Derivatization with girard reagent t combined with LC-MS/MS for the sensitive detection of 5-formyl-2 '-deoxyuridine in cellular DNA

Nucleoside 5-formyl-2'-deoxyuridine (FodU) is a major thymidine lesion generated by reactive oxygen species. In vitro and in vivo replication studies revealed that FodU can be mutagenic. A reliable and sensitive quantification method is, therefore, important for assessing the biological implications of this lesion. However, the detection limit of FodU by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was relatively poor compared with those of other oxidative DNA base damages. In this paper we described a new approach for the highly sensitive detection of FodU. We derivatized FodU with Girard reagent T to form a hydrazone conjugate harboring a precharged quaternary ammonium moiety, which enabled the facile detection of the resulting conjugate by positive-ion electrospray ionization MS. We also showed that the combination of derivatization with LC-MS/MS on a linear-ion-trap mass spectrometer could allow for the quantification of FodU at a detection limit of 3-4 fmol, which is similar to 20-fold better than that for the direct analysis of the underivatized compound. By using isotope-labeled FodU as the internal standard and this derivatization method, we further quantified, by using LC-MS/MS, the yield of FodU formed in cellular DNA.