Help on: Assessing Peak Purity Data

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Question:

“I am developing a HPLC indicating assay for a hormonal drug. I am using the peak purity option to analysis the purity of my peak once the sample has been subjected to 20% degradation under various conditions. The purity results show that my peak is not pure although its 5 overlaid spectrums are more or less identical. The resolution of my drug from its degradation products is greater than 2. I need to know how I can accurately interpret my peak purity results in order to ensure that no other degradation product is co-eluting at the same time as my main peak. I would appreciate any suggestions with regards to this matter.”

Answer:

"I assume that the results you are referring to are from the software that you are using for peak purity analysis. The purpose of this software is to compare the spectra obtained at time points across the peak and detect differences which cannot be observed by eye. Therefore it is possible that there are spectral differences which may be explained by the presence of another peak eluting at a similar time as your drug. When a drug is degraded, the degradation products are often very similar in structure leading to similar retention times and UV spectra. This means that the spectral differences observed during peak purity analysis may be very small.

To investigate whether your purity result is actually due to another peak or is just a result of the way the spectra have been compared by the software requires knowledge of how the purity result was generated. I recommend that you read through any available information on how the software compares the spectra.

Things to consider:

• What reference spectrum is being used, particularly important if it is a gradient method;
• Compare the peak purity plot observed for your forced deg sample with that for a pure reference standard;
• Loss of linearity can result in spectral differences which are not due to another peak, try diluting your sample and compare the peak purity plot obtained with the original.

A final note, peak purity can never be proven. You can only gain confidence that no other peaks have been detected. This is because the UV spectrum for similar compounds may be identical, also if the apex of both co-eluting peaks is at the same time then the spectra across the peak are likely to be identical.”