Developing a Stability-Indicating Method: Forced Degradation

At Velesco, we are occasionally asked to explain the process of developing a stability-indicating HPLC method.  One very important step in determining whether a method is stability-indicating is to perform Forced Degradation experiments.  Here, I will explain these experiments and how we approach the process.

It’s important to challenge your analytical method for its ability to accurately quantitate the active ingredient without interference from degradation products. Forced degradation experiments can be conducted to deliberately degrade your sample in order to see where the degradation peaks elute in your HPLC method. These experiments are normally performed in early development and can help you understand the degradation pathways of your API as well as helping you to develop a stability-indicating method.

The main pharmaceutically relevant degradation mechanisms are thermal, hydrolysis, oxidation, and photolysis. By exposing your sample to extreme conditions such as heat, light, a wide pH range and oxidizing agent, you can produce partially degraded samples which can then be analyzed by your HPLC method to determine if there is any interference with the active or related compound peaks. The goal is to degrade the API around 10%. Anything over 30% leads to questions of secondary degradation, so keeping the degradation below this amount is recommended.

Conditions that we tend to use as a starting point are:

Sample:   Exposure Condition

Control:   Solid state, RT

Thermal:   Solid state, 70°C

Hydrolysis:   Water, heat

Acid:   0.1N HCl, heat

Base:   0.1N NaOH, heat

Oxidation:   0.3% H2O2, 250 w/m2, 30 min

Photodegradation:   Solid state, 250 w/m2, 48 hrs

Samples are usually prepared between 0.5 mg/mL – 1 mg/mL in the above vehicles and are stored at high temperature (~70°C) to promote degradation. Some organic may be used to help dissolve the compound into solution. A blank is also prepared for each of the exposure conditions in order to compare the background to the degraded sample. Leaving the samples at these extreme conditions for 24 to 48 hours is often enough to see adequate degradation. API stored at room temperature is used as a control and should be prepared in diluent on the day of HPLC analysis. Be sure to quench (typically by dilution) the peroxide and neutralize the acidic and basic solutions prior to injection.

To demonstrate selectivity of your HPLC method, degradation product peaks should be well separated from the active ingredient, ideally with a resolution factor of at least 1.5. Alterations to your HPLC method (column temperature, gradient, mobile phase, column type, or a combination of these) may be needed to obtain adequate separation. Once your API is separated from all potential degradation peaks, you are well on your way to an analytical method that is specific and stability-indicating.

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