title: Conditions for sample preparation and quantitative HPLC/MS-MS analysis of bulky adducts to serum albumin with diolepoxides of polycyclic aromatic hydrocarbons as models.
abstract: Stable adducts to serum albumin (SA) from electrophilic and genotoxic compounds/metabolites can be used as biomarkers for quantification of the corresponding in vivo dose. In the present study, conditions for specific analysis of stable adducts to SA formed from carcinogenic polycyclic aromatic hydrocarbons (PAH) were evaluated in order to achieve a sensitive and reproducible quantitative method. Bulky adducts from diolepoxides (DE) of PAH, primarily DE of benzo[a]pyrene (BPDE) and also DE of dibenzo[a,l]pyrene (DBPDE) and dibenzo[a,h]anthracene (DBADE), were used as model compounds. The alkylated peptides obtained after enzymatic hydrolysis of human SA modified with the different PAHDE were principally PAHDE-His-Pro, PAHDE-His-Pro-Tyr and PAHDE-Lys. Alkaline hydrolysis under optimised conditions gave the BPDE-His as the single analyte of alkylated His, but also indicated degradation of this adduct. It was not possible to obtain the BPDE-His as one analyte from BPDE-alkylated SA through modifications of the enzymatic hydrolysis. The BPDE-His adduct was shown to be stable during the weak acidic conditions used in the isolation of SA. Enrichment by HPLC or SPE, but not butanol extraction, gave good recovery, using Protein LoBind tubes. A simple internal standard (IS) approach using SA modified with other PAHDE as IS was shown to be applicable. A robust analytical procedure based on digestion with pronase, enrichment by HPLC or SPE, and analysis with HPLC/MS-MS electrospray ionisation was achieved. A good reproducibility (coefficient of variation (CV) 11 %) was obtained, and the achieved limit of detection for the studied PAHDE, using standard instrumentation, was approximately 1 fmol adduct/mg SA analysing extract from 5 mg SA.
authors.affiliation: Division of Environmental Chemistry, Department of Materials and Environmental Chemistry, Stockholm University, Svante Arrhenius väg 16c, 10691, Stockholm, Sweden.
authors.forename: Emelie
authors.initials: E
authors.lastname: Westberg
authors.forename: Ulla
authors.initials: U
authors.lastname: Hedebrant
authors.forename: Johanna
authors.initials: J
authors.lastname: Haglund
authors.forename: Tomas
authors.initials: T
authors.lastname: Alsberg
authors.forename: Johan
authors.initials: J
authors.lastname: Eriksson
authors.forename: Albrecht
authors.initials: A
authors.lastname: Seidel
authors.forename: Margareta
authors.initials: M
authors.lastname: Törnqvist
mesh_terms: D001551:Benz(a)Anthracenes / Q000737:chemistry*; D001564:Benzo(a)pyrene / Q000737:chemistry*; D001580:Benzopyrenes / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D010446:Peptide Fragments / Q000032:analysis*; D011402:Pronase / Q000737:chemistry; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
chemical_list: D001551:Benz(a)Anthracenes; D001580:Benzopyrenes; D010446:Peptide Fragments; D012709:Serum Albumin; D001564:Benzo(a)pyrene; D011402:Pronase; C041517:dibenzo(a,l)pyrene; C026486:1,2,5,6-dibenzanthracene