Forced degradation

[addy]

Is there any scope for reduction test of compound as we do oxidation (In 30 % H2O2) in forced degradation study in validation.

[reyoungs]

You do not provide much detail in your question, so I will provide some general guidelines and see which part helps answer your question, if any.

Guidelines on the amount of degradation to look for:
Product is unmarketable beyond 10 degradation, so that becomes a target. Typically many people try to achieve 10-30% degradation to challenge a method. You don't want to go past this range much, because you will be generating degradants of degradants, which are truly unlikely to ever appear in your product, will have very different chromatographic behavior, and may make your method look worse than it really is.

Guidelines on the oxidant:
30% peroxide is generally a very strong oxidant. Most people work with 3% peroxide for one to eight hours at RT and find that they can achieve the target degradation with this (or even more dilute oxidant). Some molecules are very resistant to oxidation, such as flurbiprofen. I would stay away from bleach or free radical oxidants unless you are specially looking for strange oxidation effects. You are typically trying to simulate the types of stress the product may see during the normal product distribution lifetime, and these don't meet that criteria.

Guidelines on quenching:
With oxidation stress, it is important to quench the reaction after the time point, so that you stop the reaction from continuing while you are testing the samples. Sodium bisulfite or sodium thiosulfate are a good reducing agents that won't interfere with subsequent HPLC analyses, with the bisulfite best. Once you define your oxidation conditions, you can calculate an equimolar amount of the quenching agent and put it in (as a solid, if you have an aqueous mixture that will allow it to dissolve) at the designated time and your sample should stay stable. You generally don't have to worry about having a little extra bisulfite, because it is such a weak reducing agent that it is unlikely to do anything to your molecule. (Run a control if you are concerned.)

I hope that this helps. If not, please be more specific in what you are asking and we can try to be more specific in our response.

[Santosh Gandhi]

Hello Mr.Reyoungs,
I want to ask you a question regrading forced degradation, I found that a compound was so resistant to base degradation that even after using 5N NaOH at RT and even after heating the sample solution to 50°C for 15mins the degradation was minimal. In such cases what is the alternative. Can I use some other reagents like triethylamine and rise the sample solution pH to higher side (say around pH=11.0) and allow it to stand at RT and then analyse.
Thank You,
Santosh

[adam]

Reyoungs: Can you tell me exactly what is your procedure for quenching the reaction. How much bisulfite do you add? I have heard that Methionine can also be used for this purpose. Have you heard of this?

Santosh: In a situation like you describe you do not want to keep "beating the molecule to death" to get it to degrade. If you do this you will generate degradation products that you will never see in the actual course of the drug products shelf life. So there would be no point to that. Getting 10-30% degradation is one rule of thumb. The other is to apply stressing conditions that mimick what the drug would encounter in its shelf life - and not to exceed this by too much.