Analysis of 8-hydroxy-2'-deoxyguanosine in rat urine and liver DNA by stable isotope dilution gas chromatography/mass spectrometry.

A previously developed gas chromatographic/mass spectrometric method was applied to the measurement of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in rat liver DNA and in rat urine. For DNA samples, the method included: (i) fortification of samples with [15N]DNA (internal standard), (ii) enzymatic hydrolysis of DNA to deoxynucleosides, (iii) degradation of native nucleosides by treatment with trifluoroacetic acid and hydrazine, (iv) purification by C18 solid-phase extraction (SPE), (v) derivatization (acetylation followed by pentafluorobenzylation, Ac-PFB), and (vi) GC/MS analysis of the derivatives. For urine, the following methodology was used: (i) fortification of the samples with 8-18OHdG, (ii) prepurification by C18/OH SPE, (iii) derivatization, (iv) high-performance liquid chromatography purification of the Ac-PFB derivatives, and (v) GC/MS analysis. The precision of the method was demonstrated by carrying out replicate analysis of several urine and DNA samples: within-run and between-run variability was less than 5 and 8%, respectively. The analytical approaches were sufficiently sensitive to quantitate the urinary excretion of 8-OHdG (490 +/- 70 pmol/kg/24 h; sample size, 600 microliters urine) and to measure the level of 8-OHdG in liver DNA (20 8-OHdG/10(6) deoxynucleosides; sample size, 30 micrograms DNA) of rats not deliberately exposed to oxidative stress. Major advantages over previous methods are increased precision due to the use of proper isotopically labeled internal standards, and increased sensitivity due to the optimization of cleanup procedures. The simultaneous analysis of standards of three different oxidized nucleosides, namely 8-OHdG, thymidine glycol, and 5-hydroxy-methyl-2'-deoxyuridine, is shown.