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 AJAC  Vol.2 No.3 , July 2011
A Validated Liquid Chromatography-Mass Spectrometry Method for the Detection and Quantification of Oxidative Metabolites of 2,2',4,4'-Tetrabromodiphenyl Ether in Rat Hepatic Microsomes
Abstract: In the present study, we developed and validated an analytical method using ultra performance liquid chromatography-mass spectrometry (UPLC/MS) for the quantitative determination of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) metabolism by rat hepatic microsomes. BDE-47 is a brominated flame retardant that was widely used in a variety of consumer products and has subsequently been identified as a ubiquitous environmental contaminant. Hydroxy-bromodiphenyl ethers (OH-BDEs) were isolated from rat hepatic microsomes by liquid-liquid extraction. Chromatographic separation was achieved by UPLC on a C18 column with gradient elution using a mobile phase consisting of methanol and water, each containing 0.1% formic acid, at a flow rate of 0.2 mL/min. Detection and quantification were performed using a mass spectrometer in single ion recording mode with negative electrospray ionization. The UPLC/MS method was validated for linearity, limit of quantification (LOQ), accuracy, precision and recovery. The weighted calibration curves (1/X2) were linear over a concentration range of 5 - 250 nM with LOQ values between 5 nM and 50 nM for the individual OH-BDEs. Intra- and inter- day accuracy (%DEV) and precision (%RSD) values ranged from –11.7% to 9.5% and 5.9% to 16.5%, respectively. Recovery values of 70% to 90% were obtained for all OH-BDEs. The validated method allowed us to successfully analyze metabolite formation following incubation of BDE-47 with hepatic microsomes prepared from phenobarbital-treated rats. Results demonstrate that the UPLC/MS method has sufficient sensitivity and reproducibility to fully characterize the in vitro metabolism of BDE-47 and possibly other PBDEs.
Cite this paper: nullS. Moffatt, P. Edwards, A. Szeitz and S. Bandiera, "A Validated Liquid Chromatography-Mass Spectrometry Method for the Detection and Quantification of Oxidative Metabolites of 2,2',4,4'-Tetrabromodiphenyl Ether in Rat Hepatic Microsomes," American Journal of Analytical Chemistry, Vol. 2 No. 3, 2011, pp. 352-362. doi: 10.4236/ajac.2011.23043.
References

[1]   P. Vonderheide, K. E. Mueller, J. Meija and G. L. Welsh, “Polybrominated Diphenyl Ethers: Causes for Concern and Knowledge Gaps Regarding Environmental Distribution, Fate and Toxicity,” Science of the Total Environment, Vol. 400, No. 1-3, 2008, pp. 425-436. doi:10.1016/j.scitotenv.2008.05.003

[2]   M. Alaee, P. Arias, A. Sj?din and ?. Bergman, “An Overview of Commercially Used Brominated Flame Retardants, Their Applications, Their Use Patterns in Different Countries/Regions and Possible Modes of Release,” Environment International, Vol. 29, No. 6, 2003, pp. 683-689. doi:10.1016/S0160-4120(03)00121-1

[3]   R. C. Hale, M. Alaee, J. B. Manchester-Neesvig, H. M. Stapleton and M. G. Ikonomou, “Polybrominated Diphenyl Ether Flame Retardants in the North American Environment,” Environment International, Vol. 29, No. 6, 2003, pp. 771-779. doi:10.1016/S0160-4120(03)00113-2

[4]   M. J. La Guardia, R. C. Hale and E. Harvey, “Detailed Polybrominated Diphenyl Ether (PBDE) Congener Composition of the Widely Used Penta-, Octa-, and Deca-PBDE Technical Fame-Retardant Mixtures,” Environmental Science and Technology, Vol. 40, No. 20, 2006, pp. 6247-6254. doi:10.1021/es060630m

[5]   R. C. Hale, M. J. La Guardia, E. Harvey and T. M. Mainor, “Potential Role of Fire Retardant-Treated PolyureThane Foam as a Source of Brominated Diphenyl Ethers to the US Environment,” Chemosphere, Vol. 46, No. 5, 2002, pp. 729-735. doi:10.1016/S0045-6535(01)00237-5

