Anal. Chem. 2004, 76, 1921-1927
Semiautomated High-Throughput Extraction and Cleanup Method for the Measurement of Polybrominated Diphenyl Ethers, Polybrominated Biphenyls, and Polychlorinated Biphenyls in Human Serum Andreas Sjo 1 din,* Richard S. Jones, Chester R. Lapeza, Jean-Franc¸ ois Focant, Ernest E. McGahee, III, and Donald G. Patterson, Jr.
Organic Analytical Toxicology (OAT), Division of Laboratory Sciences (DLS), National Center for Environmental Health (NCEH), Centers for Disease Control and Prevention (CDC), 4770 Buford Highway Northeast Mail Stop F-17, Atlanta, Georgia 30341
A semiautomated extraction and cleanup method has been developed for the measurement of eight polybrominated diphenyl ethers (PBDEs), 2,2′,4,4′,5,5′-hexabromobiphenyl (BB-153) and 2,2′,4,4′,5,5′-hexachlorobiphenyl (CB-153). The method employs automated addition of internal standards (13C-labeled), addition of formic acid (denaturation agent), and dilution with water prior to automated overnight extraction using a modular solidphase extraction (SPE) system. Removal of coextracted biogenic materials was performed on a two-layered 3-mL disposable cartridge containing activated silica gel and a mixture of silica gel and sulfuric acid. Sample cleanup was automated using the same modular SPE system. Reproducibility and precision of the liquid handler used for internal standard additions were shown to be 2 and 4%, respectively. Overall reproducibility during processing of eight batches of samples (N ) 30/batch, including methods blanks) was below 10% for most analytes. Mean recoveries of the 13C-labeled internal standards ranged from 69 to 95% for the seven monitored PBDEs; 76 and 98% were recovered for BB-153 and CB-153, respectively. Brominated flame retardants (BFRs) and, in particular, polybrominated diphenyl ethers (PBDEs) are environmental contaminants that have been present in the environment for decades; they were originally identified in fish caught in the river Viskan in Sweden in the 1980s.1 PBDEs have since then been identified as environmental contaminants with global distribution, as shown by the identification of these compounds in both aquatic and terrestrial compartments in Europe,2-4 North America,5-7 and Asia8 * Corresponding author. E-mail:
[email protected]. (1) Andersson, O ¨ .; Blomkvist, G. Chemosphere 1981, 10, 1051-1060. (2) Sellstro ¨m, U.; Kierkegaard, A.; de Wit, C.; Jansson, B. Environ. Toxicol. Chem. 1998, 17, 1065-1072. (3) Sellstro ¨m, U.; Jansson, B.; Kierkegaard, A.; de Wit, C.; Odsjo¨, T.; Olsson, M. Chemosphere 1993, 26, 1703-1718. 10.1021/ac030381+ Not subject to U.S. Copyright. Publ. 2004 Am. Chem. Soc.
