title: Metal free columns for determination of deoxynucleotide monophosphate by liquid chromatography/mass spectrometry and application to oligonucleotide.
abstract: We have developed a highly sensitive method for the analysis of deoxynucleotide monophosphates (dNMPs), which involves the use of liquid chromatography/mass spectrometry (LC/MS) and a new metal-free column. The new column solves the problem that the phosphate group in dNMPs interacts with the metal portion of the device or column. After optimization of the analytical conditions, the limits of detection (LODs) of dNMPs were from 5.4ng/g to 6.3ng/g. Those values were 10 times lower than the LODs of previous methods. We applied the method to the determination of the base composition and the quantification of 20-mer oligonucleotide. Despite use of a very small sample amount of 14.5ng, we were able to determine the base composition, and the result was consistent with theoretical values. We were also able to quantify the mass fraction of oligonucleotide with 8.2% expanded uncertainty (k=2). By means of the developed method, we were able to analyze dNMPs with high sensitivity as well as determine the base composition and quantify the mass fraction of oligonucleotide despite use of a small amount of sample.
authors.affiliation: 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.
authors.forename: Sachie
authors.initials: S
authors.lastname: Shibayama
authors.affiliation: Chemicals Evaluation and Research Institute, 1600 Shimotakano, Sugito, Kitakatsushika, Saitama 345-0043, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Sakamaki
authors.affiliation: National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki 305-8563, Japan.
authors.forename: Taichi
authors.initials: T
authors.lastname: Yamazaki
authors.affiliation: National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki 305-8563, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Takatsu
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002853:Chromatography, Liquid; D015226:Dinucleoside Phosphates / Q000032:analysis; D057230:Limit of Detection; D013058:Mass Spectrometry; D008670:Metals / Q000737:chemistry; D009841:Oligonucleotides / Q000032:analysis*
chemical_list: D015226:Dinucleoside Phosphates; D008670:Metals; D009841:Oligonucleotides
keywords: Deoxynucleotide monophosphate; High sensitivity; LC/MS; Metal-free column; Oligonucleotide base composition; Oligonucleotide quantification