N9- and N7-substituted (guaninyl)acetic esters were studied by electrospray ionization tandem mass spectrometry (ESI-MS/MS) in order to determine their ratio in alkylation reactions. The loss of ammonia is significantly different for the N9- and N7-alkylated guanine regioisomer pairs. More importantly, the abundance of the [MH-17]+ ion is in linear correlation with the N9-isomer content. Therefore, the ratio of regioisomers can be determined in a mixture containing these compounds.