Measurement of 8-hydroxy-2 '-deoxyadenosine in DNA by liquid chromatography/mass spectrometry

8-Hydroxyadenine (8-OH-Ade) is one of the major lesions, which is formed in DNA by hydroxyl radical attack on the C-8 position of adenine followed by oxidation. We describe the measurement of the nucleoside form of this compo und, 8-hydroxy-2'-deoxyadenosine (8-OH-dAdo) in DNA by liquid chromatograph y/mass spectrometry (LC/MS). The developed methodology enabled the separati on by LC of 8-OH-dAdo from intact and modified nucleosides in enzymic hydro lysates of DNA. Measurements by MS were performed using atmospheric pressur e ionization-electrospray process. Isotope-dilution MS was applied for quan tification using a stable isotope-labeled analog of 8-OH-dAdo. The level of sensitivity of LC/MS with selected-ion monitoring (SIM) for 8-OH-dAdo amou nted to approximately 10 femtomol of this compound on the LC column. This l evel of sensitivity is similar to that previously reported using LC-tandem MS (LC/MS/MS) with multiple-reaction monitoring mode (MRM) (7.5 femtomol). This compound was quantified in DNA at a level of approximately one molecul e/10(6) DNA bases using amounts of DNA as low as 5 mug. The results suggest ed that this lesion may be quantified in DNA at even lower levels, when mor e DNA is used for analysis. In addition, gas chromatography/isotope-dilutio n mass spectrometry with SIM (GC/IDMS-SIM) was applied to measure 8-OH-Ade in DNA following its removal from DNA by acidic hydrolysis. The background levels of 8-OH-dAdo and 8-OH-Ade measured by LC/IDMS-SIM and GC/IDMS-SIM, r espectively, were nearly identical. In addition, DNA samples, which were ex posed to ionizing radiation at different radiation doses, were analyzed by these techniques. Nearly identical results were obtained, indicating that b oth LC/IDMS-SIM and GC/IDMS-SIM can provide similar results. The level of s ensitivity of GC/TMS-SIM for 8-OH-Ade was also measured and found to be sig nificantly greater than that of LC/MS-SIM and the reported sensitivity of L C/MS/MS-MRM for 8-OH-dAdo. The results show that the LC/MS technique is wel l suited for the measurement of 8-OH-dAdo in DNA. (C) 2001 Elsevier Science Inc.