Quantitation of DNA by nuclease P1 digestion and UPLC-MS/MS to assess binding efficiency of pyrrolobenzodiazepine

摘要

Accurate DNA quantitation is a prerequisite in many biomedical and pharmaceutical studies. Here we established a new DNA quantitation method by nuclease P1 digestion and UPLC-MS/MS analysis. DNA fragments can be efficiently hydrolyzed to single deoxyribonucleotides by nuclease P1 in a short time. The decent stabilities of all the four deoxyribonucleotides were confirmed under different conditions. Deoxyadenosine monophosphate (dAMP) was selected as the surrogate for DNA quantitation because dAMP showed the highest sensitivity among the four deoxyribonucleotides in the UPLC-MS/MS analysis. The linear range in DNA quantitation by this method is 1.2–5000 ng/mL. In the validation, the inter-day and intra-day accuracies were within 90%–110%, and the inter-day and intra-day precision were acceptable (RSD