Chromatography Forum

Hi,

I need to develop a 2nd ID test for a peptide dissolved in water with 5% mannitol. Any good suggestions? 1st ID test is just retention time vs a standard.

I assume that diode-array spectra will be very hard to use since most related compounds have the same spectrum. Same with FT-IR, where also the water in the product will kill the signal?

MS is not an option since the CMO does not have it. Can optical rotation be selective enough?

Or is it that bad that TLC is the only way?

Electrophoresis ?

Peter

If the firt chromatography is reversed-phase, you could try HILIC or IC as the second test.

Or mixed-mode on Promix columns

Thank you for your suggestions!!

The first ID test is reversed-phase (which I should have written)

This peptide has no acidic or basic functions whatsoever (nine amino acids with modified C and N terminus). This will exclude any kind of ion-exchange (pure IC or mixed-mode). I assume that electrophoresis will also be hard to use for the same reason.

Will HILIC be possible, and can that be considered orthogonal to reversed-phase? The peptide is fairy hydrophobic, and need about 30% acetonitrile to be eluted from a C18 column.

I've seen Tris-Tricine SDS-PAGE work even for 5-7 AA peptides, although if N-terminus is blocked you may not be able to properly fix it in gel before staining. Capillary electrophoresis may work just fine though.

You may also try looking at some low MW range SEC/GPC column instead.

CE would probably work, but I doubt that the CMO has a CE. SEC is probably not specific enough. The related molecules weighs about the same.

HILIC sounds like the way to go, but I have no feeling for that technique. Is there a risk that the molecule is too hydrophobic? Where would you start ( e.g. column and mobile phase)? The drug is in water solution, but I assume that it would work if the injection volume is very low? If needed, I can collect on SPE and elute in acetonitrile before injection.

If there is an aromatic aminoacid, you could go for a fluorescence detection. Much more specific than UV, sice there are 2 waveleghts involved (i.e. excitation and emission).

Best Regards

The peptide contains phenylalanine, which has some intrinsic fluorescence. The problem is that so does all other peptides from this API manufacturer. This will not be specific enough!

All HILIC gurus out there! Would it be possible to develop a HILIC method for this peptide?

It's still much more specific than HILIC with UV detection for instance. All other peptides may contain phenylalanine but you see fluorescence is very much dependent on the rest of the molecule – not least the conformation. So, even though the molecule is excited at the same wavelength, say 280 nm, the emission would vary a lot from a peptide to a peptide.

Anyway I just tried to help. Good luck.

Best Regards

It's still much more specific than HILIC with UV detection for instance. All other peptides may contain phenylalanine but you see fluorescence is very much dependent on the rest of the molecule – not least the conformation. So, even though the molecule is excited at the same wavelength, say 280 nm, the emission would vary a lot from a peptide to a peptide.

Anyway I just tried to help. Good luck.

Best Regards

Thanks Danko, it is appreciated! Sorry if I sounded rude.

In my experience the excitation and emission spectra of peptides with aromatic residues are very similar, independant on the rest of the peptide. They are also quite broad (you get excitation and emission within at least 20 nm from the maximum excitation/emission lambda).

.......emission spectra of peptides with aromatic residues are very similar, independant on the rest of the peptide

I'm afraid it's not quite so. There are tons of literature discussing that aspect.
Also, even though the spectrum (the peak) is broad, the lambda max is very narrow/specific (within a single nm.). In addition to that, it's the combination of the wavelength specificity and the retention time that makes it much more specific, as mentioned earlier.

Best Regards

I think I understand now!

Are you thinking of using a "stand-alone" fluorescence spectrophotometer and compare the excitation/emission spectra of the product with that of a standard (i.e. no LC)?

That could be interesting to try! I just have to make sure that the CMO has a fluorescence spectrophotometer.

Hi again,

Stand-alone apparatus is a good option. In that case the mass (the wight of the sample compared to the standard) could be included as a specificity factor, just like the extinction coefficient in conventional UV spectrophotometric ID tests (USP, PhEur, JP etc.). HPLC combined with Fluorometric detector is another good option and in that case the retention time could be the extra specificity factor/dimension.
All depends on what you've got available in the lab.

Best Regards