Resolution and Quantitation of Ribonucleosides and Deoxyribonucleosides in Digested Genomic DNA by UHPLC–MS/MS

Deoxyuridine (dUrd) is often misincorporated into genomic DNA in cancer patients receiving antimetabolite chemotherapy. This activates base excision repair and recruits ataxia telangiectasia-mutated and Rad3-related (ATR), which coordinates DNA repair with nucleotide metabolism by facilitating ribonucleotide reductase (RNR) activity. Ceralasertib (AZD6738) is the most clinically advanced ATR inhibitor (ATRi) and may abrogate this regulatory pathway and diminish RNR-mediated deoxynucleotide triphosphate synthesis. To further understand ATRi-induced dUrd misincorporation, we developed a highly sensitive LC-MS/MS method to quantitate: deoxyuridine, uridine, guanosine, adenosine, cytidine, thymidine, deoxyguanosine, deoxyadenosine, and deoxycytidine from digested genomic DNA. Assay application was demonstrated with genomic DNA digested from multiple murine cell lines treated with ceralasertib. Chromatographic separation was achieved with an Inertsil ODS-3 (150 × 2.1 mm, 3 μm) column and a gradient elution program of 0.1% formic acid in water and 0.1% formic acid in methanol over an 18-min run time. Detection was performed on a SCIEX 6500+ tandem mass spectrometer. The method proved to be accurate (90.8%-114.2%) and precise (< 7.73% CV) across analytes. Freeze-thaw stability (106.4%-114.3%), stability for 12 months at -80°C (88.7%-113.7%), and stability for 4 h at room temperature (104.1%-114.0%) were acceptable across QCs.