Overcome oligonucleotide analysis challenges with 2D-LC/MS

Oligonucleotides are typically analyzed and purified using ion-pair reversed-phase liquid chromatography (IP-RPLC) and anion-exchange chromatography. Since highly salty mobile phases are not compatible with mass spectrometry, and samples need to be desalted to participate in ion-pairing before IP-RPLC analysis, investigators can either manually prepare samples or consider using 2D-LC/MS to obtain a mass measurement from salty first dimension separations.

In this SelectScience webinar, now available on demand, Patrick Cronan, LC applications scientist at Agilent Technologies, and Dr. Matthew L. Turner, biocolumns product manager at Agilent Technologies, discuss the common challenges associated with oligonucleotide analysis and purification and how to overcome these with 2D-LC/MS and method optimization.

Think you’d benefit, but missed the live event? Register now to watch the webinar at a time that suits you or read on for highlights from the Q&A session.

What criteria do you recommend following when ordering oligonucleotides to have better chances of obtaining reliable results?

MT: A key thing to consider is what the use case is for that oligo. If you're just using them for diagnostics or maybe PCR-based methodologies, then you could probably deal with a little bit lower purity. Though, I'd still recommend at least a reverse-phase purification of those oligos because we don't want any off-target binding. If it's for an actual API, then the purity becomes critical.

The impurities with the synthesis of these oligos are so highly related to the API that we want to minimize as many interactions as possible. Luckily Agilent has a nucleic acid synthesis division where we specialize in producing APIs of oligonucleotides and you can also order guide RNAs as well from our genomics decision.

You mentioned different reverse phase columns for unpairing in the second dimension. What happens to the separation when you change the pore size or you switch to the oligonucleotides column?

PC: As your oligos get larger, the resolution of the impurities becomes clearer with the larger pore size, PLRP-S. What I found in switching between a PLRP-S column and the AdvancedBio Oligonucleotide Column is that we seem to get more retention on the column using the AdvancedBio Oligo Column. So, you hopefully can get some more separation on the ion-pair side of the 2D analysis by using the oligo column.

Is it possible to get good ion exchange separation with ammonium hydroxide and how does that compare to sodium hydroxide?

MT: Ammonium hydroxide is commonly used for deep protection during synthesis. Therefore, I don't see any issue with using ammonium hydroxide. It is not something that we've tested however, one factor to consider is that at one molar the pH would be a little lower than with sodium hydroxide so you may get a little less denaturing. It could be a good alternative, especially if you're trying to avoid things like sodium adducts.

Does using 2D-LC/MS, ion exchange, and IP-RPLC result in the formation of more ion adducts compared to using only IP-RPLC?

PC: When you're using a salty buffer in the first dimension, you could see there were a lot of sodium adducts in that data because you have the sodium in the first dimension. And even though you're desalting that diluent that you've trapped from the first dimension, you still have sodium kind of surrounding your molecules.

I've done some work with sodium hydroxide and ion exchange, and it works well but using those ammonium hydroxide ion exchange methods can help. I've used different sulfates and tartrates and you have to pick a buffer in the first dimension that's going to aid your second-dimension separation. You can deal with a little bit of the sodium adduct but if you want to start to push your oligo mass spec to the negative charge envelope, you want to start to play around with different buffers in the first dimension.

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