Reactive metabolites are estimated to be one of the main reasons behind 
unexpected drug-induced toxicity, by binding covalently to cell proteins or DNA. 
Due to their high reactivity and short lifespan, reactive metabolites are 
analyzed after chemical trapping with nucleophilic agents such as glutathione or 
cyanide. Recently, unexplained and uncharacterized methylated reaction products 
were reported in a human liver microsome based reactive metabolite trapping 
assay utilizing potassium cyanide as a trapping agent. Here, a similar assay was 
utilized to produce mono- or dimethylated and further cyanide-trapped reaction 
products from propranolol, amlodipine and ciprofloxacin, followed by 
ultra-performance liquid chromatography/time-of-flight mass spectrometry 
(UPLC/TOF-MS) and ultra-performance liquid chromatography/tandem mass 
spectrometry (UPLC/MS/MS) experiments for their more detailed structural 
elucidation. Formation of all observed cyanide-trapped products was clearly 
NADPH-dependent and thus metabolism-mediated. The suggested reaction pathways 
included N-methylation leading to iminium formation in primary and/or secondary 
amines preceded by cytochrome P450 (CYP)-mediated reactions. As the methylation 
reaction was suggested to be involved in formation of the actual reactive 
iminium ion, the observed cyanide-trapped products were experimental artifacts 
rather than trapped reactive metabolites. The results stress that to avoid 
overestimating the formation of reactive metabolites in vitro, this methylation 
phenomenon should be taken into account when interpreting the results of 
cyanide-utilizing reactive metabolite trapping assays. This in turn emphasizes 
the importance of identification of the observed cyano conjugates during such 
studies. Yet, metabolite identification has a high importance to avoid 
overestimation of in vitro metabolic clearance in the cases where this kind of 
metabonate formation has a high impact in the disappearance rate of the 
compound.