Received for publication December 8, 2006.
Revised May 16, 2007.
Accepted for publication May 16, 2007.

LC-Tandem MS 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 (ADRs) and idiosyncratic drug reactions (IDRs). 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 LC-Tandem MS. We chose acetaminophen as a model compound because this drug is known to induce covalent binding to proteins when bioactivated by cytochrome P450s to its reactive N-acetyl-p-benzoquinoneimine metabolite. First, by microsomal incubations of acetaminophen in presence of CPF and/or intact albumin, in vitro reference adducts were generated in order to determine the mass spectrometric characteristics of the expected CPF adducts and to confirm their formation upon pronase E digestion of the alkylated protein. When applying this methodology to albumin isolated from blood of patients exposed to acetaminophen, we were indeed able to detect the corresponding CPF adducts. This strategy could therefore be seen as a potential bio-monitoring tool to detect in vivo reactive intermediates of drugs and drug candidates, e.g. in the preclinical and clinical development phase.

Key words: bioactivation, covalent drug binding, protein binding, reactive intermediate