m2 for 4 min. In cars, only the surface of the seats with which occupants would have direct contact (i.e., not including seat backs), the front panel, and the steering wheel were sampled for 2 min. Samples were collected using nylon sample socks (25 µm pore size) that were mounted in the furniture attachment tube of the vacuum cleaner. After sampling, socks were closed with a twist tie, sealed in a plastic bag, and stored at -20 °C. Before and after sampling, the furniture attachment was cleaned thoroughly using an isopropanol-impregnated disposable wipe. Potential interferences from the sampling device were checked by collecting and analyzing 5 “field blanks”. These consisted of sodium sulfate (0.2 g) “sampled” using the vacuum cleaner according to the standard protocol and treated as a sample. Sample Preparation and Extraction. Dust samples were homogenized and sieved through a 500 µm mesh size sieve. Accurately weighted aliquots (typically between 100 and 300 mg of dust) were loaded into precleaned 66 mL cells containing (14) Harrad, S.; Ibarra, C.; Abdallah, M. A. E.; Boon, R.; Neels, H.; Covaci, A. Environ. Int. 2008, 34 (8), 1170–1175. (15) Harrad, S.; Wijesekera, R.; Hunter, S.; Halliwell, C.; Baker, R. Environ. Sci. Technol. 2004, 38 (8), 2345–2350.

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Table 1. Optimized APPI Source Parameters parameter

value

curtain gas collision (CAD) gas ion transfer voltage (IS) APPI temperature probe nebulizer gas auxillary gas

25 psi high 1250 V 400 °C 60 psi 30 psi

1.5 g of Florisil and Hydromatrix (Varian Inc., U.K.) to fill the void volume of the cells and spiked with 25 ng of each of BDE 128, 13C-labeled BDEs 47, 99, and 153, and 100 ng of 13CBDE 209 as internal (surrogate) standards (i.e., standards used for determination of analyte concentrations) prior to pressurized liquid extraction (ASE 300, Dionex Europe, U.K.) using hexane/dichloromethane (1:9, v/v) at 90 °C and 1500 psi. The heating time was 5 min, static time 4 min, purge time 90 s, flush volume 50%, with three static cycles. Cleanup. The crude extracts were concentrated to 0.5 mL using a Zymark Turbovap II then purified by loading onto SPE cartridges filled with 8 g of precleaned acidified silica (44% concentrated sulfuric acid, w/w). The analytes were eluted with 25 mL of hexane/dichloromethane (1:1, v/v). The eluate was evaporated to dryness under a gentle stream of nitrogen, then reconstituted in 100 µL of 13C-BDE 100 (25 pg µL-1 in methanol) used as a recovery determination (or syringe) standard, used to determine the recoveries of internal standards for QA/QC purposes. LC-NI-APPI-MS/MS Analysis. Target PBDEs (47, 85, 99, 100, 153, 154, 183, 196, 197, 203, 206, 207, 208, and 209) were separated using a dual pump Shimadzu LC-20AB Prominence liquid chromatograph (Shimadzu, Kyoto, Japan) equipped with a SIL-20A autosampler, a DGU-20A3 vacuum degasser, and a Varian Pursuit XRS3 (Varian, Inc., Palo Alto, CA) C18 reversed phase analytical column (250 mm × 4.6 mm i.d., 3 µm particle size). A mobile phase program based upon (mobile phase A) 85:10:5 methanol/toluene/water and (mobile phase B) 1:1 methanol/water at a flow rate of 500 µL min-1 was applied for elution of the target compounds; starting at 85% (mobile phase A), then increased linearly to 100% (mobile phase A) over 30 min, and then held for 25 min. The column was equilibrated with 85% (mobile phase A) for 5 min between runs. Mass spectrometric analysis was performed using a Sciex API 2000 triple quadrupole mass spectrometer (Applied Biosystems, Foster City, CA) equipped with an APPI ion source operated in negative ion mode. Toluene was used as a doping agent introduced via a separate dopant port of the APPI source using a dedicated isocratic HPLC pump (Jasco PU-2800, Easton, MD) at 12% of the flow rate of the mobile phase. Direct infusion of the target compounds into the MS/ MS system was performed using a built-in Harvard syringe pump at a flow rate of 10 µL min-1. The infusion experiments served for tuning and adjusting the source and the compoundspecific parameters during method development. Statistical Analysis. Statistical analysis of the data was conducted using Excel (Microsoft Office 2003) and SPSS version 13.0. 7462

