Case Study #5

LC Method Development for a Complex Mixed Product

The Problem:
This company had a product that consisted of a small molecule and a peptide mixture, along with other, non-critical components. A method was needed to confirm the amounts of each component in the formulation.

An existing LC method suffered from the following problems:

• The method specified a special use column that cost $1,110.
• The mobile phase required the use of trifluoroacetic acid (TFA).
• The separation of the two possible small molecule isomers was difficult.
• The method did not include any procedures for identifying and quantifying the peptide fraction.

A representative chromatogram is shown below.

The Solution:
ACCTA, Inc. conducted a thorough study of numerous column options from our list of preferred phases, and then identified conditions that allowed the quantification of both the peptide and small molecule.

The Results:

An efficient method was developed that easily separated the small molecule isomers and produced several characteristic peaks for the peptide mixture. The method even allowed quantification of a second peptide mixture that was present in other formulations.

The final procedure offered the following advantages:

• The column contained a conventional reversed phase-type packing, available from a major manufacturer for less than $400.
• The mobile phase did not require TFA, but used the less expensive and less hazardous phosphoric acid.
• The separation of the small molecule isomers was greater on this column than the original specialty column and in less time.
• Both the small molecule and peptide could be quantified in the final formulations.

See the difference in the following chromatogram.

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