Published on Web 02/24/2004
and also in European air.9 Increasing levels have been found in environmental matrixes in Sweden10,11 and also in human milk from Sweden12-14 and in human blood from Norway and Germany.15,16 More recently, levels of PBDEs up to 2 orders of magnitude higher than in Europe have been identified in North America;17,18 these levels have also been shown to increase over time in a retrospective time trend study which used samples from the southeastern United States.19 Actual steps to ban future use of the lower brominated pentaBDE and octaBDE technical mixtures due to observed high persistence and bioaccumulation or biomagnification have oc(4) Jansson, B.; Andersson, R.; Asplund, L.; Litze´n, K.; Nylund, K.; Sellstro ¨m, U.; Uvemo, U.-B.; Wahlberg, C.; Wideqvist, U.; Odsjo¨, T.; Olsson, M. Environ. Toxicol. Chem. 1993, 12, 1163-1174. (5) Norstrom, R. J.; Simon, M.; Moisey, J.; Wakeford, B.; Weseloh, D. V. C. Environ. Sci. Technol. 2002, 36, 4783-4789. (6) She, J.; Petreas, M.; Winkler, J.; Visita, P.; McKinney, M.; Kopec, D. Chemosphere 2002, 46, 697-707. (7) Manchaster-Neesvig, J. B.; Valters, K.; Sonzogni, W. C. Environ. Sci. Technol. 2002, 35, 1072-1077. (8) Watanabe, I.; Kashimoto, T.; Tatsukawa, R. Chemosphere 1987, 16, 23892396. (9) Lee, R. G. M.; Thomas, G. O.; Jones, K. C. Organohalogen Compd. 2002, 58, 193-196. (10) Kierkegaard, A.; Sellstro ¨m, U.; Bignert, A.; Olsson, M.; Asplund, L.; Jansson, B.; de Wit, C. Organohalogen Compd. 1999, 40, 367-70. (11) Sellstro ¨m, U. Ph.D. Thesis, Department of Environmental Chemistry and Institute of Applied Environmental Research, Stockholm University, Stockholm, Sweden, 1999. (12) Meironyte´ Guvenius, D. Ph.D. Thesis, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden, 2002. (13) Nore´n, K.; Meironyte´, D. Chemosphere 2000, 40, 1111-1123. (14) Meironyte´, D.; Nore´n, K.; Bergman, A° . J. Toxicol. Environ. Health 1999, 58 (Part A), 329-341. (15) Thomsen, C.; Lundanes, E.; Becher, G. Organohalogen Compd. 2001, 52, 206-209. (16) Schro ¨ter-Kermani, C.; Helm, D.; Herrmann, T.; Pa¨pke, O. Organohalogen Compd. 2000, 47, 49-52. (17) Sjo ¨din, A.; Patterson, D G., Jr.; Bergman, A° . Environ. Int. 2003, 29, 829839. (18) Schecter, A.; Pavuk, M.; Pa¨pke, O.; Ryan, J. J.; Birnbaum, L.; Rosen, R. Environ. Health Perspect. 2003, 111, 1723-1729. (19) Sjo ¨din, A.; Jones, R. S.; Lapeza, C. R.; Focant, J.-F.; Wang, R.; Turner, W. E.; Needham, L. L.; Patterson, D. G., Jr. Organohalogen Compd. 2003, 61, 1-4. (20) Cox, P.; Efthymiou, P. Off. J. Eur. Union 2003, OJ L 42, 45-46.
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curred only recently in Europe20 and in the state of California in the United States. The PBDE manufacturing industry has recently agreed to withdraw the pentaBDE and octaBDE mixtures from the U.S. market after negotiations with the Environmental Protection Agency (EPA). Consumption of the pentaBDE mixture in North America has been estimated to be 7100 tons in 2001, which corresponds to 95% of the total world demand.21 Because of the high North American consumption, the decision was made at the CDC to develop a high-throughput analytical method aimed at targeting BFRs, and PBDEs in particular. One persistent polychlorinated biphenyl (PCB) congener, 2,2′,4,4′,5,5′hexachlorobiphenyl, CB-153, was also included among the target analytes to act as a comparative analyte to the BFRs. Although the aim was to develop a high-throughput method, the method should also continue to improve accuracy and reproducibility, as compared to existing methodologies.22,23 An SPE method was deemed to be the most beneficial approach to reach these goals and to perform the necessary cleanup on silica: silica/H2SO4 packed into an SPE