Evaporation standard stock solution

Discussions about sample preparation: extraction, cleanup, derivatization, etc.

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Hello everyone,

I post this message because i have a question about standard stock solution used to therapeutic drug monitoring by LC-MS/MS.

In our laboratory, we use standards in ampulla, generally with one molecule solubilised in methanol at 1 mg/ml with a final volume of 1 ml. After opening, the solution is transferred into amber glass with teflon cap, and stored at freezer.
I have a problem because i suspect methanol evaporation of my standard solution leading to higher concentration that expected.

I would like to know if somebody has already noticed the same problem in its laboratory ? Or eventually if somebody has suggestions or advices to solve my problem ?

Thank you in advance.
I think that 1 ml of solution is too small an amount to be stored. Any small loss of solvent (evaporation, droplets on the cap, etc) will lead to a large error. Probably preparing a larger amount of standard (e.g. 25 ml) in a narrow neck volumetric flask with a ground glass cap is the best option.
carlo.annaratone wrote:
I think that 1 ml of solution is too small an amount to be stored. Any small loss of solvent (evaporation, droplets on the cap, etc) will lead to a large error. Probably preparing a larger amount of standard (e.g. 25 ml) in a narrow neck volumetric flask with a ground glass cap is the best option.


I would also parafilm when storing. I've an increase in standard concentrations due to people either not parafilming or poorly parafilming a ground glass stoppered flask.
Yes, if there's any solvent evaporation, concentration of the standard will be higher than you expect, so results of the sample will be lower than actual.

I'd tell my boss that I'd need to open a new ampule each time. Compared to your salary and upkeep to keep that sophisticated instrumentation going, should be a drop in the bucket, the cost of doing business.

The volumetric flask idea: I'd mark the liquid level with a Sharpie like an hour after putting into the freezer, and compare to that before withdrawing any for subsequent use.

We actually did a study of our OTC pharmaceutical standard solutions stored in the refrigerator for two years in bottles with Polyseal cone seals, to establish real data to demonstrate 90 day storage life, and even with opening/withdrawal like once per week, area/concentration compared to fresh standard was within 2% RSD.
At freezer temperatures with a properly sealed solid cap (not a center-hole cap as used for autosamplers) methanol evaporation is less likely than several other possible causes of unexpected results.

What evidence do you have that methanol has evaporated ?

An easy way to check for evaporation is to weigh the sealed vial before it goes into the freezer, then when you want to use it let it warm to room temperature, wipe it dry and weigh it again.

Peter
Peter Apps
I think that evaporation might not be the source of methanol loss - rather it could be selective fractionation in the droplets. Think about this scenario: vial is half empty so there is a lot of headspace. Methanol established vapour pressure equilibrium. Some droplets containing solvent only form on the top of the vial and on the cap. The vial is opened: the methanol on the cap evaporates, concentrating the standard in the bottom. Repeat multiple times, and there you go! A larger amount of standard in a narrow neck flask should minimize this issue as the headspace/liquid content ratio is negligible
Thank you very much for your answers, it helps me to understand the possible causes of my problem.

Peter, i think that methanol has evaporated in my calibration standrad stock solution because during my lasts levetiracetam quantitation, quality control have fall at aroud -15% at the 3 level we use. Generally we have not more than +/-5% deviation. After that, i have made a stability test compared to a new ampulla and found +10% levetiracteam in the old standard compared to the new. And my last serie i have made this morning with the new calibration standard gave me CQ deviation < 5% at each level.

And Carlo, there is effectively a lot of headspace in our vials after repeted pipetting during time. It could support your hypothesis.

Clearly i think we have to revise our way of making.
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