title: The analysis of major impurities of lipophilic-conjugated phosphorothioate oligonucleotides by ion-pair reversed-phase HPLC combined with MALDI-TOF-MS.
abstract: A simple and rapid ion-pair reversed phase high-performance liquid chromatography (IP-RP-HPLC) method was developed to analyse the major impurities of lipophilic-conjugated phosphorothioate oligonucleotides (ODNs), which provided better separation performance than capillary gel electrophoresis and ion exchange chromatograph methods. The study showed that covalent conjugations of lipophilic group (docosanyl, C(22)) to ODN at 5'-termini (denoted as 5'C(22)-Flu) or 3'-termini (denoted as 3'C(22)-Flu) exhibited similar chromatographic retention behavior. Some important analytical conditions of IP-RP-HPLC, including column type, ion-pairing buffer composition, and separation temperature, were investigated for the effects on the separation of crude 5'C(22)-Flu. As expected, the method developed was successfully applied to the analysis of crude 3'C(22)-Flu and both purified products. Furthermore, the related impurities derived from the synthetic process were identified by matrix-assisted laser desorption-ionization time-of-flight mass spectrum. These MS results are of benefit to understanding the major process-related impurities in lipophilic-ODN conjugates synthesis, thereby elevating the quality of target products.
authors.affiliation: Chinese PLA Postgraduate Medical School, Beijing, China.
authors.forename: Cai-Hong
authors.initials: CH
authors.lastname: Liu
authors.forename: Dan-Dan
authors.initials: DD
authors.lastname: Lu
authors.forename: Xin-Xiu
authors.initials: XX
authors.lastname: Deng
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Jing-Yu
authors.initials: JY
authors.lastname: Zhang
authors.forename: Yu-Lin
authors.initials: YL
authors.lastname: Zhang
authors.forename: Sheng-Qi
authors.initials: SQ
authors.lastname: Wang
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D054735:Phosphorothioate Oligonucleotides / Q000032:analysis* / Q000138:chemical synthesis; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D054735:Phosphorothioate Oligonucleotides