Chromatography Forum

Hello to everyone!

This is my first post on this forum and I kinda need a help from you concerning validation by LC-MS, especially in the stock solution stability issue.

I am relatively new in the bioanalysis field - I graduated as an organic/medicinal chemist - and I have to admit that there are still a couple of things in my mind tagged with question marks, after 4 months intensive... "self-study"...

One of them happens to deal with stock solution stability. When looking at literature, it's stated more or less in this way almost of the time "Prepare fresh solution from the reference material and comparing the absolute response of the fresh solution with that of the stored solution".

OK. I understood what values to compare. But the problem is "how well"... I mean, how is it actually carried out in a PK lab to be scientifically and regulatorily "sound"? You make stock solutions, and you run the LC-MS, you get the peak area at 0h and - let's say - 6 hours later you run the same test but only with samples that were left at RT?

OR, you make stock solutions, you store a set of stock aliquots in the refrigerator/freezer and another set aliquots are left on the bench at room temperature and you make a batch with both types of aliquots to analyze them in a single LC-MS run?

Which way is more correct? I have to say I'm quite confused and this question makes me bite my nails

Another "subsidiary" question would be: why in other stabilities tests in biomatrices we use standard calibration curves to generate concentration values that'll be compared, and for stock stability test we use peak areas?

Thanks for any help you can provide...

OR, you make stock solutions, you store a set of stock aliquots in the refrigerator/freezer and another set aliquots are left on the bench at room temperature and you make a batch with both types of aliquots to analyze them in a single LC-MS run?

Not quite. You would prepare a stock solution from your primary reference material which would be stored in a fridge/freezer, in the absence of light or at room temperature, whichever is appropriate. Sometime later (a day/week/month/year) you would prepare another stock solution using the same (not a different batch) primary reference material you used to make the original. By running the two solutions together you can determine the stability of your solution under particular storage conditions. The difference in peak areas will indicate how much your of your target compound has disappeared over time.

Basically.

As for the "how well". That really depends on which regulatory body is applicable to your line of work.

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Not quite. You would prepare a stock solution from your primary reference material which would be stored in a fridge/freezer, in the absence of light or at room temperature, whichever is appropriate. Sometime later (a day/week/month/year) you would prepare another stock solution using the same (not a different batch) primary reference material you used to make the original. By running the two solutions together you can determine the stability of your solution under particular storage conditions. The difference in peak areas will indicate how much your of your target compound has disappeared over time.

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Thank you richiekich for your reply. If I'm following you correctly, for a "basic" solution stability test, you should:

1. Prepare a stock solution, and leave it at room temperature for 6h (for exm)
2. After 6h, prepare another stock solution
3. Run a single LC/MS run with both of the stock solutions above being analyzed, same batch

Correct? If so, I am beginning to wonder how much primary reference material you need to lead a full validation to end. 100 mg Maybe?

Correct? If so, I am beginning to wonder how much primary reference material you need to lead a full validation to end. 100 mg Maybe?

That would depend on the number of intervals you wished to test, as well as the amount in your standard preparation.

For many APIs we analyze, we have very good stability in solution, so we will test at 24 hours, 1 week, 2 week, 3 week, 1 month. This would only require a total of 6 standard preparations.

For others with less stability, we might do 12 hours, 24 hours, 48 hours, 72 hours.

We typically continue the study until we see values that are greater than 2% different. We'll then dial back one test point and call that our expiration. For example if we tested 24 48 72 and 96 hours, and the 72 passed, but the 96 failed, we would list 48 as our expiration.

That would depend on the number of intervals you wished to test, as well as the amount in your standard preparation.

Thank you paulw for your reply.

I wish I had an actual value of the reference material though (full validation).