Sample degradation in autosampler

[carls]

As you know troubleshooting these types of problems with protiens and peptides are especially difficult due to the depedance of many processes on many different variables including sequence, proximity to another specific amino acid, pH, ionic strength, etc, etc.

Its interesting to follow this problem since there can be many different "weird" causes.

[Mattias]

Just to update with the last results: The ampoules that were spiked with EDTA (0.1% approx of disodium EDTA) and left in the autosampler were all oxidised this morning. There was a pH increase from the EDTA (from pH 4.0 to pH 4., which could also promote oxidation though.

I have now added a true antioxidant to some open ampoules over the weekend. I will also place one open ampoule in my fathers wine cellar (in front of his cooling fan). That environment has nothing to do with the lab environment...

In case the antioxidant works, I can add this to a lot of ampoules and see if there are any oxidised ampoules coming from the production (to solve the one or two phenomena question)

[HW Mueller]

Mattias, I would like to oblige, but this is a science forum which is open to the public, is apparently indexed by google, etc., so that I would rather not get the reputation to participate in tea parties (coffee klatsch).
Thus, for those who want to learn something from this, me included I thought, it would be essential to clear up another nebulous statement on your part, namely that you did "light stress", but you do not say whether that was done under the exclusion of air (capped or open vial?). Peter was apparently talking about air oxidation which is initiated and/or propagated by light. I might add here that visible light can do this also if one has some visible light absorber (dirt) in there.

[Peter Apps]

Peter was apparently talking about air oxidation which is initiated and/or propagated by light. I might add here that visible light can do this also if one has some visible light absorber (dirt) in there.

Yes, that was what I had in mind. I recall an interesting study in which someone studied the oxidation of vegetable oils with various contaminants. Since they had 1l of oil in sealed bottles with about 2 ml of headpsace they did not see much difference between treatments, and their oil was remarkably resistant to oxidation !

Peter

[Mattias]

HW Mueller> It must be very hard for anyone to write anything in this short format without leaving any information behind. It is OK to ask for more information, not to insinuate an "evil" motive or fraud. Why would I ask for help then? Not all information was even known when this post was started. I think you need to think of your tone in your answers, since you otherwise seem to be a perosn of high competence and experience.

1. I don't see the point in randomising samples when having so many replicates showing the same thing in each condition. How do you randomise samples in front of a fan?

2. The light stress was performed on closed ampoules. On the other hand, the amount of UV-light inside the autosampler must be minute. The ampoules contain, except the solution, at least the same volume of normal air.

3. The experiment that showed 5% oxidised ampoules was performed in US. Since nine individual ampoules are tested for impurities in the release test, it would mean that about 23% of the batches would be OOS (fortunately that is not the case). I still believe that the oxidation is caused during the analysis, but where?

4. The ampoules have now been opened in front of the fan in the wine cellar. That is a quite interesting expriment since the wine cellar is dark, cold, no fluorescent light and an environment far away from solvents and heavy traffic (our lab is at the intersection of the two largest highways in Denmark.)

On Monday I will get the results of the antioxidant test and the winecellar samples.

[Don_Hilton]

A couple of side notes: First - I had a professor, years ago, who would look at you intently as you asked a question and there was no smile. I feared the man, knowing that he though me an idiot (which still may be true) and I expected him to shoo me away before hearing my question. Worse yet he would answer you directly as if the answer to your question were obvious. Years later I realize that the stare and lack of expression were simply the man putting the effort into listening - to get it right. And his answer was simple and straightforward. I was probably actually not the star idiot - just another of many students. I just misread the fellow. And I have learned that you are more likely to read your fears than the other person’s body language out of electronic communication.

Other: I submitted a paper and got back replies from three referees. Two of the replies were favorable and asked for one or two things. The third was a reply that ran three or four pages and was kind enough not to point out that better papers are written by school children – in wax crayon. As I looked at one of the questions I asked "How could anyone misunderstand what I said." And then I as I added a bit of detail, I saw how a bit of detail made the matter clearer. I fact, it had remove some serious ambiguity that I had not seen. And then the paragraph that he said was unnecessary - if I dropped it the information was... Never mind. Redundant! And so on. This person asked about 50 stupid questions. But when the manuscript was adjusted to keep anyone from being able to ask the stupid questions – it was much, much better. The referee was harsh, but he/she could not write what I was supposed to have communicated, but simply guide me to do it.

