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.