Optimization of Separating Conditions for Cytosine and 5-Methylcytosine in Flue-cured Tobacco Leaves

A reversed-phase high performance liquid chromatographic(RP-HPLC) method for the determination of cytosine and 5-methylcytosine in flue-cured tobacco leaves were developed and optimized in mobile phase,detecting temperature and flow rate.The DNA was extracted by CTAB method and hydrolyzed with perchloric acid.Then the pH value was adjusted to between 3 and 5 with KOH.Cytosine and 5-methylcytosine were separated on a reversed-phase C18 column(250 mm×4.6 mm,5 μm),with the mobile phase of methanol+ 5 mmol/L pentane sodium(10︰90,V/V) at a flow rate of 0.7 ml/min.The ultraviolet detector was operated at 273 nm at 30℃.The injection volume was 20 μl.The recoveries of cytosine and 5-methylcytosine in flue-cured tobacco leaves were 103.50% and 96.67% respectively with the relative standard deviation of 2.35% and 2.80% respectively.This method was applied to determine the cytosine and 5-methylcytosine changes in flue-cured tobacco leaves during growth period and the satisfactory results were obtained.