Development and validation of a gas chromatography/mass spectrometry method for the assessment of genomic DNA methylation

A method for the determination of DNA global methylation, taken as the ratio (%) of 5‐methylcytosine (5mCyt) versus the sum of cytosine (Cyt) and 5mCyt, via gas chromatography/mass spectrometry (GC/MS), was developed and validated. DNA (2.5 µg) was hydrolyzed with aqueous formic acid 88%, spiked with cytosine‐2,4‐¹³C₂,¹⁵N₃ and 5‐methyl‐²H₃‐cytosine‐6‐²H₁ as internal standards, and derivatized with N‐methyl‐N‐(tert‐butyldimethylsilyl)trifluoroacetamide and 1% tert‐butyldimethylchlorosilane, in the presence of acetonitrile and pyridine. GC/MS, operating in single ion monitoring mode, separated and specifically detected all nucleobases as tert‐butyldimethylsilyl derivatives, without interferences, with the exception of guanosine. The method was linear throughout the range of clinical interest and had good sensitivity, with a limit of quantification of 3.2 pmol for Cyt and 0.056 pmol for 5mCyt, the latter corresponding to a methylation level of 0.41%. Intra‐ and inter‐day precision and accuracy were below 4.0% for both analytes and methylation. The matrix absolute effect, process efficiency and coefficient of variation ranged from 96.5 to 101.2%. The matrix relative effect was below 1%. The method was applied to the analysis of different human DNAs, including: nonmethylated DNA from PCR (methylation 0.00%), hypermethylated DNA prepared using M.SssI CpG methyltransferase (methylation 18.05%), DNA from peripheral blood leukocytes of healthy subjects (N = 6, median methylation 5.45%), DNA from bone marrow of leukemia patients (N = 5, 3.58%) and DNA from myeloma cell lines (N = 4, 2.74%). Copyright © 2009 John Wiley & Sons, Ltd.