Quantitation of DNA by nuclease P1 digestion and UPLC-MS/MS to assess binding efficiency of pyrrolobenzodiazepine
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. Th...
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doaj-6f745856436d46438683f302eb3e6d912021-04-02T14:25:14ZengElsevierJournal of Pharmaceutical Analysis2095-17792020-06-01103247252Quantitation of DNA by nuclease P1 digestion and UPLC-MS/MS to assess binding efficiency of pyrrolobenzodiazepineYong Ma0Buyun Chen1Donglu Zhang2Corresponding author.; Drug Metabolism and Disposition, Genentech, 1 DNA Way, South San Francisco, CA, 94080, USADrug Metabolism and Disposition, Genentech, 1 DNA Way, South San Francisco, CA, 94080, USACorresponding author.; Drug Metabolism and Disposition, Genentech, 1 DNA Way, South San Francisco, CA, 94080, USAAccurate 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.http://www.sciencedirect.com/science/article/pii/S2095177920303981Nuclease P1UPLC-MS/MSDNA quantitationDNA alkylationPyrrolobenzodiazepine (PBD-Dimer) |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Yong Ma Buyun Chen Donglu Zhang |
spellingShingle |
Yong Ma Buyun Chen Donglu Zhang Quantitation of DNA by nuclease P1 digestion and UPLC-MS/MS to assess binding efficiency of pyrrolobenzodiazepine Journal of Pharmaceutical Analysis Nuclease P1 UPLC-MS/MS DNA quantitation DNA alkylation Pyrrolobenzodiazepine (PBD-Dimer) |
author_facet |
Yong Ma Buyun Chen Donglu Zhang |
author_sort |
Yong Ma |
title |
Quantitation of DNA by nuclease P1 digestion and UPLC-MS/MS to assess binding efficiency of pyrrolobenzodiazepine |
title_short |
Quantitation of DNA by nuclease P1 digestion and UPLC-MS/MS to assess binding efficiency of pyrrolobenzodiazepine |
title_full |
Quantitation of DNA by nuclease P1 digestion and UPLC-MS/MS to assess binding efficiency of pyrrolobenzodiazepine |
title_fullStr |
Quantitation of DNA by nuclease P1 digestion and UPLC-MS/MS to assess binding efficiency of pyrrolobenzodiazepine |
title_full_unstemmed |
Quantitation of DNA by nuclease P1 digestion and UPLC-MS/MS to assess binding efficiency of pyrrolobenzodiazepine |
title_sort |
quantitation of dna by nuclease p1 digestion and uplc-ms/ms to assess binding efficiency of pyrrolobenzodiazepine |
publisher |
Elsevier |
series |
Journal of Pharmaceutical Analysis |
issn |
2095-1779 |
publishDate |
2020-06-01 |
description |
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. |
topic |
Nuclease P1 UPLC-MS/MS DNA quantitation DNA alkylation Pyrrolobenzodiazepine (PBD-Dimer) |
url |
http://www.sciencedirect.com/science/article/pii/S2095177920303981 |
work_keys_str_mv |
AT yongma quantitationofdnabynucleasep1digestionanduplcmsmstoassessbindingefficiencyofpyrrolobenzodiazepine AT buyunchen quantitationofdnabynucleasep1digestionanduplcmsmstoassessbindingefficiencyofpyrrolobenzodiazepine AT dongluzhang quantitationofdnabynucleasep1digestionanduplcmsmstoassessbindingefficiencyofpyrrolobenzodiazepine |
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