Analysis of Oligonucleotides by HPLC−Electrospray Ionization Mass Spectrometry

Abstract

A new interface procedure has been developed that allows, for the first time, the high-efficiency analysis of synthetic oligonucleotides up to 75 bases by reversed-phase HPLC and on-line electrospray ionization mass spectrometry. For oligonucleotides up to 30 bases in length, single-base resolution can be obtained with low levels of cation adduct formation in the negative ion electrospray mass spectra. A key part of the method uses 1,1,1,3,3,3-hexafluoro-2-propanol as an additive to the HPLC mobile phase, adjusted to pH 7.0 with triethylamine. This novel additive results in both good HPLC separation and efficient electrospray ionization. The broad potential of this new method is demonstrated for synthetic homopolymers of thymidine (PolyT), fragments based on the pBR322 plasmid sequence, and phosphorothioate ester antisense oligonucleotides. This approach will be of particular utility for the characterization of DNA probes and PCR primers and quality control of antisense compounds such as phosphorothioates and their metabolites, as well as of materials used in clinical trials.

Modification of the universally unmodified uridine-33 in a mitochondria-imported edited tRNA and the role of the anticodon arm structure on editing efficiency

“…Total digestion of tRNA Trp with RNase T1 and analysis of the products by negative-ion LC/ESI-MS (Figs+ 1 and S2) was conducted essentially as described (Felden et al+, 1998), except for the addition of a second scan function at elevated cone voltage (65 V vs+ 37 V) to release modified bases (Crain et al+, 1999)+ tRNA Trp and its gel-purified RNase T1-derived 13-mer fraction were digested to nucleosides with nuclease P1, phosphodiesterase I, and bacterial alkaline phosphatase (BAP; Crain, 1990) and analyzed by positive ion LC/ESI-MS (Felden et al+, 1998; Figs+ S1 and S3)+ The gel-purified 13-mer fraction was totally digested with RNase T2 (Sigma R6398) for 1 h at 37 8C (1 U/20 pmol RNA); 0+6 U BAP was added and the incubation continued for 1 h (Fig+ 2)+ All of these LC/MS studies were conducted using a Hewlett-Packard (Palo Alto, California) 1090 DAD liquid chromatograph interfaced to a Micromass (Beverly, Massachusetts) Quattro II triple quadrupole mass spectrometer with MassLynx v+ 3+2 instrument control and analysis data system+ High-resolution mass measurements (Fig+ 5) and sequencing of unmodified (synthetic) ct and mt tRNA Trp 13-mers (Fig+ 3) and selected RNase T2-derived dinucleotides were conducted with a Micromass Q-Tof 2 tandem mass spectrometer, using ESI with negative ions detected+ Sample introduction for mass measurements and CID analyses of gel-purified 13-mers was by loop injection with the sample dissolved in MeOH ϩ H 2 O, 1:1+ The Ϫ4 charge state molecular ions were mass selected for dissociation (collision energy 30 eV Elab for all 13-mers) to derive sequences (Fig+ 3)+ A Waters (Milford, Massachusetts) Cap LC liquid chromatograph was interfaced to the Q-Tof 2 for LC/MS/MS analysis of the RNase T2-derived dinucleotides ⌿mp-s 2 Up (from the edited ASL) and ⌿mpCp (ct ASL; Figs+ S4 and S5)+ The HPLC eluents were the 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)/TEA/MeOH solvents, prepared as described (Apffel et al+, 1997), except that the A and B buffers were 0+2 M HFIP+ The gradient was developed using curve 6 as follows: 0-5 min, 100% A; 40 min, 75% A; 50 min, 0% A; 50-55 min, 0% A+ The column (Phenomenex Luna C18; 150 ϫ 0+5 mm) was eluted at a flow rate of 6 mL/min+ For these and the LC/MS analyses using the Quattro II instrument, all digests were injected directly onto the columns, without prior clean-up, and all eluents were directed into the mass spectrometer (s) without splitting+…”

A Novel and Intuitive Method of Displaying and Interacting with Mass Difference Information: Application to Oligonucleotide Drug Impurities

“…Capillary gel electrophoresis (CGE) [7], strong anionexchange (SAX) chromatography [8], 31 P NMR spectroscopy [8,9], matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) [10,11], liquid chromatography coupled to electrospray ionization (ESI) mass spectrometry [12,13], and tandem mass spectrometry (MS/MS) [13][14][15] are among the analytical methods that have been used to determine the quality of oligonucleotides and characterize their impurities. Ion pair-reversed phase (IP-RP) HPLC coupled with a UV detector and electrospray ionization has been identified as one of the most suitable methods because of its ability to provide increased chromatographic resolution and reliable qualitative and quantitative information [6].…”

Development of generic liquid chromatography-mass spectrometry methods using experimental design

“…A solution to the compatibility issue has been to use more volatile amines, such as dimethylhexylamine, which has been used in the analysis of a metabolite of clodronate [9]. There have been methods developed for the analysis of oligonucleotides and nucleotides by LC-MS using a 1,1,1,3,3,3-hexafluoro-2-isopropanoltriethylamine as the ion-pairing agent [10,11]. However, hexafluoroisopropanol is highly corrosive, relatively expensive and must be handled with care.…”