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Table 2. Optimized Compound-Specific Parameters, Linearity, LODs, and LOQs of Target Compounds BDE no.

precursor (m/z)

fragment (m/z)

DPa (V)

CEb (V)

47 85 99 100 153 154 183 196 197 203 206 207 208 209

420.8 500.8 500.8 500.8 578.8 578.8 658.6 738.6 738.6 738.6 816.6 816.6 486.6 486.6

78.8 78.8 78.8 78.8 78.8 78.8 78.8 78.8 78.8 78.8 78.8 78.8 78.8 78.8

-12 -8 -11 -14 -8 -8 -12 -14 -12 -15 -11 -12 -10 -8

-55 -60 -60 -63 -65 -65 -61 -71 -71 -71 -75 -69 -71 -75

a

linearity (0.02-5 ng µL-1) r2 r2 r2 r2 r2 r2 r2 r2 r2 r2 r2 r2 r2 r2

) ) ) ) ) ) ) ) ) ) ) ) ) )

0.997 0.996 0.996 0.998 0.996 0.997 0.998 0.996 0.997 0.997 0.996 0.996 0.997 0.996

LOD (pg)

LOQ (pg)

29.8 26.1 12.3 15.2 18.3 20.1 16.2 15.7 14.9 14.2 13.6 13.3 13.8 12.7

125.1 100.2 52.3 57.6 77.6 84.7 70.6 64.9 60.4 59.6 53.6 53.0 56.9 54.5

Declustering potential. b Collision energy.

RESULTS AND DISCUSSION Optimization of APPI Parameters. Toluene was used as the doping agent in preference to acetone, due to its more favorable ionization energy and lower proton affinity that combine to facilitate better proton transfer reaction with the analytes. Maximum sensitivity was obtained upon introduction of dopant to the APPI source at 12% of the mobile phase flow rate (Figure SI-1 in the Supporting Information). The use of methanol in the mobile phase produced higher sensitivity than acetonitrile which is in agreement with previous reports.12,13 This is probably due to the much lower yield of acetonitrile ions (about an order of magnitude less) than for methanol mobile phase caused by the higher ionization potential (IP) of acetonitrile (IP ) 12.2 eV) resulting in less ionization by ion-analyte reactions than for methanol.16 The use of mobile phase modifiers (e.g., ammonium acetate 1-10 mM) had no significant effect on the intensity of the produced peaks. Therefore, no mobile phase modifiers were used. The optimized source parameters are given in Table 1. Optimization of Compound-Specific Parameters. Stable [M-Br + O]- ions (Figure SI-2 in the Supporting Information) were formed for all the studied PBDEs via a substitution reaction between [M-Br]- ions and O2 present in the ambient air of the APPI source or produced by thermal decomposition of oxygen-containing solvent molecules according to the following mechanism:17 M + O2-• f [M-Br + O]- + OBr• The [M-Br + O]- ions were monitored as precursor (qualifier) ions at Q1 (first quadrupole of the MS/MS system) for all the studied compounds except for BDE-208, BDE-209, and 13CBDE-209 where higher intensity ion clusters were observed between m/z 483 and 493 corresponding to [C6Br5O]- ions (Figure SI-3 in the Supporting Information), which were thus selected as precursor ions for these three compounds. The [M-Br + O]- ions formed can fragment to produce [M′-HBr]- and/or [M′-Br2]- ions with varying intensities depending on the pattern and degree of bromine substitution

Figure 1. Separation of target PBDEs on a RP-C18 column (250 mm × 4.6 mm; 3 µm).

on the two phenyl rings of the studied PBDE congener. However, maximum sensitivity was obtained when using a high collision energy to obtain [Br]- ions (Figure SI-4 in the Supporting Information) which were thus monitored as fragment (quantifier) ions at Q3 for all target compounds (Table 2). The collision energy (CE) and declustering potential (DP) were optimized for each target compound to produce maximum sensitivity (Table 2).

