title: Derivatization with Girard reagent T combined with LC-MS/MS for the sensitive detection of 5-formyl-2'-deoxyuridine in cellular DNA.
abstract: 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 approximately 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.
authors.affiliation: Environmental Toxicology Graduate Program and Department of Chemistry, University of California at Riverside, Riverside, California 92521, USA.
authors.forename: Haizheng
authors.initials: H
authors.lastname: Hong
authors.forename: Yinsheng
authors.initials: Y
authors.lastname: Wang
mesh_terms: D001622:Betaine / Q000031:analogs & derivatives* / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000378:metabolism; D003857:Deoxyuridine / Q000031:analogs & derivatives* / Q000032:analysis; D006367:HeLa Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D006835:Hydrazones / Q000737:chemistry; D013058:Mass Spectrometry / Q000379:methods*; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors
chemical_list: D006835:Hydrazones; D000644:Quaternary Ammonium Compounds; D001622:Betaine; C061519:5-formyl-2'-deoxyuridine; D004247:DNA; C012332:Girard's reagent T; D003857:Deoxyuridine