Analysis of PBDEs, PCBs and their metabolites in environmental and biological samples by gas and liquid chromatography with mass spectrometric detection
Lupton, Sara J.
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Polychlorinated biphenyls (PCBs) are a class of persistent organic pollutants that are found in the environment since the 1970’s due to its heavy usage in transformers and paints. PCBs are highly lipophilic and tend to bioaccumulate in tissues of animals. PCBs are also known to biomagnify through the food web. On the other hand, polybrominated diphenyl ethers (PBDEs) are contaminants that only recently emerged in the last decade. PBDEs also have the ability to bioaccumulate and biomagnify, but studies have shown that PBDEs are more readily metabolized than PCBs. The most likely route of PBDE metabolism is through initial transformation by cytochrome P450’s (CYPs). Some of the main metabolic products are hydroxylated PBDEs that resemble thyroxin and other hormones in the body. Hence, these metabolites could potentially act as endocrine disruptors. Therefore, there is heightened concern when these compounds accumulate in the body. In order to understand the mechanism of transport, accumulation, and metabolism of these halogenated compounds in the environment and in biota, it is important to be able to analyze them in different matrices and measure both the parent compounds and metabolic products at low concentrations. For these reasons, methods were developed for the analysis of these compounds in environmental and biological samples. Specifically, extraction and gas chromatographic/mass spectrometric (GC/MS) methods were developed to analyze PCBs and PBDEs in common carp muscle, plasma and liver. Extractions were either completed by liquid-liquid extraction (LLE) for liquid samples or pressurized liquid extraction (PLE) for solid samples. Two GC/MS instruments were used and evaluated, one with single quadrupole MS and the other with a triple quadrupole MS, both with electron impact ionization. To determine the extent of metabolism of PBDEs in humans, incubations of BDEs 47, 99 or 153 were prepared with human liver microsomes. A method was also developed to analyze for 14 C-labeled BDEs 47, 99, 153 and their metabolites after incubation with human liver microsomes which contain CYPs. Analysis of radiolabeled PBDE and metabolites was achieved using a high performance liquid chromatograph with an on-line radiochemical detector. Non-labeled compounds and metabolites were also analyzed by GC/MS. In addition, a method for the analysis of hydroxylated-PBDEs (OH-PBDEs) by liquid chromatography/with ion-trap mass spectrometry (LC/MS) was developed to facilitate the identification of novel metabolites in biological samples. The LC/MS method was developed using atmospheric pressure chemical ionization (APCI) to allow for negative ionization of OH-PBDEs. The fragmentation pattern obtained from tandem MS analysis by ion-trap MS provided valuable structural information to allow identification of metabolites. Common carp analyzed from Eastern Lake Erie have high levels of PCBs and detectable levels of PBDEs. PCB concentrations ranged from non-detect to 12,000 ng/g lipid in muscle where PCBs 138 and 153 were the most detected. PBDE concentrations in muscle ranged from 1.5 to 100ng/g lipid, where PBDE 47 was the most detected compound. Incubations of BDE 47, 99 and 153 with human liver microsomes showed hydroxylation and cleavage of BDEs 47 and 99. BDE 153 had no detectable metabolites. BDEs 47 and 99 were dihydroxylated or cleaved into bromophenols. BDE 99 was metabolized at a faster rate than BDE 47 indicating that metabolism of 99 is one possible reason for the increased abundance of BDE 47 in tissues. Results indicated that LC/MS with APCI ionization is suitable for the detection of low levels of OH-PBDEs, which allowed for the separation of nine OH-PBDEs ranging from tribrominated to hexabrominated. LC/MS is more advantageous than GC/MS in the analysis of polar metabolites because there is no need to derivatize the hydroxylated metabolites prior to analysis.