Formic acid hydrolysis/liquid chromatography isotope dilution mass spectrometry: An accurate method for large DNA quantification.
Liquid chromatography-isotope dilution mass spectrometry (LC-IDMS) with formic acid hydrolysis was established for the accurate quantification of λDNA. The over-decomposition of nucleobases in formic acid hydrolysis was restricted by optimizing the reaction temperature and the reaction time, and accurately corrected by using deoxynucleotides (dNMPs) and isotope-labeled dNMPs as the calibrator and the internal standard, respectively. The present method could quantify λDNA with an expanded uncertainty of 4.6% using 10fmol of λDNA. The analytical results obtained with the present method were validated by comparing with the results of phosphate-base quantification by inductively coupled plasma-mass spectrometry (ICP-MS). The results showed good agreement with each other. We conclude that the formic acid hydrolysis/LC-IDMS method can quantify λDNA accurately and is promising as the primary method for the certification of DNA as reference material.
National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki, 305-8563, Japan. Electronic address: s-shibayama@aist.go.jp.
Sachie
S
Shibayama
National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki, 305-8563, Japan.
Shin-Ichiro
SI
Fujii
National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki, 305-8563, Japan.
Kazumi
K
Inagaki
National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki, 305-8563, Japan.
Taichi
T
Yamazaki
National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki, 305-8563, Japan.
Akiko
A
Takatsu
D010582:Bacteriophage lambda / Q000235:genetics; D002138:Calibration; D002623:Chemistry Techniques, Analytical / Q000379:methods* / Q000592:standards; D002853:Chromatography, Liquid; D004247:DNA / Q000032:analysis*; D005561:Formates / Q000737:chemistry*; D006868:Hydrolysis; D007553:Isotope Labeling; D013058:Mass Spectrometry
D005561:Formates; C030544:formic acid; D004247:DNA
DNA quantification; Formic acid hydrolysis; Isotope dilution mass spectrometry; LC/MS/MS; Nucleobases