undefined peaks after 2 years of stability test for product

[krystynakk]

hello,

I am doing validation of method (224 nm/254 nm) for the product (ointment gel type); I have got 2 undefined peaks, which areas are aroud 1-2% according to main peak (according to API substance) at chromatogram for product after 2 years stability tests at 25C;

the 2 undefined peaks are only at 254 nm, I have no observed them at 224 nm at all;
main ingredient of the product and its (possible) impurities absorbed generally at 224 nm (maximum)
the rest of ingredients are preservatives (aseptines and benzyl alcohol) absorbed mainly at 254 nm;

I have tried stressed conditions-
I have checked degradation products fpr preservatives: for aseptines I have tried hydroxybenzoic acid, for benzyl alcohol I have tried benzoic acid in addition to benzaldehyde;

I have tried different mixtures of preservative with/without their degradation products in water/ethanolic solutions with or without temperature without no results;

I need to mentioned that I am working at small company without as 'inventions' as: LSMS, LCMS - NMR and so on; by the way, I do not think that 'separation' of the previous described 2 undefined peaks would be no reasonable, because they are only 1-2% of main peak (i.e. per 1g of product there is 10 mg of API substance, therefore, that undefined peaks are around 0,1-0,2 mg pear peak)

any constructive idea?

[tom jupille]

Before we can make any suggestions, we need to know what is the question?

If the question is "how do I identify those peaks", then the answer is most likely to be "by MS". In your case, that means sending samples to an outside lab.

[unmgvar]

i know that i is the dummy question of the obvious but it needs to be asked at the earliest stage possible.

have you tried to show that it is not a case of "contamination" of your sample during one of the stages of your work while doing the tests in the lab?
reagents, tools, flasks, hplc, analyst and so on?

[Bryan Evans]

Do you have access to any other data (maybe some stressed sample chromatograms)? You can look to see if there is a trend - growing
degradate peaks over time.

Other wise - you have to do some investigation. Same suggestions as everyone else - mobile phase, reagents, glassware, ect. You can try
the following and see if you find the extra peaks:

- inject the mobile phase
- inject the sample and/or standard diluent
- rinse all glassware with diluent - and redo the analysis

If its not local contamination - send it out for LC-MS analysis.

[vickig]

Do the peaks show up in stressed placebo?

[krystynakk]

hello,

i did stressed tests using variations of main ingredients/preservatives/known impurities with no results;
i did stressed test using different 'conditions' for doing samples (higher temperatures, uv light, oxidisers. reductors, acidic and basic media) with no results;
i have checked placebo, phase, and methanol (its a cosolvent for samples) - there was no peaks which i was looking for
i checked solvents, cosolvents, glassware (i even used brand new ones) with no results
i checked containers in which product is kept and its influence on the product (i put some of the container to the sample which i prepared for hplc) with no results;

as i said i have no access to lc-ms-nmr systems, morover i think that difference between hplc and lc-ms-nmr system can be to large for the analysis; i mean, the product has around more or less 20 peaks (substrates in according to preservatives and known contaminations) in addition to comlicated gradien method of analysis; these circumstaces can cause impossibility in simple transfer hplc to lc-ms-nmr method;

that why i asked you for advice; because i have been working on that product so long i may not notice simple solutions; thats why i had asked you if you had any ideas simpler than ms analysis;

maybe you have some great solutions for stressing tests? I mean reactants which can make stress quicker?

[unmgvar]

Ok i have a story from a very good collegue of mines that he told me several years ago. it has probably nothing to do with the case at hand but the abnormal solution might help you think better for your solution.

there were having the same problem then you. even worst. the new peak was growing fast. all the tests they did like yours showed nothing and they were at lost. the stability job was origanlly outsourced to another lab and nobody in both companies could understand what was the problem.
after a lot of thinking my friend decided to get the samples back to his labs.
he received the samples in small tight sealed plastics bags.
and that was part of the problem. the other lab was doing a procedure of "grounding" the bags before getting a sample.
turns out that the plastic polymers had a chromophor in them that after several of those cycles and from general aging started to brake away and got into the sample.
chromophor intensity was very big and the actual amount was in the ppb.
they took a new bag put it in front of a television screen for 24 hours and showed that the mystery peak was from brokendown material in the bag

we never know from where it might come

[vickig]

Similarly, we have seen ink from product labels leach through polypropylene packaging to contaminate a product.

[mingqin]

There may be an interaction between benzyl alcohol and container materials (if it is plastic). The problem is very common. I suggest that you check the concentration of benzyl alcohol to see if it is constant. The degradation of benzyl alcohol is fast in some cases. I had the problem before.