title: Comparing ion-pairing reagents and counter anions for ion-pair reversed-phase liquid chromatography/electrospray ionization mass spectrometry analysis of synthetic oligonucleotides.
abstract: BACKGROUND
  Ion-pair reversed-phase liquid chromatography/electrospray ionization mass spectrometry (IP-RP-LC/ESI-MS) has been widely used for the quality control of oligonucleotides. However, researchers are still looking to improve mobile phase systems for IP-RP-LC/ESI-MS analysis of oligonucleotides. This study compared the performance of six ion-pairing reagents with three different counter anions for IP-RP-LC/ESI-MS analysis of oligonucleotides.
  
  
  METHODS
  The study was performed using a Waters ultra-performance liquid chromatography (UPLC®) system coupled to a Waters LCT Premier XE mass spectrometer by using a UPLC® OST column (2.1 mm × 100 mm, 1.7 µm). Buffer systems containing acetate, bicarbonate, and hexafluoroisopropanolate salts of six ion-pairing reagents (triethylamine, tripropylamine, hexylamine, N,N-dimethylbutylamine, dibutylamine, N,N-diisopropylethylamine), respectively, were optimized for IP-RP-LC/ESI-MS analysis of oligonucleotides, and then the optimized conditions were applied for the separation of oligonucleotides.
  
  
  RESULTS
  Counter anions definitely play a role in IP-RP-LC/ESI-MS analysis of oligonucleotides. Buffer containing 30 mM diisopropylethylamine and 200 mM hexafluoroisopropanol provided the highest separation of unmodified heterogeneous oligonucleotides, but tripropylammonium hexafluoroisopropanolate achieved the most enhanced separation of sequence isomers. However, triethylammonium acetate and bicarbonate had equally the highest separation for positional isomers.
  
  
  CONCLUSIONS
  IP-RP-LC/ESI-MS separation of oligonucleotides is mainly sequence dependent, but it is also dependent on both the type of ion-pairing reagent and counter anion present in the mobile phase.
authors.affiliation: Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK.
authors.forename: Lingzhi
authors.initials: L
authors.lastname: Gong
mesh_terms: D000838:Anions / Q000032:analysis / Q000737:chemistry; D056148:Chromatography, Reverse-Phase / Q000379:methods*; D005021:Ethylamines / Q000737:chemistry; D009841:Oligonucleotides / Q000032:analysis* / Q000737:chemistry*; D020005:Propanols / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D006845:Hydrocarbons, Fluorinated
chemical_list: D000838:Anions; D005021:Ethylamines; D009841:Oligonucleotides; D020005:Propanols; C001337:hexafluoroisopropanol; D006845:Hydrocarbons, Fluorinated; C027070:N,N-diisopropylethylamine