Chromatographic Separation. Different C18-RP columns were studied for separation of target PBDEs. Columns with 3 µm particle size were preferred, because BDE-207 and BDE-208 could not be well-separated on 5 µm particle size columns of different dimensions (Shimadzu, 150 mm × 4.6 mm, Kyoto, Japan, and Thames-Restek, Pinnacle, 250 mm × (16) Short, L. C.; Cai, S. S.; Syage, J. A. J. Am. Soc. Mass Spectrom. 2007, 18 (4), 589–599.

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Figure 2. Representative example of two MRM (multiple reaction monitoring) scans for BDE-209 and between the two signals at a 100:1 ratio.

13

C-BDE-209 showing no interference

Figure 3. Comparison of certified and indicative19 PBDE concentrations (nanograms per gram) in SRM 2585 with the average values (n ) 5) obtained in this study. Error bars represent standard deviations.

4.6 mm, Saunderton, U.K.). Although the number of theoretical plates is similar in (150 mm × 2.1 mm) and (250 mm × 4.6 mm) 3 µm C18-RP columns, the latter was preferred due to the very high pressure (>3000 psi) required to achieve the desired 0.5 mL min-1 flow rate when using the (150 mm × 2.1 mm) column. This caused several leaks in the HPLC and autosampler system, and hence the larger dimensioned column was used in this study. (17) Basso, E.; Marotta, E.; Seraglia, R.; Tubaro, M.; Traldi, P. J. Mass Spectrom. 2003, 38 (10), 1113–1115.

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The 14 target PBDEs were well-resolved from each other on a retention time basis (Figure 1) and from the 13C-labeled analogues on a MRM basis (e.g., BDE-99 was monitored at MRM 500.8 f 78.8, while 13C-BDE-99 was monitored at 512.4 f 78.8). The identity of each analyte was confirmed via injection of individual standards using the same analytical conditions. No interference between the native PBDEs and their 13C-labeled analogues was observed even at a 100:1 concentration ratio (Figure 2). Addition of toluene to the mobile phase program was necessary for elution of the higher

Table 3. Summary of Concentrations (ng g-1) of PBDEs in Indoor Dust Samples (n ) 25) Analyzed by Different Techniques GC-EI/MS14 GC-ECNI/MS15 parameter/ PBDE no. 47 100 99 154 153 183 197 203 196 209 average SDa median minimum maximum

50.6 72.0 22.1 1.4 306.5

16.7 22.6 9.3 0.5 88.4

93.7 122.0 44.7 3.7 495.7

parameter/ PBDE no. 47 100 99 average SDa median minimum maximum a

50.0 69.8 19.8 1.8 289.7

16.7 21.4 7.8 0.9 78.5

94.4 123.8 49.2 4.6 511.3

12.6 20.2 7.5 0.5 98.3

30.1 75.5 10.2 0.5 377.0

36.7 108.0 8.2 1.0 548.9

25.5 40.9 15.1 1.0 203.8

45.5 65.8 12.9 1.4 254.7

71.9 77.7 40.7 3.8 311.8

98 350.3 157 430.5 21 486.7 267.7 519 668.2

LC-APPI-MS/MS 154 153 183 197 203 196 13.3 22.9 7.2 0.9 112.4

29.0 71.4 9.2 0.8 357.9

35.2 103.3 10.4 1.3 525.9

25.0 38.3 14.2 0.9 188.7

46.4 68.1 12.5 0.9 269.6

71.5 78.4 37.8 3.2 323.1

209 98 327.8 157 624.5 20 933.9 314.2 519 721.0

Table 4. Statistical Comparisona between Concentrations of PBDEs in Dust Samples (n ) 25) Obtained by GC-EI/MS14 (Tetra- to Hexa-BDEs), GC-ECNI-MS15 (Hepta- to Deca-BDEs), and LC-APPI-MS/MS BDE no.

t testb paired for means

F-testc for variance

Pearson correlation (r)

slope GC vs LC

47 100 99 154 153 183 197 203 196 209

0.49 0.23 0.48 0.96 1.11 1.31 0.46 0.59 0.26 0.30

1.07 1.10 0.97 0.78 1.12 1.09 1.14 0.94 0.98 0.99

0.997 0.992 0.998 0.993 0.999 0.999 0.994 0.993 0.995 0.999

0.966 0.954 1.013 1.121 0.954 0.956 0.931 1.027 1.004 1.001

a

Confidence level set at 95%. b t-critical ) 2.06. c F-critical ) 1.98.