I read some of HW Millers comments and questions across many postings as being like this referee. Organize your data and show the steps in logic so that even he cannot misunderstand you. He may push you to organize the information in such a way that your next post will be: “Hey folks, you’d never believe how simple it was…â€

[carls]

Don Hilton+Peter Apps> We have done light stress (10*ICH) on the product, and the impurities that were (slightly) increased were not the oxidation products. We have clear glass ampoules with a transparent plastic label on the outside, since the product was proven not light sensitive.

Don Hilton: Is the question then:

Were the UV stability tests done on samples in sealed ampoules or samples that were open to air?

[carls]

Since we have digressed somewhat into the rules of etiquette:

I agree that it's not possible to provide all of the information everyone would want to know in a post on a forum such as this and it should not be necessary. In fact, it may not be possible due to confidentiality concerns.

If others are providing useful responses, enough information was given for the majority of people.

I also believe that those responding to posts have responsibilities such as reading the entire thread before responding and being clear in their responses.

[Don_Hilton]

As the issue seems to be out of the environment - what could fall into the sample? And my mind goes to the Lambic beers. You have shown much air flow leads to degredation. Little or no air flow - infrequent degredation. The issue will not be the major comonents of air: oxygen, nitrogen, CO2, etc - these are present in all samples of room air at slimilar concentration. But particulate matter is not distributed evenly.

An off the wall idea: Take a couple of capped LC vials that have shown to have no oxidation after standing and transfer into them an alequot from a vial that has shown degredation. If the capped vial shows degredation, suspect something biological. (I had started to suggest a microliter syringe here, but metal may or may not interfere. So, I am editing to suggest either a microleter syringe or pipetter with a plastic tip. If one does one does not show the transfer, try the other. Or, someone who works with biological contamination issues might jump in here and give a better idea.)

[HW Mueller]

Don, I had a very tough schooling especially as graduate student (Boulder, CO). I am actually quite civil, here. You are completely right in assuming that I am trying to pry people into being logic and systematic. . . . .

Mattias,
your statement, "I forgot to tell that all analyses in this investigation have been performed on the same product batch (the batch was rejected due to high level of oxidation products in one of nine ampoules during QC analysis). In QC, the solutions are always transferred to LC-vials.", is indicative to me that something is wrong with the batch you used for your analyses, or that it was handled wrongly in the analysis. To me it is not clear whether you knew which of these samples had already been oxidized and whether you took them out before doing your experiments. My statement on randomization has to do with that and also with not knowing whether groups of samples were handled differently before doing your actual experiments (an example: maybe the samples came to you in trays, one tray went into the drawer, the other in front of the fan, the latter was oxidized prior to you receiving it, of course, if you did this experiment several times, randomization could have come about "automatically"). Also, besides previous suggestions it could have helped to check into a time dependence on the oxidation. Also repeating the oxygenation test on some of these samples would have been helpful.
One more thing comes to mind. it helps immensly if one knows the scope of the reaction .

[Mattias]

carls> The UV-stress test was performed on sealed ampoules.

Don Hilton> I have never heard of oxidation caused by biological processes but I am willing to test any theory soon. One obvious thing that I should test is to transfer only half of the solution to LC-vials. When I find an oxidised solution, I can go back to that ampoule and analyse the remaining solution. If this phenomenon is not product related, I should not see any oxidation in the other half. The problem is that I have not found one single OOS result in the batch I have access to now (which was rejected for just oxidation), using capped LC-vials.

HW Mueller> I have only access to the last found OOS batch (the one out of nine batch), and I have just got a plastic bag with hundreds of ampoules. There is no possibility to see if any of the ampoules have been treated differently. All freshly opened ampoules have contained a low amount of oxidation products, typically 0.5% (sum of the two peaks), which is also present in the substance. After a night exposed to airflow, the level is about 5-10% depending how close to the fan they stand.

