Validation Challenges for 'Area Percent' Methods

Analytical methods which use area normalisation, or ‘area percent’, as a calibration model are common in the analysis of biomolecules by chromatography and electrophoresis. They present particular challenges when it comes to validation.

The approach is to assign a value of 100% to the area of all the peaks in a chromatogram or electropherogram and then quantify individual peaks as a percentage of that total. Although a very simple idea, the reality is that these methods can be quite complex. In particular, the interpretation of what the percentage value assigned to each peak actually means. If a correlation between this value and the amount of the component present in the sample for analysis is desired then it has to be assumed that the size of the peaks relates to how much is actually there.

This may not actually be the case. For example, if using the popular detector of UV, then the size of the peaks will relate to not just how much is there but also the type of chromophores present in the molecules. Additionally, an area percent approach assumes that there is a linear correlation from zero to 100% which allows each peak to be expressed as a percentage, this may not be the case if the size of the peaks are not within the dynamic linear range of the detector.

When it comes to method validation, it is desirable to address these assumptions and demonstrate that they are justified and this may be achieved if there are reference materials for the components in the sample. However, often the reason for using this approach is that external reference materials for the sample components separated by the method are not available. In this case method validation is challenging.

If you want to learn more about validating methods then you may be interested in the following courses from Mourne Training Services Ltd:

Validation and Transfer of Methods for Biopharmaceutical Analysis (applied to large molecules)

Validation and Transfer of Methods for PharmaceuticalAnalysis (applied to small molecules)