Sensitive detection of 5-methylcytosine and quantitation of the 5-methylcytosine/cytosine ratio in DNA by gas chromatography--mass spectrometry using multiple specific ion monitoring.

A method combining gas chromatography and mass spectrometry (GC-MS) with multiple specific ion monitoring has been developed for the detection of 5-methylcytosine and the quantitation of the ratio of methyleytosine to cytosine in DNA. The trimethylsilyl derivatives of cytosine and 5-methylcytosine obtained from DNA hydrolysates are separated by isothermal elution on an OV-225 column and detected by specific ion monitoring in a DuPont 321 mass spectrometer. As little as 1.6 pmol of 5-methylcytosine in Φ_χ174 DNA can be detected, corresponding to a tenfold improvement in sensitivity over that obtained by conventional techniques. The ratio of 5-methylcytosine to cytosine of DNA from φ_χ174, calf thymus, salmon sperm, and several mouse tissues has also been determined. The results agree well with those obtained by other methods.