Quantitation of 5-(hydroxymethyl)uracil in DNA by gas chromatography with mass spectral detection

5-(Hydroxymethyl)uracil is a product of oxidative DNA damage. This hydroxylated base was quantified in DNA by GC-MS using either acid or enzymatic hydrolysis of the DNA and isotopically labeled internal standards. Both 5-(hydroxymethyl)uracil and thymine were quantified in each DNA sample and the results expressed as a ratio. This procedure controlled for possible errors in the quantitation of DNA prior to hydrolysis and derivatization. In addition, quantitation of thymine was important due to possible variations in DNA hydrolysis efficiency for each sample. The isotopically labeled internal standards controlled for compound instability through the procedure and for variations in derivatization efficiency. The conditions used for acid hydrolysis of the DNA resulted in considerable degradation of 5-(hydroxymethyl)uracil; however, since isotopically labeled 5-(hydroxymethyl)uracil was added prior to acid treatment, 5-(hydroxymethyl)uracil still could be quantified. The degradation of 5-(hydroxymethyl)uracil was avoided using enzymatic hydrolysis of the DNA. In DNA that had been treated with hydrogen peroxide and iron in the presence of EDTA, the observed level of 5-(hydroxymethyl)uracil using enzymatic hydrolysis was 1.6-fold higher than when using acid hydrolysis of the DNA. With analysis of 2 micrograms of DNA, the detection limit for 5-(hydroxymethyl)uracil was 3/10(5) thymines.