Highlights

• A novel method was developed involving alkylation of the phosphonic acid group, resulting in decreased polarity and enhanced peak shape. This approach also improved the response in mass spectrometry (MS) with an electrospray ionization source (ESI).
• The method’s novelty is supported by the consistent findings across various nucleotide derivatives, and it offers detailed mass spectra analysis for all 24 analytes.
• This innovative method allows for quantifying 24 nucleotides in a concise analysis cycle of 18 minutes, surpassing the performance of previous ion-pairing and hydrophilic interaction chromatography (HILIC) techniques. Moreover, it maintains column performance, making it superior to ion-pairing methods. The method’s potential applications span a wide range of studies requiring the analysis of endogenous nucleotides, nucleoside analogs, metabolites, and other complex sample components like sugar bisphosphates.

Summary

A novel approach for quantifying nucleotides through a derivatization reaction followed by LC-MS/MS analysis has been developed. This method involves alkylation of the phosphonic acid group, which reduces polarity and enhances peak shape.

Compared to previous techniques, this method allows for quantifying 24 nucleotides in a shorter runtime. The derivatized nucleotides exhibit distinct ion transitions in LC-MS/MS analysis. Notably, this method holds promise for analyzing endogenous nucleotides and other metabolites. The selection of derivatization reagents, such as TMSD and bis(trimethylsilyl)trifluoroacetamide, was based on their advantages of small sample sizes, cost-effectiveness, and low toxicity. TMSD successfully derivatized AMP, ADP, and ATP, while BSTFA partially derivatized ADP and ATP. The derivatized nucleotides exhibit longer retention times and unique ion transitions, reducing LC-MS/MS analysis interference.

Li and colleagues assert that this validated method for nucleotide analysis in cell samples has broad applications in future research endeavors.

Z. Li et al., “Method for Quantification of Ribonucleotides and Deoxyribonucleotides in Human Cells Using (Trimethylsilyl)diazomethane Derivatization Followed by Liquid Chromatography–Tandem Mass Spectrometry,” Anal. Chem., vol. 91, no. 1, pp. 1019–1026, Jan. 2019, doi: .