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Quantitation of DNA by nuclease P1 digestion and UPLC-MS/MS to assess binding efficiency of pyrrolobenzodiazepine.
J Pharm Anal. 2020 Jun; 10(3):247-252.JP

Abstract

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.

Authors+Show Affiliations

Drug Metabolism and Disposition, Genentech, 1 DNA Way, South San Francisco, CA, 94080, USA.Drug Metabolism and Disposition, Genentech, 1 DNA Way, South San Francisco, CA, 94080, USA.Drug Metabolism and Disposition, Genentech, 1 DNA Way, South San Francisco, CA, 94080, USA.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

32612871

Citation

Ma, Yong, et al. "Quantitation of DNA By Nuclease P1 Digestion and UPLC-MS/MS to Assess Binding Efficiency of Pyrrolobenzodiazepine." Journal of Pharmaceutical Analysis, vol. 10, no. 3, 2020, pp. 247-252.
Ma Y, Chen B, Zhang D. Quantitation of DNA by nuclease P1 digestion and UPLC-MS/MS to assess binding efficiency of pyrrolobenzodiazepine. J Pharm Anal. 2020;10(3):247-252.
Ma, Y., Chen, B., & Zhang, D. (2020). Quantitation of DNA by nuclease P1 digestion and UPLC-MS/MS to assess binding efficiency of pyrrolobenzodiazepine. Journal of Pharmaceutical Analysis, 10(3), 247-252. https://doi.org/10.1016/j.jpha.2020.05.011
Ma Y, Chen B, Zhang D. Quantitation of DNA By Nuclease P1 Digestion and UPLC-MS/MS to Assess Binding Efficiency of Pyrrolobenzodiazepine. J Pharm Anal. 2020;10(3):247-252. PubMed PMID: 32612871.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Quantitation of DNA by nuclease P1 digestion and UPLC-MS/MS to assess binding efficiency of pyrrolobenzodiazepine. AU - Ma,Yong, AU - Chen,Buyun, AU - Zhang,Donglu, Y1 - 2020/05/26/ PY - 2020/04/02/received PY - 2020/05/24/revised PY - 2020/05/25/accepted PY - 2020/7/3/entrez PY - 2020/7/3/pubmed PY - 2020/7/3/medline KW - DNA alkylation KW - DNA quantitation KW - Nuclease P1 KW - Pyrrolobenzodiazepine (PBD-Dimer) KW - UPLC-MS/MS SP - 247 EP - 252 JF - Journal of pharmaceutical analysis JO - J Pharm Anal VL - 10 IS - 3 N2 - 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. SN - 2214-0883 UR - https://www.unboundmedicine.com/medline/citation/32612871/Quantitation_of_DNA_by_nuclease_P1_digestion_and_UPLC-MS/MS_to_assess_binding_efficiency_of_pyrrolobenzodiazepine L2 - https://linkinghub.elsevier.com/retrieve/pii/S2095-1779(20)30398-1 DB - PRIME DP - Unbound Medicine ER -
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