Quantification of modified nucleotides and nucleosides by isotope dilution mass spectrometry

Abstract

Epigenetic modifications are closely related to certain disorders of the organism, including the development of tumors. One of the main epigenetic modifications is the methylation of DNA cytosines, 5‐methyl‐2′‐deoxycycytidine. Furthermore, 5‐mdC can be oxidized to form three new modifications, 5‐(hydroxymethyl)‐2′‐deoxycytidine, 5‐formyl‐2′‐deoxycytidine, and 5‐carboxy‐2′‐deoxycytidine. The coupling of liquid chromatography with tandem mass spectrometry has been widely used for the total determination of methylated DNA cytosines in samples of biological and clinical interest. These methods are based on the measurement of the free compounds (e.g., urine) or after complete hydrolysis of the DNA (e.g., tissues) followed by a preconcentration, derivatization, and/or clean‐up step. This review highlights the main advances in the quantification of modified nucleotides and nucleosides by isotope dilution using isotopically labeled analogs combined with liquid or gas chromatography coupled to mass spectrometry reported in the last 20 years. The different possible sources of labeled compounds are indicated. Special emphasis has been placed on the different types of chromatography commonly used (reverse phase and hydrophilic interaction liquid chromatography) and the derivatization methods developed to enhance chromatographic resolution and ionization efficiency. We have also revised the application of bidimensional chromatography and indicated significant biological and clinical applications of these determinations.

Accurate Quantification of Ten Methylated Purine Nucleosides by Highly Sensitive and Stable Isotope-Diluted UHPLC–MS/MS

“…Quantification of the other free modified nucleosides, involving methylated modifications on the purine nucleobase or methoxy group substitutions on the ribose, was corrected based on the internal standard [D 3 ]-m6A. This correction was based on their shared chemical resemblance, as most of their stable isotope-labeled standards need to be chemically or biologically synthesized and are commercially unavailable , (Scheme S1). Regular purines without chemical modification on their nucleobase exhibited lower recovery rates, with 79.1 ± 3.7% for [ 15 N 5 ]-rA and 72.0 ± 8.8% for [ 15 N 5 ]-rG, due to their higher hydrophilicity (Table S4).…”