title: Analysis of global DNA methylation by hydrophilic interaction ultra high-pressure liquid chromatography tandem mass spectrometry.
abstract: We developed and validated a rapid, sensitive, and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) method for determination of global DNA methylation in tissue. DNA was extracted by phenol-chloroform, hydrolyzed using 88% formic acid at 140°C, spiked with cytosine-2,4-(13)C(15)N(2) as internal standard, evaporated under nitrogen, reconstituted in methanol, and analyzed by LC-MS/MS in multiple reaction monitoring mode to reflect the global DNA methylation of the tissue. The method was linear throughout the range of clinical interest and had good sensitivity, with a limit of quantification of 0.5pg for both cytosine (Cyt) and 5-methylcytosine (5mCyt). The linear range of calibration curve was 1-50 and 1-100ng/ml for 5mCyt and Cyt, respectively, with a correlation coefficient higher than 0.99. The relative standard deviation (RSD) was 0.70-4.09% and 0.60-4.81% for Cyt and 5mCyt, respectively. The intraday precision expressed as RSD ranged from 1.86% to 4.67%, whereas the interday values ranged from 3.72% to 4.68%. The recovery of the method varied from 86.52% to 105.14%. This yielded a simple and reliable LC-MS/MS assay for detection of Cyt and 5mCyt, thereby enabling the evaluation of global DNA methylation.
authors.affiliation: Guangdong Medical College, Zhanjiang, Guangdong 524023, People's Republic of China.
authors.forename: Jun-jie
authors.initials: JJ
authors.lastname: Zhang
authors.forename: Lijian
authors.initials: L
authors.lastname: Zhang
authors.forename: Keyuan
authors.initials: K
authors.lastname: Zhou
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Ye
authors.forename: Chunan
authors.initials: C
authors.lastname: Liu
authors.forename: Liangtao
authors.initials: L
authors.lastname: Zhang
authors.forename: Jingxuan
authors.initials: J
authors.lastname: Kang
authors.forename: Chun
authors.initials: C
authors.lastname: Cai
mesh_terms: D044503:5-Methylcytosine / Q000737:chemistry / Q000378:metabolism; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D015179:Colorectal Neoplasms / Q000235:genetics / Q000378:metabolism; D003562:Cytidine / Q000737:chemistry / Q000378:metabolism; D019175:DNA Methylation; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D057230:Limit of Detection; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D003562:Cytidine; D044503:5-Methylcytosine