Standard deviation.

brominated congeners within a reasonable retention time without the need to use flow rates above 0.5 mL min-1 that can cause a marked decrease in NI-APPI signal due to production of large solvent ion clusters having excessive solvation energies.18,19 A minimum of 5% water in the mobile phase together with a 5 min column equilibration at 85% mobile phase A between runs was found necessary to maintain the LC column conditions for optimum separation and obtain reproducible retention times in successive runs. A 100% organic mobile phase composition at any time during the run was found to affect the separation of octa- and nona-BDEs, while eliminating the equilibration step would affect both separation and retention times of the analytes. Validation. The developed analytical method was validated according to the ICH guidelines.20 The accuracy of the developed method was assessed by applying it to the determination of PBDEs in NIST SRM 2585 (Organics in House Dust). SRM 2585 has certified values for BDEs 47, 85, 99, 100, 153, 154, 183, 203, 206, and 209, while two “indicative values” for BDEs 196 and 197 were reported.21 The results obtained compared favorably to the certified and indicative values (Figure 3) with internal standard recoveries ranging from 71-87% and RSD < 10% for all target PBDEs indicating good accuracy and precision of the developed method. This is likely a result of the use of 13C-labeled PBDEs as internal standards which both (a) compensate for any variability in instrumental response of the mass spectrometer between injections and (b) compensate for matrix related ion suppression or enhancement effects that can occur in the ion source.8 Linearity was assessed by plotting five-point calibration curves for each of the studied PBDE congeners in the concentration range 20-5000 pg µL-1. All the calibration (18) Robb, D. B.; Covey, T. R.; Bruins, A. P. Anal. Chem. 2000, 72 (15), 3653– 3659. (19) Kauppila, T. J.; Kotiaho, T.; Kostiainen, R.; Bruins, A. P. J. Am. Soc. Mass Spectrom. 2004, 15 (2), 203–211. (20) ICH Topic Q 2 (R1) Validation of Analytical Procedures: Text and Methodology. http://www.emea.europa.eu/pdfs/human/ich/038195en.pdf, 1995 (accessed July 18, 2009). (21) Stapleton, H. M.; Harner, T.; Shoeib, M.; Keller, J. M.; Schantz, M. M.; Leigh, S. D.; Wise, S. A. Anal. Bioanal. Chem. 2006, 384 (3), 791–800.

plots displayed good linearity (r2 ) 0.996-0.998) within the studied range. Interday (repeatability) and intraday (intermediate precision) variability were assessed via replicate injections of the same standards on the same day (five concentrations, three replicates each) and different days (five concentrations, three replicates each) and found to be minimal (RSD < 10%) indicating good precision of the developed methodology. None of the target compounds were detected in the analyzed field blanks. Therefore, method limits of detection (LOD) were calculated based on a 3:1 signal-tonoise (S/N) ratio and ranged from 12 to 30 pg per target compound on column (Table 2). Interestingly, the LOD for BDE-209 (13 pg) was similar to those of the lower PBDE congeners. This contrasts with GC-ECNI/MS methods where the LOD for BDE-209 is commonly an order of magnitude higher than the lower congeners.8 The enhanced sensitivity for BDE-209 may be attributed to the lack of thermal degradation problems with our LC-based method compared to GC methods. Method limits of quantification (LOQ) were estimated based on a S/N ratio of 10:1 and ranged from 52 to 125 pg (Table 2). Evaluation of the method robustness was considered during the development phase. Temperature changes between 18-27 °C had no significant effect on the analysis results of calibration standards (RSD