HPLC-MS/MS quantification of genomic deoxyuridine via differential protonation

Precise quantification of trace genomic deoxyuridine (dU) is critical for elucidating DNA damage mechanisms. This analytical process is often hampered by severe isobaric mass spectrometric interference originating from the M + 1 isotope of highly abundant deoxycytidine (dC). Herein, we established a highly sensitive HPLC-MS/MS analytical strategy for the quantitative analysis of dU. By utilizing an acidic mobile phase system on a Zorbax SB-C18 column, dC and dU exhibited distinct protonation states, enabling their successful baseline separation. Methodological validation demonstrated a low limit of detection of 0.5 nM (2.5 fmol on-column with 5 μL injection), with excellent linearity, precision, and accuracy. Analysis of biological samples revealed significant dU basal heterogeneity across seven cell lines, with the highest accumulation observed in A549 cells, and precisely captured the dynamic increase in dU within 293T cells following 5-fluorouracil (5-FU) exposure. This study provides a reliable quantitative tool for investigating nucleotide metabolic imbalances and DNA damage repair.