We have always thought this product is very stable, and it is handled without any protection in the production. The ampoules are filled without nitrogen, so the solution is always exposed to air (50% of the ampoule volume is air). If it was sensitive to air, we would not be able to produce it like this.

That is why I got very surprised to see that the solution was so heaviliy oxidised just because the ampoules were standing open in the autosampler (no one had tried this before). This behaviour does not fit with our experience of this peptide. Thus, it seemed like a clue to our occasional OOS problem which we have not been able to solve. I still don't know if these problems are connected though.

[HW Mueller]

Where does that ~0.5% oxidation product come from? You get two products?
This still needs systematic, well thought through experimentation if you want to find out whether this batch is more succeptible to oxidation or whether the cause is a different treatment of the samples than before. For instance does this batch oxidize under O2 bubbling in the light in the drawer? . . . . . . . Such things should be done with samples that have been checked for content at the start and best at the same time the fan experimnt is repeated.
Is the temp. controled?
If you have not done this you should find out as much about this oxydation as you can, seems a bit strange to me, maybe it´s really a biological process.

[Mattias]

Yes, the oxidation creates two oxidation products (isomers). The substance that we buy from another company always contain a small amount of oxidation products (acc. to their certificate), approx 0.5% in total. This is also what I see when I analyse the solution directly (or from capped LC-vials).

I can agree that the investigation so far has been more of a trial-and-error type. It started off as a wish from the production if I could screen this batch for the frequency of OOS-ampoules (it has always been considered to be a production problem, not an analytical problem). This is when I filled the autosampler with opened ampoules (I was lazy) and let the system run overnight (which resulted in 100% OOS ampoules).

Regarding my last crazy experiments: the ampoule that had been standing open I front of a cooling fan in the winecellar the entire weekend did not show any increase of oxidation products. It is only one ampoule, but this is on the other hand the first good result I have seen from any opened ampoule exposed to moving air. Why does it not oxidise in the wine cellar?

[krickos]

Hi

I find this topic very intriging I will try to not stray away to much, here we go.

"air quality":
If i understand you correctly you have observed the following.
-exposure to moving laboratory air=> oxidation
-exposure to non moving laboratory air-no oxidation ie drawer-no exposure to particles "only" volatile stuff in laboratory air.
-no oxidation in wine cellar, well only one vial but still

Regardless of type (sterile, aseptic other..) of your product is, the production line should should be in a "classed area" with regard to maximal permitted particles in rest, so it would be intresting to see if exposure to production quality air gives the same result (or if you place it in a glove box with a HEPA filtering unit overnight).

An organic chemist might also be helpful. "in my world" those types of oxidation products usually is produced under harsher conditions and not at rooom temperature but there are at least 2 exceptions but hardly likely:

A redox reaction variant of the Jones reaction, school example is an alchol reacting with Cr(VI) just above room temp, giving a ketone, a tiol bound is weaker than the -OH bound and might react faster.

Consistant with your stress testing according to ICH, peroxides (H2O2) exposure typically do not produce much sulfoxides unless there is a catalyst present, then it can give a higher yeild at room temperature.

I have even seen "leach" issues from glue on lables on plastic ampoules, causing oxidation, but that seems non relevant in this case.

[Mattias]

Krickos> The idea of placing the open ampoules in a controlled area sounds interesting. I will discuss this with my US collegues! In the forced degradation we added H2O2, and the amount of oxidation products went sky high. So there must be enough catalysts present for a reaction (the product contains also NaCl for isotonicity, which can bring some metals to the solution)

My observations: If we assume that the result of the wine cellar sample is true, which I believe it is (the air velocity in the wine cellar is way higher than in the autosampler at least), the reason for the solution not oxidising must either be the "chemistry" in the cellar or the darkness (the wine cellar is completely dark). The temperature in the wine cellar is about the same as in the autosampler (10C). One observation: The solution from the wine cellar contained quite a lot of particles (easliy observed when holding the ampoule against the light)

To exclude the light, I have placed a number of open ampoules in the Acquity autosampler (at 10C) but this time with the small lamp inside shut off. I have also covered the entire autosampler with Alu-foil. Tomorrow morning we will know the result.