- 第3日 6月24日（金） P会場（501，502，503）
Modified nucleic acids used in oligonucleotide therapeutics exhibit specific low-mass fragment ions in MS/MS. By focusing on specific low-mass fragments, it is possible to identify and quantify similar sequences.
Two oligonucleotide therapeutics mixtures were prepared with varied concentrations. The samples were measured using Nano-flow liquid chromatography-tandem mass spectrometry (LC-MS/MS) in multiplexing parallel reaction monitoring mode. The analysis was performed using "AQXeNA."(Fig.1) The software was developed with two algorithms and graphical user interface: 1. SpiceCmd (Mitsui Knowledge Industry Co. Ltd.) for extracting LC-MS/MS peaks and 2. Ariadne (1) for characterizing precursor and product ions.
As a demonstration, the mixed sample of two oligonucleotide therapeutics was prepared and quantified. Calibration curves of the specific low-mass fragment ions were made with each oligonucleotide therapeutics using normalized peak area at ten calibration points with different concentrations, 0.25, 0.5, 1, 2.5, 5,10, 25, 50, 250, and 500 fmol/uL. Correlation coefficients of the curves were obtained in 0.9946–0.9973 with each specific low-mass fragment ions, even though the samples involve another oligonucleotide therapeutic. Quantification based on specific low-mass fragment ions is an excellent method for quantifying oligonucleotide therapeutics because it is less affected by internal standards and analogues than fragment ions derived from the backbone.
1) Nakayama H et al. Nucleic Acids Res. 2009;37(6): e47.