cartridge. Automation of the procedure to the largest practical extent gives us high throughput and at the same time reduces variability originating from several laboratorians performing the analyses. EXPERIMENTAL SECTION Safety. The experimenter undertaking this work with human specimens (serum) and other potentially pathogen containing samples must understand the potential of exposure and limit this risk of exposure with proper use of personal protective equipment, such as lab coat, protective glasses, and laboratory gloves. Any potential spills of human serum are decontaminated with 10% bleach or 70% ethanol solution, allowing a contact time for decontamination of 15 min. The experimenter also must be aware of the presence of 13C6-1,2,3,4-tetrachlorodibenzo-p-dioxin (13C61,2,3,4-TCDD) in the recovery standard used and the potential health effects of exposures to TCDD.24 Certified Reference Standards. Two internal standard spiking solutions obtained from Cambridge Isotope Laboratories (Andover, MA) were used for BFRs and PCBs, respectively. The spiking standard for BFRs contained the following 13C12-labeled congeners at a concentration of 7.5 pg/µL in methanol: 2,4,4′tribromodiphenyl ether (BDE-28); 2,2′,4,4′-tetraBDE (BDE-47); 2,2′,4,4′,5-pentaBDE (BDE-99); 2,2′,4,4′,6-pentaBDE (BDE-100); 2,2′,4,4′,5,5′-hexaBDE (BDE-153); 2,2′,4,4′,5,6′-hexaBDE (BDE154); 2,2′,3,4,4′,5′,6-heptaBDE (BDE-183); decaBDE (BDE-209), and 2,2′,4,4′,5,5′-hexabromobiphenyl (BB-153). The spiking standard for PCBs contained 2,2′,4,4′,5,5′-hexachlorobiphenyl (CB-153) at a concentration of 7.5 pg/µL in methanol. The recovery spiking standard was also obtained from Cambridge Isotope Laboratories (Andover, MA) and contained the following labeled compounds: 13C -1,2,3,4-TCDD (2.5 pg/µL); 3,3′,4,4′-tetraBDE (13C -BDE-77, 6 12 7.5 pg/µL); 2,2′,3,4,4′,6-hexaBDE (13C12-BDE-139, 7.5 pg/µL), and 2,2′,3,3′,4,5,5′,6,6′-nonaCB (13C12-CB-208, 10 pg/µL). The solvent (21) Bromine Science and Environmental Forum, BSEF. Available: http:// www.bsef-site.com/docs/BFR_vols_2001.doc. [accessed 16 October 2003]. (22) Sandau, C. D.; Sjo ¨din, A.; Davis, M. D.; Barr, J. R.; Maggio, V. L.; Waterman, A. L.; Preston, K. E.; Preau, J. L., Jr.; Barr, D.; Needham, L. L.; Patterson, D. G., Jr. Anal. Chem. 2003, 75, 71-77. (23) Hovander, L.; Athanasiadou, M.; Asplund, L.; Klasson-Wehler, E. Organohalogen Compd. 1998, 35, 115-118. (24) Myers, G. L.; Patterson, D. G., Jr. Professional Safety 1987, 32, 30-37.
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for the recovery standard was n-hexane containing 10 and 2 vol % of nonane and dodecane, respectively. A 10-point calibration curve spanning the concentration range 0.2-2000 pg/µL containing the 13C-labeled BFRs and CB-153 at a concentration of 75 pg/µL was used for the gas chromatography/ isotope dilution high-resolution mass spectrometry (GC/IDHRMS) analyses. The calibration standard contained the following native analytes: 2,2′,4-triBDE (BDE-17); BDE-28; BDE-47; 2,3′,4,4′tetraBDE (BDE-66); 2,2′,3,4,4′-pentaBDE (BDE-85); BDE-99; BDE100; BDE-153; BDE-154; BDE-183; 2,2′,3,4,4′,5,5′,6-octaBDE (BDE203); BDE-209; BB-153, and CB-153. Chemicals. Reagents and solvents used in the current inventory were of the highest possible grade available or intended for pesticide residue analysis and were only used after verification by GC/IDHRMS analysis monitoring for the BFRs listed above. Dichloromethane (DCM), n-hexane, and methanol were of pesticide grade and purchased from TEDIA (Fairfield, OH); water of HPLC grade was also purchased from TEDIA. Hydrochloric acid (37% in water 99.999%) and sulfuric acid (99.999%) were purchased from Aldrich (Milwaukee, WI). Formic acid (min 88%, ACS grade) and dodecane (
