Determination of 5-methylcytosine in DNA by gas chromatography-electron-capture detection

The following sequence of analytical steps was used to determine the amount of 5-methylcytosine (mol-%) in calf thymus and human lymphocyte DNA:acid hydrolysis of the DNA, derivatization (pentafluorobenzyl bromide, solid phase extraction, pivalic anhydride), internal standard addition, solid phase extraction, high-performance liquid chromatography, and gas chromatography with electron-capture detection. The steps were carefully optimized, leading to a recovery of 30 +/- 1.0% starting with a nucleobase standard containing 1.25 ng of 5-methylcytosine. A second analysis of this sample gave a 30 +/- 0.3%, demonstrating a high precision for the method. In good agreement with earlier work by others, 1.2 +/- 0.10 mol-% of 5-methylcytosine was then found in a 350 ng sample of calf thymus DNA, and values of 0.9 +/- 0.07 and 0.8 +/- 0.04 mol-% (two runs) were found in hyman lymphocyte DNA.