Analysis of global DNA methylation by hydrophilic interaction ultra high-pressure liquid chromatography tandem mass spectrometry

• Published in: Analytical biochemistry 2011-06, Vol.413 (2), p.164-170
• Main Authors: Zhang, Jun-jie, Zhang, Lijian, Zhou, Keyuan, Ye, Xiaoxia, Liu, Chunan, Zhang, Liangtao, Kang, Jingxuan, Cai, Chun
• Format: Article
• Language: English
• Subjects: 5-Methylcytosine - chemistry; 5-Methylcytosine - metabolism; Calibration; Chromatography, High Pressure Liquid - methods; Colorectal Neoplasms - genetics; Colorectal Neoplasms - metabolism; correlation; Cytidine - chemistry; Cytidine - metabolism; cytosine; DNA; DNA Methylation; formic acid; high performance liquid chromatography; Humans; Hydrophilic interaction chromatography (HILIC); hydrophilicity; Hydrophobic and Hydrophilic Interactions; Limit of Detection; Mass spectrum; methanol; methylation; monitoring; nitrogen; tandem mass spectrometry; Tandem Mass Spectrometry - methods; Ultra high-pressure liquid chromatography (UHPLC)
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2011.01.029

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- 13C 15N 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 0.5 pg for both cytosine (Cyt) and 5-methylcytosine (5mCyt). The linear range of calibration curve was 1–50 and 1–100 ng/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.