Highly sensitive and selective analytical methods are required for the development and the quality control of nucleic acid drugs, which are expected to be the next-generation pharmaceuticals. Conventionally, the ion pair reverse phase mode and the ion exchange mode are frequently used for analysis of oligonucleotides. The ion pair agent tends to remain in the device and the ion exchange mode, which requires high concentration salt as the eluent, is not suitable for MS detection. In this application, unpurified oligo DNA synthesis product (ATACCGATTAAGCGAAGTTT) was analyzed using HILICpak VN-50 2D, a polymer-based HILIC mode column, with LC/UV/MS detection. The ion pair reagent is not required in this condition using HILIC mode, and good oligomer separation was obtained from dimer to 20mer, a main component, by gradient elution of 50mM ammonium formate aqueous solution and acetonitrile. Furthermore, it was confirmed that the calibration curve with MS detection has high linearity, quantification with high sensitivity and high selectivity was also possible.
Sample : 1µL
Synthesized oligo DNA 20mer (ATACCGATTAAGCGAAGTTT; crude)
2.2mg/mL (in H2O)
Column : Shodex HILICpak VN-50 2D (2.0mmI.D. x 150mm) Eluent : (A)50mM HCOONH4 aq./(B) CH3CN Linear gradient ; (B%) 60% (0 to 10min), 60% to 55% (10 to 15min), 60% (15 to 20min) Flow rate : 0.2mL/min Detector : PDA(260nm), ESI-MS (SIM Negative) Column temp. : 40°C