Determination of 5-hydroxymethyl-2′-deoxycytidine in Rice by High-performance Liquid Chromatography–Tandem Mass Spectrometry with Isotope Dilution

5-hydroxymethyl-2′-deoxycytidine (5-hmdC), which plays important roles in the epigenetic remodeling in mammals, has been recently determined in plant genomes at trace concentrations. In this study, a method for the sensitive, specific, and rapid assessment of 5-hmdC in rice is reported using high-performance liquid chromatography–tandem mass spectrometry (HPLC–tandem MS) with isotope dilution. The recovery of this method was from 75.6% to 81.3% for 5-hmdC and the intra- and inter-day precision values were within 5.81%. The quantification limit for 5-hmdC was 5.0 pM. Genomic DNA from rice was extracted and enzymatically digested for nucleotides for the HPLC–tandem MS assay. The 5-hmdC levels were 1.32 ± 0.08, 1.64 ± 0.12, and 1.98 ± 0.07 modifications per million nucleosides in the Shengdao-18, Shengxiang-145, and Jindao-263 rice cultivars. The application of HPLC–tandem MS for 5-hmdC demonstrated its presence in the genomic DNA of rice and is useful in further studying the role of modified cytosine in the epigenetic regulation of rice.