Method for the analysis of oxidized nucleosides by gas chromatography/mass spectrometry

A method for sensitive analysis of the oxidatively modified nucleosides 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 5-hydroxymethyl-2'-deoxyuridine (HMdU) is described. The method combines acetylation and pentafluorobenzylation of the nucleosides followed by analysis by gas chromatography/electron capture negative ion chemical ionization-mass spectrometry. The detection limits of the method for aqueous standards of HMdU and 8-OHdG were 12 and 18 fmol of starting material (signal-to-noise ratio, 3:1), respectively. The method was linear for 8-hydroxy-2'-deoxyguanosine over five orders of magnitude and gave satisfactory reproducibilities (intraassay RSD < or = 5%) for the analysis of 8-OHdG in both aqueous standards and urine fortified at the level of 35 nM. The limit of detection for the analysis of 8-OHdG in urine was 1.8 pmol, corresponding to a level of 8-OHdG in urine of 35 nM (10 micrograms/liter) at a urine sample volume of 50 microliters. Besides urine the method was applied to the analysis of 5-hydroxy-methyl-2'-deoxyuridine isolated from genomic DNA of Bacillus subtilis bacteriophage H1. Results obtained indicate that the method is potentially suitable for the determination of oxidized nucleosides in biological samples. The selectivity of the method should be enhanced in order to lower the limit of detection in biological samples.