title: Liquid chromatography/tandem mass spectrometry detection of covalent binding of acetaminophen to human serum albumin.
abstract: Covalent binding of reactive electrophilic intermediates to proteins is considered to play an important role in the processes leading to adverse drug reactions and idiosyncratic drug reactions. Consequently, both for the discovery and the development of new drugs, there is a great interest in sensitive methodologies that enable the detection of covalent binding of drugs and drug candidates in vivo. In this work, we present a strategy for the generation and analysis of drug adducts to human serum albumin. Our methodology is based on the isolation of albumin from blood, its digestion to peptides by pronase E, and the sensitive detection of adducts to the characteristic cysteine-proline-phenylalanine (CPF) tripeptide by liquid chromatography/tandem mass spectrometry. We chose acetaminophen (APAP) as a model compound because this drug is known to induce covalent binding to proteins when bioactivated by cytochromes P450 to its reactive N-acetyl-p-benzoquinoneimine metabolite. First, by microsomal incubations of APAP in presence of CPF and/or intact albumin, in vitro reference adducts were generated to determine the mass spectrometric characteristics of the expected CPF adducts and to confirm their formation on pronase E digestion of the alkylated protein. When applying this methodology to albumin isolated from blood of patients exposed to APAP, we were indeed able to detect the corresponding CPF adducts. Therefore, this strategy could be seen as a potential biomonitoring tool to detect in vivo reactive intermediates of drugs and drug candidates, e.g., in the preclinical and clinical development phase.
authors.affiliation: LACDR, Division of Molecular Toxicology, Department of Pharmacochemistry, Vrije Universiteit, de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
authors.forename: Micaela C
authors.initials: MC
authors.lastname: Damsten
authors.forename: Jan N M
authors.initials: JN
authors.lastname: Commandeur
authors.forename: Alex
authors.initials: A
authors.lastname: Fidder
authors.forename: Albert G
authors.initials: AG
authors.lastname: Hulst
authors.forename: Daan
authors.initials: D
authors.lastname: Touw
authors.forename: Daan
authors.initials: D
authors.lastname: Noort
authors.forename: Nico P E
authors.initials: NP
authors.lastname: Vermeulen
mesh_terms: D000082:Acetaminophen / Q000737:chemistry / Q000378:metabolism*; D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000032:analysis; D000818:Animals; D016227:Benzoquinones / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid / Q000379:methods*; D003545:Cysteine / Q000031:analogs & derivatives / Q000032:analysis; D006801:Humans; D007097:Imines / Q000737:chemistry / Q000378:metabolism; D008862:Microsomes, Liver / Q000378:metabolism; D008954:Models, Biological; D015394:Molecular Structure; D009842:Oligopeptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D015203:Reproducibility of Results; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism*; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D016227:Benzoquinones; D007097:Imines; D009842:Oligopeptides; D012709:Serum Albumin; D000082:Acetaminophen; C028473:N-acetyl-4-benzoquinoneimine; D003545:Cysteine; D000111:Acetylcysteine