Journal
ANALYTICAL CHEMISTRY
Volume 91, Issue 22, Pages 14569-14576Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.9b03625
Keywords
-
Categories
Funding
- Intramural NIST DOC [9999-NIST] Funding Source: Medline
Ask authors/readers for more resources
Accurate, traceable quantification of ribonucleotide or deoxyribonucleotide oligomers is achievable using acid hydrolysis and isotope dilution mass spectrometry (ID-MS). In this work, formic acid hydrolysis is demonstrated to generate stoichiometric release of nucleobases from intact oligonucleotides, which then can be measured by ID-MS, facilitating true and precise absolute quantification of RNA, short linearized DNA, or genomic DNA. Surrogate nucleobases are quantified with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) workflow, using multiple reaction monitoring (MRM). Nucleobases were chromatographically resolved using a novel cation-exchange separation, incorporating a pH gradient. Trueness of this quantitative assay is estimated from agreement among the surrogate nucleobases and by comparison to concentrations provided for commercial materials or Standard Reference Materials (SRMs) from the National Institute of Standards and Technology (NIST). Comparable concentration estimates using NanoDrop spectrophotometry or established from droplet-digital polymerase chain reaction (ddPCR) techniques agree well with the results. Acid hydrolysis-ID-LC-MS/MS provides excellent quantitative selectivity and accuracy while enabling traceability to mass unit. Additionally, this approach can be uniquely useful for quantifying modified nucleobases or mixtures.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available