Validation, and Use of Early Phase Methods

Following the development of a candidate separation method there are many method factors, such as detection wavelength, sample 

FIGURE 8 Critical regions in a chromatogram to evaluate ( I) co-elution of impurities, (2) highly retained compounds (apolarity issue), (3) non-retained compounds (polarity issue), and (4) co-elution with the API (isomers). 

FIGURE 9 Use of non-aqueous conditions to separate a highly retained compound (x).The rest of the chromatogram remains comparable. 

The ultimate goal of an assay method is the separation and visualization of all components in a single chromatogram. Proper selection of detection wavelength is a critical part of method development. When choosing a detection wavelength, the following factors need to be taken into consideration  

A diode array detector is well suited to achieve these goals. Full range spectra should be collected for the method development samples and evaluated with two-dimensional or three-dimensional visualization to determine the best detection wavelength. 

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