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 [ ¹³ C ₁₀ , ¹⁵ N ₂ ]-5-methyl-2'-deoxycytidine from [ ¹³ C ₁₀ , ¹⁵ N ₂ ]-2'-deoxythymidine. We also describe an approach for the oxidation of [ ¹³ C ₁₀ , ¹⁵ N ₂ ]-5-methyl-2'-deoxycytidine and [ ¹³ C ₁₀ , ¹⁵ N ₂ ]-2'-deoxythymidine with Na ₂ S ₂ O ₈ , leading to the generation of [ ¹³ C ₁₀ , ¹⁵ N ₂ ]-5-formyl-2'-deoxycytidine, [ ¹³ C ₁₀ , ¹⁵ N ₂ ]-5-carboxy-2'-deoxycytidine or [ ¹³ C ₁₀ , ¹⁵ N ₂ ]-5-(hydroxymethyl)-2'-deoxyuridine, correspondingly. Moreover, we provide optimized protocols for the oxidation of [ ¹³ C ₅ , ¹⁵ N ₂ ]-thymine to [ ¹³ C ₁₀ , ¹⁵ N ₂ ]-5-hydroxymethyluracil, [ ¹³ C ₁₀ , ¹⁵ N ₂ ]-5-formyluracil, and [ ¹³ C ₁₀ , ¹⁵ N ₂ ]-5-carboxyuracil using Na ₂ S ₂ O ₈ .