How to identify peaks observed by UV-HPLC in stability studies

HPLC analysis of process related impurities by reverse phase mass spectrometry ( LC-MS analysis )
Alphalyse
Jan 24. 2018

Question:

We have developed a Reversed-Phase (RP) UV- HPLC chromatography method. It is used for stability studies of our new pharmaceutical protein.

How do we identify the peaks observed by UV to confirm they are product-related impurities? Can we ensure that UV-peaks contain single molecules?

 

Answer:

The best way I know of is to connect the HPLC directly to a mass spectrometer (LC-MS). This provides a simple characterization of peaks observed by UV-HPLC. By doing this, you have an additional detection method that can distinguish molecules by their molecular mass. – Which is perfect for stability studies.

You probabably already know that sometimes it is not possible to separate degradation products by RP chromatography alone. However, combining HPLC analysis and LC-MS analysis makes it quite easy to use molar mass to identify degradation products. For example deamidations, oxidation, and truncated molecules.

Another benefit I’d like to highlight of combining HPLC analysis and Mass Spectrometry analysis is that you can confirm that single peaks only contain one molecule.

 

Combine HPLC with LC-MS  >>

 

The problems of using Reversed-Phase HPLC analysis with UV detection alone is:

  • It is not always possible to separate the drug substance (DS) from product-related impurities.
  • You might see a shoulder. But you have no way of knowing if it is an artifact or a DS related molecule (degradation product).
  • New peaks appearing in the chromatogram cannot be characterized directly by UV detection.

 

Fortunately, there’s a simple solution

Coupling an HPLC instrument directly to a high-resolution mass spectrometer provides you with an additional detection level for product-related impurities.

Reversed-phase chromatography typically uses buffers that are directly compatible with mass spectrometry. Therefore it is quite straight-forward to connect the two instruments.

 

So when should you use LC-MS analysis in stability studies?

You can apply LC-MS analysis to all types of biomolecules. Hence, both synthetic peptides, recombinant proteins and antibodies. Therefore, I suggest you use the analysis to confirm product-related impurities whenever your chromatogram shows an unexpected peak.

In most cases, you will find that the LC-MS signal is much more sensitive than UV detection. In addition, you will see that the accuracy of MW determination for all types of molecules is in the low ppm range.

This enables confident identification of the molecule.

 

Learn more about combining your UV-HPLC method with Mass Spectrometry  >>

About Alphalyse

Alphalyse uses expertise in the field of protein chemistry and bioinformatics, combined with top-of-the-line mass spectrometry equipment, to provide protein analysis services globally.

Alphalyse aims to deliver high quality data and customer service to all of our clients in a fast and convenient way.

www.alphalyse.com

Newsletter Subscribe to The Alphalyse Newsletter and get more
information on protein analysis methods and applications
© 2019 Alphalyse, Inc.