Preparation of Internal Standards for 2D-UPLC-MS/MS Quantification of Noncanonical DNA Bases.

Reliable quantitative analysis of DNA modification using liquid chromatography coupled with tandem mass spectrometry requires stable isotope-labeled internal standards. Only some of them are commercially available. Here we present a method allowing for the synthesis of [13C10,15N2]-5-methyl-2'-deoxycytidine from [13C10,15N2]-2'-deoxythymidine. We also describe an approach for the oxidation of [13C10,15N2]-5-methyl-2'-deoxycytidine and [13C10,15N2]-2'-deoxythymidine with Na2S2O8, leading to the generation of [13C10,15N2]-5-formyl-2'-deoxycytidine, [13C10,15N2]-5-carboxy-2'-deoxycytidine or [13C10,15N2]-5-(hydroxymethyl)-2'-deoxyuridine, correspondingly. Moreover, we provide optimized protocols for the oxidation of [13C5,15N2]-thymine to [13C10,15N2]-5-hydroxymethyluracil, [13C10,15N2]-5-formyluracil, and [13C10,15N2]-5-carboxyuracil using Na2S2O8.