[6]   F. Rahman, K. H. Langford, M. D. Scrimshaw and J. N. Lester, “Polybrominated Diphenyl Ether (PBDE) Flame Retardants,” Science of the Total Environment, Vol. 275, No. 1-3, 2001, pp. 1-17. doi:10.1016/S0048-9697(01)00852-X

[7]   H. M. Stapleton, A. Sj?din, R. S. Jones, S. Niehüser, Y. Zhang and D. G. Patterson, “Serum Levels of PolybroMinated Diphenyl Ethers (PBDEs) in Foam Recyclers and Carpet Installers Working in the United States,” Environmental Science and Technology, Vol. 42, No. 9, 2008, pp. 3453-3458. doi:10.1021/es7028813

[8]   D. O. Odusanya, J. O. Okonkwo, and B. Botha, “PolyBrominated Diphenyl Ethers (PBDEs) in Leachates From Selected Landfill Sites in South Africa,” Waste Management, Vol. 29, No. 1, 2009, pp. 96-102.

[9]   M. No?l, N. Dangerfield, R. A. S. Hourston, W. Belzer, P. Shaw, M. B. Yunker and P. S. Ross. “Do trans-Pacific Air Masses Deliver PBDEs to Coastal British Columbia, Canada?” Environmental Pollution, Vol. 157, No. 12, 2009, pp. 3404-3412. doi:10.1016/j.envpol.2009.06.025

[10]   K. Law, T. Halldorson, R. Danell, G. Stern, S. Gewurtz, M. Alaee, C. Marvin, M. Whittle, and G. Tomy, “BioacCumulation and Trophic Transfer of Some Brominated Flame Retardants in a Lake Winnipeg (Canada) Food Web,” Environmental Toxicology and Chemistry, Vol. 25, No. 8, 2006, pp. 2177-2186. doi:10.1897/05-500R.1

[11]   N. G. Dodder, B. Strandberg and R. A. Hites, “Concentrations and Spatial Variations of Polybrominated Diphenyl Ethers and Several Organochlorine Compounds in Fishes From the Northeastern United States,” Environmental Science and Technology, Vol. 36, No. 2, 2002, pp. 146-151. doi:10.1021/es010947g

[12]   M. A. McKinney, I. Stirling, N. J. Lunn, E. Peacock and R. J. Letcher, “The Role of Diet on Long-Term Concentration and Pattern Trends of Brominated and Chlorinated Contaminants in Western Hudson Bay Polar Bears, 1991-2007,” The Science of the Total Environment, Vol. 408, No. 24, 2010, pp. 6210-6222. doi:10.1016/j.scitotenv.2010.08.033

[13]   C. Kelly, M. G. Ikonomou, J. D. Blair and F.A.P.C. Gobas, “Hydroxylated and Methoxylated Polybrominated Diphenyl Ethers in a Canadian Arctic Marine Food Web,” Environmental Science and Technology, Vol. 42, No. 19, 2008, pp. 7069-7077. doi:10.1021/es801275d

[14]   A. Mazdai, N. G. Dodder, M. P. Abernathy, R. A. Hites and R. M. Bigsby, “Polybrominated Diphenyl Ethers in Maternal and Fetal Blood Samples,” Environmental Health Perspectives, Vol. 111, No. 9, 2003, pp. 1249-1252. doi:10.1289/ehp.6146

[15]   B. Johnson-Restrepo, K. Kannan, D.P. Rapaport and B.D. Rodan, “Polybrominated Diphenyl Ethers and PolyChlorinated Biphenyls in Human Adipose Tissue from New York,” Environmental Science and Technology, Vol. 39, No. 14, 2005, pp. 5177-5182. doi:10.1021/es050399x

[16]   A. Schecter, M. Pavuk, O. P?pke, J. J. Ryan, L. Birnbaum and R. Rosen, “Polybrominated Diphenyl Ethers (PBDEs) in U.S. Mothers' Milk,” Environmental Health Perspectives, Vol. 111, No. 14, 2003, pp. 1723-1729. doi:10.1289/ehp.6466

[17]   J. R. Gee and V. C. Moser, “Acute Postnatal Exposure to Brominated Diphenylether 47 Delays Neuromotor Ontogeny and Alters Motor Activity in Mice,” Neurotoxicology and Treatment, Vol. 30, No. 2, 2008, pp. 79-87.

