Separation of some polypeptide hormones by high-performance liquid chromatography

Area of Science:

• Analytical Chemistry
• Biochemistry
• Chromatography

Background:

• Peptide hormones such as adrenocorticotropic hormone (ACTH), luteinizing hormone-releasing hormone (LH-RH), and insulins are crucial biomolecules.
• Accurate separation and purity assessment of these peptides are essential for research and clinical applications.
• Existing chromatographic methods may face challenges in resolving closely related peptide analogues.

Purpose of the Study:

• To develop and validate a novel reversed-phase chromatography (RPC) method for the separation of diverse peptide hormones.
• To investigate the influence of amino acid composition and sequence on peptide elution order.
• To establish a rapid technique for assessing peptide hormone purity and resolving complex peptide mixtures.

Main Methods:

• Utilized a commercial reversed-phase material with a mobile phase consisting of tartrate buffer-acetonitrile systems.
• Incorporated sodium 1-butanesulphonate and sodium sulphate as mobile phase additives.
• Separated and analyzed analogues of ACTH (n=21), LH-RH (n=3), and insulins (n=4).

Main Results:

• Successfully separated 21 ACTH analogues, 3 LH-RH analogues, and 4 insulins using the developed RPC method.
• Demonstrated that elution order is primarily governed by the hydrophobicity of constituent amino acid residues.
• Showcased the method's efficacy in distinguishing between closely related peptides, including diastereomers and positional isomers, based on overall molecular hydrophobicity.

Conclusions:

• The developed tartrate buffer-acetonitrile based RPC method provides effective separation of various peptide hormones.
• This chromatographic technique is highly valuable for rapid purity assessment of peptide hormones.
• The method facilitates the separation of closely related peptides up to approximately 6000 molecular weight, offering significant analytical utility.