Application of LC-MS technique in quantitative analysis of biological samples of oligonucleotides

      Oligonucleotides are one of the most promising drug fields in biomedical research and development. These drugs are oligonucleotides composed of 12 to 30 nucleotides synthesized chemically. At present, the oligonucleotides listed in the world mainly include antisense nucleic acid (ASO), small interfering RNA (siRNA) and nucleic acid aptamer. The biological sample analysis methods mainly include molecular hybridization (such as: nucleic acid molecular hybridization - enzyme-linked immunosorbent assay and real-time fluorescence quantitative PCR), liquid chromatography, molecular hybridization - high performance liquid chromatography fluorescence, high performance liquid chromatography - mass spectrometry and so on. LC-MS, which combines the high separation efficiency of HPLC with the high sensitivity and selectivity of MS, has become one of the powerful analytical tools for the quantitative study of oligonucleotides and their metabolites in biological substrates.

    Electrospray ion source (ESI) negative ion mode is often used for LC-MS analysis of oligonucleotides. The selection of sample treatment method, liquid chromatography and mass spectrometry conditions is also closely related to the retention mechanism, ionization method and ionization efficiency of liquid chromatography.

    Difficulties and solutions of LC-MS analysis of oligonucleotides:

    (1) Select liquid chromatography conditions compatible with ESI-MS (reversed-phase ion pair chromatography or HILIC)

    (2) ESI-MS parameter optimization: wide charge distribution, low signal strength, reduce the formation of cationic adducts

    (3) Sample processing methods: liquid-liquid extraction (phenol-dichloromethane or chloroform-isopropyl alcohol), solid-phase extraction (Clarity OTX SPE), liquid-liquid extraction combined with solid-phase extraction

    (4) Non-specific adsorption: The reserve solution and working solution are added to the chelating agent (EDTA), low adsorption consumables

    (5) Residue: Bioinert column and bioinert liquid phase system

    The verification of LC-MS analysis methods for oligonucleotides was carried out in accordance with the requirements of chromatographic analysis method verification.

    At present, oligonucleotides developed by Macro Pharma include antisense ASO and siRNA, and serious residues of siRNA analytes were encountered in the process of method development. The 1260 Infinity II Prime bioinert liquid chromatography system for the elimination of residues, Clarity OTX reagent for the rapid extraction of oligonucleotides by SPE (products of the United States), with a recovery rate of 50-60%; With the TRIPLE QUAD 7500 mass spectrometer, the lower limit of quantitation can reach 0.500 ng/mL.