title: 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.
abstract: The purpose of this study was to develop and validate an ultra performance liquid chromatography-mass spectrometry (UPLC/MS) method to investigate the hepatic oxidative metabolism of 2,2',4,4',5-pentabromodiphenyl ether (BDE-99), a widely used flame retardant and ubiquitous environmental contaminant. Hydroxylated metabolites were extracted using liquid-to-liquid extraction, resolved on a C18 column with gradient elution and detected by mass spectrometry in single ion recording mode using electrospray negative ionization. The assay was validated for linearity, accuracy, precision, limit of quantification, range and recovery. Calibration curves were linear (R2 > or = 0.98) over a concentration range of 0.010-1.0 microM for 4-OH-2,2',3,4',5-pentabromodiphenyl ether (4-OH-BDE-90), 5'-OH-2,2',4,4',5-pentabromodiphenyl ether (5'-OH-BDE-99) and 6'-OH-2,2',4,4',5-pentabromodiphenyl ether (6'-OH-BDE-99), and a concentration range of 0.0625-12.5 microM for 2,4,5-tribromophenol (2,4,5-TBP). Inter- and intra-day accuracy values ranged from -2.0% to 6.0% and from -7.7% to 7.3%, respectively, and inter- and intra-day precision values ranged from 2.0% to 8.5% and from 2.2% to 8.6% (n=6), respectively. The limits of quantification were 0.010 microM for 4-OH-BDE-90, 5'-OH-BDE-99 and 6'-OH-BDE-99, and 0.0625 microM for 2,4,5-TBP. Recovery values ranged between 85 and 100% for the four analytes. The validated analytical method was applied to identify and quantify hydroxy BDE-99 metabolites formed in vitro. Incubation of BDE-99 with rat liver microsomes yielded 4-OH-BDE-90 and 6'-OH-BDE-99 as major metabolites and 5'-OH-BDE-99 and 2,4,5-TBP as minor metabolites. To our knowledge, this is the first validated UPLC/MS method to quantify hydroxylated metabolites of PBDEs without the need of derivatization.
authors.affiliation: Faculty of Pharmaceutical Sciences, The University of British Columbia, 2146 East Mall, Vancouver, British Columbia V6T 1Z3, Canada. erratico@interchange.ubc.ca
authors.forename: Claudio A
authors.initials: CA
authors.lastname: Erratico
authors.forename: András
authors.initials: A
authors.lastname: Szeitz
authors.forename: Stelvio M
authors.initials: SM
authors.lastname: Bandiera
mesh_terms: D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D055768:Halogenated Diphenyl Ethers / Q000032:analysis*; D006900:Hydroxylation; D016018:Least-Squares Analysis; D008297:Male; D013058:Mass Spectrometry / Q000379:methods*; D008862:Microsomes, Liver / Q000378:metabolism*; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: C477694:2,2',4,4',5-brominated diphenyl ether; D055768:Halogenated Diphenyl Ethers