It seems like everyone is hopping on the therapeutic oligonucleotide train. Whether siRNA’s or antisense, oligo therapeutics are experiencing quite the comeback. With the new resurgence in oligo interest comes a new generation of analytical chemists faced with the same challenge—HPLC analysis for synthetic oligonucleotides.

Often, a standard C18 alone won’t suffice in selectivity and one must carefully select a column that is appropriate for the rigors of oligo method development. Ion pairing-HPLC (IP-HPLC) is an absolute must, with triethylamine, tributylamine, and hexylamine being some common alkylamine ion pairing reagents used for synthetic oligonucleotide methods. Additionally, high temperature, 50°C, even 60°C, is required to disrupt the intramolecular interactions which can affect peak shape quite drastically.

So, you’ve spent some time, selected a robust column, and you have a decent method by LC-UV. Perhaps using 50-100 mM TEAA, pH 7.0 for mobile phase A, acetonitrile or methanol for mobile phase B. Running a shallow gradient at 1% B/minute under a high temperature of 50°C and achieving a partial n-1 separation (which is sometimes all we can ask for in this world). Great! But what if I told you your method could be even better? Next time, consider developing an MS-friendly method to begin with.

40nt DNA Oligo-RP 3µm Fingerprint

Now, all the protein biochemists are screaming at me because TFA methods are straightforward, but when transitioning to MS-friendly volatile acidic modifiers like formic acid, your peaks go from sharp to broad very quickly. TFA vs. FA for proteins is basically the Jekyll and Hyde of chromatography.

But in the world of oligo LC/MS methods, you’re still working with an alkylamine ion pair in it. Of course, you’ve now dedicated your mass spec to negative mode, but that’s neither here nor there. But there’s something else mandatory in LC/MS for oligos. This weird, esoteric polyfluorinated solvent additive, Hexafluoroisopranol (HFIP).

Unless you run GPC on a regular basis, you’re probably not familiar with HFIP. It’s also not a common additive in most other IP-HPLC, with most alkylamine and HFIP mobile phases being pH 7-9. When I first started working with oligos, I simply thought HFIP was a magical acidic modifier to adjust mobile phase to a reasonable pH.

However, HFIP does much more.

Primarily, HFIP is used to improve electrospray desorption, a critical part of mass spec development for large molecules. Chen et al (1) proposed that because of its low boiling point, HFIP will evaporate quickly, helping with droplet formation and leaving the solvent basic at approximately pH 10. This facilitates the ionization of the oligo. As TEA donates a proton to water, it will evaporate off, leaving the negatively charged oligo in gas phase to enter into the MS.

Of course, there is an optimal amount of HFIP. Rudge et al (2) showed that if you use too much, peak shape and sensitivity goes down. Gong (3) show that 50 mM HFIP and either N, N-diisopropylethylamine or hexylamine (respectively) gave best ionization efficiency and peak shapes. This is contrary to other literature which has concentrations as high as 400 mM HFIP (4).

The other interesting thing about HFIP is that it actually affects the solubility of the ion pair itself. Cramer (5) postulated that because of the limited solubility of alkylamine ion pair in HFIP, it improves its ion pairing capability. Essentially, it makes your TEA act like SUPER TEA. Which is actually good because you can get away with lower concentrations. So maybe your mass spec won’t be dedicated to negative mode after all. Admittedly, you’ll probably have to clean the ion source before your small molecule colleague runs their sample.

So, chromatographically, HFIP methods tend to be better than TEAA or HAA methods. In fact, at Phenomenex, we almost run exclusively alkylamine HFIP methods for all our LC-UV application work. We also tend to stick to DIEA so much so that I even wrote a tech note about it. Take a look HERE.

There are other method parameters to consider looking into for improving chromatography, not the least of which is looking at alternative particle morphologies for IP-HPLC. For example, using core-shell HPLC/UHPLC column technology can improve peak shapes and run times for oligos.

Either way, whether you’ve been in the therapeutic oligonucleotide game since the beginning, or whether you were into oliogs, then you weren’t, but now you’re back again, HPLC analysis for synthetic oligonucleotides can be challenging. Start off on the right foot and consider looking into HFIP in the mobile phase, even if you aren’t running LC/MS.

Brian Rivera
Phenomenex Product Manager, Bioseparations & Specialty LC

Mr. Brian Rivera is currently the Product Manager for Bioseparations Products at Phenomenex, Incorporated, Torrance, California, USA. In this role, Brian is responsible for seeing Bioseparations products from proof of concept through to commercialization. He is also responsible for line extensions to existing product lines and oversees the global business for this rapidly growing segment within the company’s liquid chromatography and sample preparations product lines. To facilitate the growth and better understand the needs of the global market, Brian spends a good portion of his time giving technical product seminars on the company’s latest products and interacts with scientists that are facing the most complicated Bioseparations challenges.

Prior to this role, Brian has held a variety of positions in sales and marketing related to biotechnology products. He was a Technical Sales Specialist for Phenomenex and Eppendorf. He was an Account Manager for ProZyme and a Biotechnology Product Specialist for Phenomenex. And prior to his business-facing roles, Brian worked in analytical methods development and in manufacturing support roles for a few biotechnology companies in the San Francisco Bay Area; including Avid Bioservices, ProZyme, and Chiron Corporation. He has a Bachelor’s Degree from the University of California, Davis.

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References:
1. Chen B, Mason SF, Bartlett MG. The effect of organic modifiers on electrospray ionization charge-state distribution and desorption efficiency for oligonucleotides. J Am Soc Mass Spectrom. 2013;24(2):257-64
2. Rudge, James et al. “Preparation and LC/MS Analysis of Oligonucleotide Therapeutics from Biological Matrices.” Chromatography Today. 1 Mar. 2011: 16-20. Print.
3. Gong L, Mccullagh JS. Comparing ion-pairing reagents and sample dissolution solvents for ion-pairing reversed-phase liquid chromatography/electrospray ionization mass spectrometry analysis of oligonucleotides. Rapid Commun Mass Spectrom. 2014;28(4):339-50.
4. Studzińska S, Pietrzak L, Buszewski B. The Effects of Stationary Phases on Retention and Selectivity of Oligonucleotides in IP-RP-HPLC. Chromatographia. 2014;77(23-24):1589-1596.
5. Bonilla JV, Srivatsa GS. Handbook of Analysis of Oligonucleotides and Related Products. CRC Press; 2011.