Improving Oligonucleotide Sensitivity and Separation for LC-MS Applications

Mar 01, 2009

Ion-pairing assisted reversed-phase high performance liquid chromatography (HPLC) has been a common method for purifying oligonucleotides for some time, but has not been widely used for routine quality control methods that assess purity. Ion-exchange HPLC, matrix-assisted laser desorption–ionization mass spectrometry (MALDI-MS), and capillary electrophoresis have been used more commonly for determining oligonucleotide purity. With the development of several potential human therapeutic oligonucleotide drugs, many manufacturers are now looking to use LC–MS as an identity test as well as to quantitate and identify synthetic impurities.

Oligonucleotides are organic polymers in which nucleosides (an aromatic base attached to a ribose sugar) are linked by a phosphodiester into long chains up to thousands of bases in length.

The combination of aromatic bases, polar sugars, and polyanionic phosphates can make separations of oligonucleotides very challenging; many will only differ slightly in their overall charge and hydrophobicity based upon their base composition and length. Oligonucleotides from natural sources are typically thousands of bases in length and are more amenable to separation using molecular biology techniques. Oligonucleotide therapeutics and oligonucleotide reagents are much shorter in length (from 2 to 100 bases in length) and usually are generated through synthetic methods using stepwise chemical synthesis. Such synthetic oligonucleotides usually are purified and analyzed using chromatographic techniques; often ion-exchange chromatography is used to purify the desired full-length oligonucleotides from shorter length synthesis impurities. Reversed-phase chromatography also is used for purifying synthetic oligonucleotides; however, their high polarity requires the use of an ion-pairing buffer (typically triethylamine or other alkyl amines) to increase retention and improve selectivity (1).

Interest in quality control analytical methods for oligonucleotides has grown in the last few years as oligonucleotide therapeutic candidates have been passing through the research phase of product development and into clinical trials (2). Traditionally, ion-exchange high performance liquid chromatography (HPLC), capillary electrophoresis, polyacrylamide gel electrophoresis (PAGE), and matrix-assisted laser desorption–ionization (MALDI)-MS have been used to characterize oligonucleotides; however, each method has unique difficulties in quantitating and characterizing minor impurities. Lately, manufacturers have looked at adopting reversed-phase LC–MS for characterizing impurities in oligonucleotide products (2). Reversed-phase LC–MS has unique advantages in that impurities can be separated chromatographically from a parent and then identified and quantitated based upon MS or tandem MS-MS analysis. However, using reversed-phase LC–MS is not without its limitations. Modifications to the ion-pairing mobile phase to allow for MS compatibility often result in reduced resolution of oligonucleotides compared to traditional ion-pairing reversed-phase HPLC separation methods. Sensitivity also can be an issue in that ion-pairing reagents are known suppressors of electrospray MS; however, such reagents are needed when working with oligonucleotides to achieve retention on reversed-phase columns (3,4).

Research was undertaken to improve LC–MS sensitivity by investigating different concentrations of ion-pairing buffers to achieve the best MS signal while maintaining adequate selectivity between oligonucleotides that differ by only one residue in length. Different MS systems were used as well to determine if mobile phase conditions were MS-dependent or specific to a particular interface. Because TEA–HFIP buffer mixtures are commonly used for oligo MS work, the ratio between the buffers was varied to determine the mixture that delivered the optimal MS signal while maintaining resolution.


Oligonucleotide samples were run on an 1100 HPLC (Agilent Technologies, Wilmington, Delaware) with quaternary pump module, autosampler, and a diode-array detector. The column oven temperature was set at 50 °C and various gradients were used, depending upon the sample and mobile phase used for analysis. LC–MS analysis of oligonucleotides was performed using either a 3000 triple quadrupole, 4000QTrap hybrid-triple quadrupole-LIT (Applied Biosystems, Foster City, California) or the Esquire ion-trap MS system (Bruker, Billerica, Massachusetts). All three MS systems were operated in negative ion mode (because oligonucleotides are polyanionic) and sample was introduced into each MS system using their standard electrospray interfaces with optimized operating conditions. An m/z range from 500 to 2200 was monitored in total ion current (TIC) mode for each instrument. Molecular weight deconvolution–deconstruction was performed using the Bayesian Protein Reconstruct tool within BioAnalyst (Applied Biosystems).

Figure 1
The HPLC column used for all the separations was a 150 mm x 2.0 mm Clarity 3-µm Oligo-RP column (Phenomenex, Inc., Torrance, California), an HPLC medium specifically designed for oligonucleotide separations. Mobile phase was varied run-to-run (see specific figures for composition), but consisted of a combination of TEA and HFIP in 98% water and 2% methanol for the aqueous mobile phase. For the organic mobile phase, TEA and HFIP were mixed in with 98% methanol and 2% water. The gradient was from 5% to 10% organic in 5 min followed by an increase from 10% to 30% organic in 15 min. For the last set of chromatograms, the second gradient was shallower — 10–30% organic in 60 min.

lorem ipsum