[18]   C. E. Talsness, S. N. Kuriyama, A. Sterner-Kock, P. Schnitker, S. W. Grande, M. Shakibaei, A. Andrade, K. Grote and I. Chahoud, “In Utero and Lactational Exposures to Low Doses of Polybrominated Diphenyl Ether- 47 Alter the Reproductive System and Thyroid Gland of Female Rat Offspring,” Environmental Health Perspectives, Vol. 116, No. 3, 2008, pp. 308-314. doi:10.1289/ehp.10536

[19]   T. Hamers, J. H. Kamstra, E. Sonneveld, A. J. Murk, T. J. Visser, M. J. M. Van Velzen, A. Brouwer and ?. Bergman, “Biotransformation of Brominated Flame Retardants into Potentially Endocrine-Disrupting Metabolites, with Special Attention to 2,2',4,4'-Tetrabromodiphenyl ether (BDE-47),” Molecular Nutrition & Food Research, Vol. 52, No. 2, 2008, pp. 284-298. doi:10.1289/ehp.10536

[20]   J. M. Sanders, L.-J. Chen, E. H. Lebetkin and L. T. Burka, “Metabolism and Disposition of 2,2',4,4'-Tetrabromodiphenyl Ether Following Administration of Single or Multiple Doses to Rats and Mice,” Xenobiotica, Vol. 36, No. 1, 2006, pp. 103-117. doi:10.1080/00498250500485107

[21]   G. Marsh, M. Athanasiadou, I. Athanassiadis and A. Sandholm, “Identification of Hydroxylated Metabolites in 2,2',4,4'-Tetrabromodiphenyl Ether Exposed Rats,” Chemosphere, Vol. 63, No. 4, 2006, pp. 690-697. doi:10.1016/j.chemosphere.2005.07.072

[22]   S. W. Cheng, K. Randal and, A. T. Kotchevar. “In Vitro Metabolism Studies of Polybrominated Diphenyl Ethers using Rat and Human Liver Microsomes,” American Journal of Biochemistry and Biotechnology, Vol. 4, No. 3, 2008, pp. 295-303. doi:10.3844/ajbbsp.2008.295.303

[23]   M. Athanasiadou, S. N. Cuadra, G. Marsh, ?. Bergman and K. Jakobsson, “Polybrominated Diphenyl Ethers (PBDEs) and Bioaccumulative Hydroxylated PBDE Metabolites in Young Humans from Managua, Nicaragua,” Environmental Health Perspectives, Vol. 116, No. 3, 2008, pp. 400-408. doi:10.1289/ehp.10713

[24]   S. Mas, O. Jauregui, F. Rubio, A. de Juan, R. Tauler and S. Lacorte, “Comprehensive Liquid Chromatography-Ionspray Tandem Mass Spectrometry Method for the Identification and Quantification of Eight Hydroxylated Brominated Diphenyl Ethers in Environmental Matrices,” Journal of Mass Spectrometry, Vol. 42, No. 7, 2007, pp. 890-900. doi:10.1002/jms.1224

[25]   P. R. Edwards, E. G. Hrycay and S.M. Bandiera, “DifFerential Inhibition of Hepatic Microsomal AlkoxyreSorufin O-Dealkylation Activities by Tetrachlorobiphenyls,” Chemico-Biological Interactions, Vol. 169, No. 1 2007, pp. 42-52. doi:10.1016/j.cbi.2007.05.004

[26]   T. Omura and R. Sato, “The Carbon Monoxide-Binding Pigment of Liver Microsomes I: Evidence for its Hemoprotein Nature,” The Journal of Biological Chemistry, Vol. 239, No. 7, 1964, pp. 2370-2378.

[27]   O. H. Lowry, N. J. Rosebrough, A. L. Farr and R. J. Randall, “Protein Measurement with the Folin Phenol Reagent,” The Journal of Biological Chemistry, Vol. 193, 1951, pp. 265-275.

[28]   A. Erratico, A. Szeitz and S. M. Bandiera. “Validation of a Novel in Vitro Assay Using Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC/MS) to Detect and Quantify Hydroxylated Metabolites of BDE-99 in Rat Liver Microsomes,” Journal of chromatography B, Analytical technologies in the biomedical and life sciences, Vol. 878, No. 19, 2010, pp. 1562-1568. doi:10.1016/j.jchromb.2010.04.014

 
 
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