INTRODUCTION: A number of withdrawn drugs are known to undergo bioactivation by 
a range of drug metabolizing enzymes to chemically reactive metabolites that 
bind covalently to protein and DNA resulting in organ toxicity and 
carcinogenesis, respectively. An important goal in drug discovery is to identify 
structural sites of bioactivation within discovery molecules for providing 
strategic modifications that eliminate or minimize reactive metabolite 
formation, while maintaining target potency, selectivity and desired 
pharmacokinetic properties leading to the development of efficacious and 
nontoxic drugs.
AREAS COVERED: This review covers experimental techniques currently used to 
detect reactive drug metabolites and provides recent examples where information 
from mechanistic in vitro studies was successfully used to redesign candidate 
drugs leading to blocked or minimized bioactivation. Reviewed techniques include 
in vitro radiolabeled drug covalent binding to protein and reactive metabolite 
trapping with reagents such as glutathione, cyanide, semicarbazide and DNA 
bases. Case studies regarding reactive metabolite detection using a combination 
of varied techniques, including liquid chromatography-tandem mass spectrometry 
and NMR analyses and subsequent structural modification are discussed.
EXPERT OPINION: Information derived from state-of-art mechanistic drug 
metabolism studies can be used successfully to direct medicinal chemistry 
towards the synthesis of candidate drugs devoid of bioactivation liabilities, 
while maintaining desired pharmacology and pharmacokinetic properties.