On-column degradation gets worse during sequence

[Mattias]

Hi,

I ábout to transfer a new method to our QC lab, but have run into problems.

In QC they get a 0.5-3% peak eluting just before the main peak. The peak looks like Jabba The Hut and show all characteristics of on-column degradation. The size of this peak is also corresponding to the loss of main peak area in each injection. There are no extra peaks in blank injections. I have never seen this peak in my lab, and we are both using Waters 2695 instruments.

It is quite OK when the sequence is started, but gets worse and worse over the sequence. I do not understand why? If this is related to metals from the instrument, should it not stay constant or be decreasing? When the column is cleaned or exchanged the problem is gone for a short while.

Any ideas of how to fix this problem the best way? Passivation with HNO3 seems to be out of the question (last time they did that, they whole pump assembly had to be exchanged). Product is a 10 AA peptide.

[danko]

When the column is cleaned or exchanged the problem is gone for a short while

Are you injecting from the same sample after cleaning or exchanging the column or du you take a ny/fresh sample following the column cleaning/exchange?

Best Regards

[Mattias]

Hi,

It is a new sample every time (vials from production).

I just found out that the instrument is used for another product that uses a weak sulfuric acid in the mobile phase. I guess this in not so good when the next analysis is sensitive to metal ions.

But I still don't understand why it gets worse and worse during the sequence. Can metal ions be trapped in the column (XBridge), which then can act as a "reaction vessel".

I understand that the key is to remove the leakage of metals into the mobile phase, and they will do a passivation with EDTA now.

[danko]

It is a new sample every time

So, maybe not quite "column degradation"?
Maybe the degradation is occuring in the vial/autosampler?

Try the column cleaning or column exchange procedure but inject from the same same/initial vial. And then you might see the peak already in the first run?

Best Regards

[danko]

I meant of cours "on coulmn degradation" naturally.

Best Regards

[aceto_81]

I meant of cours "on coulmn degradation" naturally.

Best Regards

@danko: what exactly did you mean???

But I think danko is right: on column degradation should be constant, not getting worse.
So I'm also think about sample degradation instead of on column degradation.
Maybe you should run your samples as you did, but immediately afterwards inject a fresh sample, to see if the peak also is that worse.

Ace

[danko]

Hi aceto_81,

I did mean "on column degradation" as you guessed it right. I just forgot the "on" word.
And I did meat too that if a degradation occurs then it happens in the vial (in the autosampler) rather than on the column.

Best regards

[drewa512]

New sample in that it is freshly made and run within a few minutes of completion? or a new vial? My immediate thought was degradation in the auto sampler. If the column was causing some sort of problem, or the column itself dying, you would expect to see some problems in the blanks.

[Mattias]

Hi,

The drug is a peptide in solution in 2R vials. The solution is directly transferred to LC-vials (i.e. no sample preparation). The drug is stable for 3 years at 2-8°C in its original container.

The problem seems to fit on-column oxidation, probably caused by leakage of metals from the instrument. My theory is that the leaked metal ions are slowly accumulated on the column (should bind well to the negative silanols), causing more and more degradation over a sequence.

The problem did not gets much better by the EDTA treatment of the instrument. I have asked QC to move another Alliance system - but I have not recieved any update from them for a while. I will let you know how it goes. I dot not see anything of this problem on my Alliance system!

[zokitano]

Hi Mattias,

Have you tried to add some EDTA in your sample/standard solvent (usually in umol or mmol range)?
I had the similar problem (developing peak deterioration gradually with time/injections) with small acidic compounds which turned to act as complexation ligands and indeed reacted with the residual metals in the HPLC system (on column). The problem with peak deterioration was solved by adding only 2 nmol EDTA (absolute amount) in the injection (sample/standard) solvent.

More information you could find in the literature. For example:

Polar Anionic Metabolome Analysis by Nano-LC/MS with a Metal Chelating Agent
Khin Than Myint , Taisuke Uehara , Ken Aoshima and Yoshiya Oda
Anal. Chem., 2009, 81 (18), pp 7766–7772

Formation of phosphopeptide-metal ion complexes in liquid chromatography/electrospray mass spectrometry and their influence on phosphopeptide detection
S. Liu, C. Zhang, J.L. Campbell, H. Zhang, K.K.–C. Yeung, V.K.M. Han, G.A. Lajoie
Rapid Commun. Mass Spectrom, 19 (2005), p. 2747

Best regards