Investigation of Sample Preparation Methods for the Analysis of PBDE and Metabolite Accumulation in Humans Using Gas Chromatography Tandem Mass Spectrometry

Abstract

Polybrominated diphenyl ethers (PBDEs) are anthropogenic flame retardants that were widely used from the 1960s to resist flame propagation and dispersion in consumer products such as electronics, appliances, and polyeurathane foam products. Based on their ubiquitous presence, PBDEs transport in the environment and bioaccumulate in humans and in wildlife. Once PBDEs are retained in humans, they are metabolized into hydroxylated brominated diphenyl ether metabolites (OH-BDEs) which are prone to have greater endocrine disrupting effects than the parent PBDEs. Furthermore, PBDEs and OH-BDEs share a relationship with their methoxylated analogs (MeO-BDEs), which have been detected at high levels in humans, however their occurrence in humans remain unclear. The first part of this work entails the development of an analytical sample preparation method to screen for a variety of PBDE, OH-BDE and MeO-BDE congeners using gas chromatography tandem mass spectrometry (GC-MS/MS). Previous extraction methods utilized multiple fractionation steps and were often labor intensive. Thus, a simultaneous extraction and clean-up method for breast milk and serum was developed herein. This “one-shot” analytical method uses selective reaction monitoring (SRM) mode in GC-MS/MS to provide excellent sensitivity and selectivity for trace analysis detection. Target analytes include a screening of 12 PBDEs, 15 OH-BDEs, and 16 MeO-BDEs and was applied to human serum and breast milk for method validation. Sheep serum and donated breast milk were used for method validation. The analytical method provided low limits of detection (LODs) ranging from 2 to 14 pg, and analyte recoveries of 60-125% for tri to hexa brominated congeners. This method was applied to 48 paired breast milk and blood serum samples from middle-aged women living in Texas, U.S. in 2005, to assess the partitioning of brominated diphenyl ethers (BDEs) in the body. PBDE-47, -153, -99, and -100 made up 95% of total PBDE loading; and the major OH-BDE congeners were tetra-brominated PBDE-47 metabolites including: 5-OH-BDE-47, 6-OH-BDE-47, 4’-OH-BDE-49, 3-OH-BDE-47, and a small presence of PBDE-99 metabolites: 5’-OH-BDE-99 and 6’-OH-BDE-99. Total individual levels of PBDE were similar in the two matrices, however it is evident that OH-BDEs retain greater in blood serum than in breast milk. This trend has been observed before in similar studies, providing evidence that OH-BDEs are being retained in the body and may be transferred prenatally to developing fetuses. Studies have shown that there is a 10 to 100 fold variability in the metabolism of PBDEs to OH-BDEs. Thus, there is an urgent need to identify the sources of the interindividual variability in PBDE metabolism in humans. In order to assess the differences in PBDE and OH-BDE concentrations, the genotype of these forty-eight individuals were processed using polymerase chain reaction (PCR). The genotypes were characterized based on their specific present allele CYP 2B6 variants *1, *4, *5, *6, *7. The majority of the volunteers expressed either the CYP 2B6 *1, *6, or *7 allele and the concentrations of PBDE-47 to the detected PBDE-47 metabolites were compared. Statistical analysis using Pearson’s correlation coefficient demonstrated that there are catalytic differences in PBDE metabolism to OH-BDEs; and that the genotype of an individual may have an effect on xenobiotic metabolism in the body. Finally, in order to guide further analytical chemists to better understand other the relationship among PBDEs, OH-BDEs, and MeO-BDEs and their subsequent routes of exposure in humans, an analytical method was developed for the analysis of these congeners in foodstuffs. The analytical method, QuEChERS (acronym for quick, easy, cheap, effective, robust, safe), was first introduced for the analysis of pesticides in fruits, however due to its universal power, it can be adapted to other classes of analytes and other matrices as well. Because these BDEs have been detected in soil, vegetation, and in the atmosphere, it is possible that humans can have alternate sources of exposure. Optimization of the previous QuEChERS methods include a more cost-effective sample preparation that can ultimately increase detection limits for the identification of trace levels of these endocrine disrupting chemicals.