Chromatography Forum

Hi again

Well do not fix to hard on metals with regard to catalysts at this stage, think I misread your earlier post a bit. If you google sufoxides and peroxides you will soon find an "organic" catalyst (MIT, MOT...?!) so it might even be "product induced" oxidation in stress test.

It is good to close out any UV issues, however any diffuse UV issues in laboratory usually shows up early and especially if you have done UV stress test according to lux requirements. However one might find some information there depending on exposure to UV on:
-"open dish with pure drug substance"
-"open dish with product solution""
-"closed ampoules "

Hi,

The samples (open ampoules) that were stored in the autosampler in complete darkness were as oxidised as without any light protection.

This should exclude the light as being the oxidation promotor. I am now left with the chemistry in the lab, I guess.

One thing I will try now is to place empty (open) LC-vials in the autosampler overnight and then add sample solution and cap the vials. If it is particles in the air, some particles should land in the empty vial causing oxidation even in capped vials?

As I have said before, there may still be a problem with the ampoules from production. But if I have a problem in the analysis as well, I will not be able to separate them from each other.

- Empty LC-vials without cap placed in autosampler for 5 hours (in the worst positions).
- Ampoules transferred to these vials and then capped. Stored overnight in autosampler.

= no increase of oxidation products.

This indicates that the problem is not particles, but some gas phase contaminant. How about ozone? Our lab is 50 m away from a four lane highway, and 2 km away from the largest airport in Scandinavia. Laser printers are also known to generate ozone, and there is one just next to the LC. Just an educated guess, but do any of you have any experience of ozone problems or an easy way to detect this gas?

Ozone and/or nitrogen dioxide seem possible although they're typically present at <1ppm. Various commercial instruments are available to measure them.

Any volatile peroxides possible in you lab? Any bleach routinely used?

Maybe these papers will help:

Peptide Ozonolysis: Product Structures and Relative Reactivities for Oxidation of Tyrosine and Histidine Residues, J. Am. Soc. Mass Spec.
2006, 17 (9), pp 1289-1298

Ozone in Ambient Air as a Source of Adventitious Oxidation. A Mass Spectrometric Study, Anal. Chem., 2006, 78 (13), pp 4352–4362

Mattias, if you want to discriminate between oxidation prior to sample handling (i.e. production related) and sample preparation (i.e. procedures in the lab) just add some methionine to a considerable number of vials and do the analysis on them. The methionine should prevent further oxidation by getting oxidized itself. Thus you’ll be able to get a status on the oxidation prior to your intrusion.

Best Regards

Thanks for your suggestions and references!

I have now purchased a ozone measuring device from Draeger (inexpensive, measuring down to 0.5 ppm in air). I have one problem with this theory though, and that is why the samples in the drawers don't oxidise? It fits quite well to the other observations though (like why 100% oxygen could not oxidise the peptide).

Danko> excellent suggestion! I have tested to add methionine, and that prevents oxidation of the opened ampoules (no matter where they are placed). Time for screening!

Mattias - any progress with this?

Hi carls>

Yes! I have now tested 107 ampoules with added antioxidant, and not one have been oxidised (that was from the OOS batch). Clearly the production is not the cause of oxidation.

I would like to do direct injection of the ampoule, but his time on the Waters Alliance system (the same as they have in QC). I believe it uses some kind of vial recognition, because I get "missing vial" when I try to inject from the ampoules.

Does anyone know if it is possible to turn this vial recognition off, or how to "fool" the autosampler to do an injection without using vial cap?

Thanks again!

This is an old thread where we discussed the oxidation of one of my peptides, due to something in the air (not the oxygen itself).

Now I have another peptide that should not be so sensitive to oxidation, and I see the same thing. I have done some new experiments.

- When I keep my samples in the airflow of the autosampler or put them in front of an electrical fan, the samples oxidise quickly.
- When I create the airflow by using vacuum and a chamber (same laboratory air that is blowing on top), nothing happens for days.

Has anyone heard of "oxidation by use of electrical fans"? It sounds very strange to me!

This is an old thread where we discussed the oxidation of one of my peptides, due to something in the air (not the oxygen itself).

Now I have another peptide that should not be so sensitive to oxidation, and I see the same thing. I have done some new experiments.

- When I keep my samples in the airflow of the autosampler or put them in front of an electrical fan, the samples oxidise quickly.
- When I create the airflow by using vacuum and a chamber (same laboratory air that is blowing on top), nothing happens for days.

Has anyone heard of "oxidation by use of electrical fans"? It sounds very strange to me!

Electric motor, sparks, ozone seems plausible.

Peter

Thanks Peter,

Do you think that sparks are generated in most fans, or only if the fan is faulty? I see the same thing at two places. We have touched on ozone before, but only the ozone coming from the outside.

My guess is that brushless motors should not produce sparks, but you can get ozone even without sparks.

From Wikkedpedia "Ozone may be formed from O2 by electrical discharges and by action of high energy electromagnetic radiation. Certain electrical equipment generate significant levels of ozone. This is especially true of devices using high voltages, such as ionic air purifiers, laser printers, photocopiers, tasers and arc welders. Electric motors using brushes can generate ozone from repeated sparking inside the unit. Large motors that use brushes, such as those used by elevators or hydraulic pumps, will generate more ozone than smaller motors." http://en.wikipedia.org/wiki/Ozone

Peter

Look at page 801 here: http://books.google.com/books?id=5tcDAA ... &q&f=false
or
page 63 here: http://books.google.com/books?id=eiYDAA ... &q&f=false