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 < 10%). The validated method was successfully applied to quantitate DNA isolated from tumors and organs of a mouse xenograft model. Compared to the quantitation methods using UV absorbance, the reported method provides an enhanced sensitivity, and it allows for the accurate quantitation of isolated DNA with contamination of RNA and ribonucleotide.

准确的DNA定量是许多生物医学和药物研究的先决条件。在这里，我们通过核酸酶P1消化和UPLC-MS / MS分析建立了一种新的DNA定量方法。DNA片段可以在短时间内通过核酸酶P1有效地水解为单个脱氧核糖核苷酸。在不同条件下证实了所有四种脱氧核糖核苷酸的体面稳定性。选择脱氧腺苷单磷酸（dAMP）作为DNA定量的替代物，因为在UPLC-MS / MS分析中，dAMP在四种脱氧核糖核苷酸中显示出最高的灵敏度。通过这种方法进行DNA定量的线性范围是1.2–5000 ng / mL。在验证中，日间和日内精度在90％–110％之内，日间和日内精度是可以接受的（RSD <10％）。验证的方法已成功应用于从小鼠异种移植模型的肿瘤和器官中分离的DNA定量。与使用紫外线吸收的定量方法相比，所报道的方法提供了更高的灵敏度，并且可以对RNA和核糖核苷酸的污染对分离的DNA进行精确定量。