Room temperature derivatization of 5-hydroxy-2 '-deoxycytidine and 5-hydroxymethyl-2 '-deoxyuridine for analysis by GC/MS

Hydroxylated DNA bases are one type of oxygen free radical-induced damage t o DNA. Such damage has been implicated in the process of carcinogenesis, an d the levels of hydroxylated DNA bases may serve as a marker of cancer risk in humans. Measurement of oxidative DNA damage can be hampered by the ease with which artifactual oxidative DNA damage can be induced via sample proc essing. In this report we describe convenient room temperature derivatizati on and stability of 5-hydroxy-2'-deoxycytidine (5-OHdCyd) and 5-hydroxymeth yl-2'-deoxyuridine (5-OHmdU) using GC/MS analysis. The derivatization reage nt was N,O-bis(trimethylsilyl)-triftuoroacetamide (BSTFA) containing 1% tri methylchloro-silane:acetonitrile, 2:1. This method avoids use of acid and i s much milder than previously reported derivatization conditions which typi cally involve heating above 100 degrees C for at least 20 min. Although hea ting has been reported to be problematic, the calculated levels of 5-OHdCyd and 5-OHmdU in enzymatically-hydrolysed calf thymus DNA were very similar in our hands with and without heating the sample for 20 min. As an example of the technique, comparison of 5-OHdCyd and 5-OHmdU levels in calf thymus DNA indicated relatively higher endogenous levels of 5-OHdCyd. In DNA treat ed with hydrogen peroxide and ferric chloride, however, the levels of 5-OHm dU increased much more than that of 5-OHdCyd. In addition to these hydroxyl ated derivatives of deoxycytidine and thymidine, the method also appears to work well with 8-oxoguanine, 4,6-diamino-5-(formylamino)pyrimidine, and 5- methyl-2'-deoxycytidine. This method may therefore be useful with a variety of modified DNA bases and nucleosides.