Highlights

• The study emphasizes the significance of endogenous ribonucleotides (RNs) and deoxyribonucleotides in vital life processes and their association with diseases like neurodegeneration, cancer, and mitochondrial depletion syndrome, highlighting the critical role of these nucleotides in health and disease.
• Maintaining a balanced nucleotide pool is essential for DNA replication fidelity, as an imbalance in dNTP/NTP ratios can lead to the erroneous incorporation of NTPs into DNA, resulting in DNA instability. This finding underscores the importance of nucleotide balance in maintaining genomic stability.
• A novel LC-MS/MS method has been developed for the simultaneous quantitative analysis of 22 endogenous RNs and dRNs in cancer cells. This method features pre-column MTBSTFA derivatization, enabling one-step cell extraction with 85% methanol, a rapid derivatization reaction at room temperature in just 5 minutes, and efficient retention of derivatives on a typical C18 column. It offers a streamlined and efficient approach for nucleotide detection and quantification, providing valuable insights into metabolites of nucleoside analogs and other phosphonate-containing drugs like bisphosphonates.

Summary

A novel and highly sensitive method employing pre-column derivatization with MTBSTFA has been developed to accurately determine ribonucleotides (RNs) and deoxyribonucleotides (dRNs) in human cells. This approach offers a straightforward, swift, sensitive, and dependable means for in vitro intracellular pharmacology research.

In a study led by Huixia Zhang and her colleagues in 2022, they introduced the MTBSTFA derivatization-LC-MS/MS technique for quantitatively analyzing endogenous nucleotides in human colorectal carcinoma cells. This newly described LC-MS/MS method is characterized by its simplicity, rapidity, specificity, and efficiency.

This method facilitates metabolite quantification and is valuable for analyzing nucleotides in cancer cells. Given the critical role of nucleotide balance in DNA replication and the potential for instability when dNTP/NTP ratios are imbalanced, this method holds significant relevance.

Moreover, endogenous ribonucleotides and deoxyribonucleotides are vital for various life processes and are closely associated with developing diseases such as neurodegeneration and cancer. Understanding the levels of these nucleotides is crucial.

Specifically, adenosine monophosphate (AMP) is pivotal in cellular energy homeostasis. Hence, developing a rapid and convenient assay for quantifying phosphorylated forms of intracellular RNs and dRNs becomes notably significant for further research.

The study’s findings align with previous research, as Zhang suggested, “Exposure to 0.1 mM 10-hydroxycamptothecine resulted in a decrease in cell population in the G0/G1 phase and an increase in the S phase, as observed in previous research.

H. Zhang et al., “MTBSTFA derivatization-LC-MS/MS approach for the quantitative analysis of endogenous nucleotides in human colorectal carcinoma cells,” Journal of Pharmaceutical Analysis, vol. 12, no. 1, pp. 77–86, Feb. 2022, doi: .