Comparison of PGC and Biphenyl stationary phases for the high throughput analysis of DNA epigenetic modifications by UHPLC-MS/MS

Epigenetic alterations such as cytosine methylation, hydroxymethylation, formylation and carboxylation are well known modifications that are frequently associated with various disease such as cancer. These modifications are usually studied at the gene level to evaluate their impact on the expression of genes but there is a need for a whole genome quantification that can be more easily used as effect biomarkers. Here, we compare two high throughput methods for the UHPLC-MS/MS analysis of these four epigenetic markers in large cohort studies. The first method uses a porous graphitic carbon stationary phase modified by surface adsorption of triethylamine while the second method uses a biphenyl reversed phase. The two developed methods are then applied to 40 blood neutrophils DNA samples of former smokers and never-smokers from an agricultural occupational biobank. The results obtained shows no differences for the evaluation of 5-methylcytosine and 5-hydroxymethylcytosine with the two methods but highlight a diminution of both DNA methylation and hydroxymethylation in former smokers when compared to a never smoking population.