Identification and quantification of mutagenic halogenated cytosines by gas chromatography, fast atom bombardment, and electrospray ionization tandem mass spectrometry

Oxidative modification of nucleic acids has been implicated in carcinogenesis. One potential mechanism involves halogenation by the myeloperoxidase and eosinophil peroxidase systems of phagocytes. In the current studies, three mass spectrometric methods for the in vitro and in vivo analysis of halogenated cytosines and deoxycytidines were compared: gas chromatography-electron ionization-mass spectrometry (GC-EI-MS) with a quadrupole instrument, fast atom bombardment or electrospray ionization (ESI) tandem MS with a four-sector magnetic instrument, and liquid chromatography ESI tandem MS (HPLC-ESI-MS/MS) with an ion-trap instrument. GC-EI-MS with selected ion monitoring of dimethyl-tert-butylsilyl derivatives of nucleobases was the most sensitive method. High-energy collisionally induced dissociation MS/MS analysis with a four-sector magnetic instrument yielded detailed structural information about halogenated nucleoside adducts but required relatively large amounts of material. The most sensitive analysis of intact halogenated deoxycytidine was achieved with extracted ion chromatograms using HPLC-ESI-MS/MS with an ion-trap instrument. Our results indicate that GC-EI-MS is the methodology of choice for ultrasensitive analysis of halogenated cytosines. HPLC-ESI-MS/MS provides greater structural detail for these compounds and may rival GC-EI-MS in sensitivity with more advanced liquid chromatography applications. The mass spectrometric methods we have developed should be useful for evaluating the role of phagocyte-derived oxidants in halogenating nucleobases, nucleosides, and DNA at sites of inflammation.