Three orthogonal chromatography methods map impurities and newly reported oligomers in a 20-amino-acid peptide

A paper published online in October 2026 in the Journal of Pharmaceutical and Biomedical Analysis sets out three complementary liquid-chromatography methods for profiling the impurities of a synthetic peptide of 20 amino acids. The authors, Andreja Vukoja, Ema Pavić, Sanja Galović and Nives Galić, report what they describe as the first detection and characterisation of oligomers of this peptide, dimers and trimers, using size-exclusion chromatography.

Why one method is not enough

The authors note that peptides are complex molecules that can carry complex impurity profiles. Before any lab work, they reviewed the peptide's sequence for weak points: sites that could fragment, lose an amide group (deamidation), oxidise, or link into covalent oligomers under stress. Each of those products behaves differently on a column, which is why they developed three separation modes rather than relying on one.

• Size-exclusion chromatography (SEC) separates molecules by size and was used to detect and characterise the dimers and trimers.
• Reversed-phase (RP) chromatography and hydrophilic interaction chromatography (HILIC) together separated 10 non-oligomeric impurities.

All three ultra-performance (UPLC) methods use mobile phases that are compatible with mass spectrometry, so the separated degradation products could be identified directly by high-resolution mass spectrometry.

How the samples were stressed

To generate degradation products, the team applied forced-degradation conditions set out in ICH guidelines: acid and base hydrolysis, oxidation with hydrogen peroxide, heat and light exposure. Method development followed Analytical Quality-by-Design principles, with risk assessment using Ishikawa diagrams and heat maps and a Design of Experiments approach. The final methods were validated as suitable for analysing the model peptide and its related impurities.

What it adds to existing methods

According to the paper, few published methods address this peptide's degradants and related substances, and the authors found no prior data on methods for its oligomeric impurities. They note that one earlier method relied on a non-MS-compatible ion-pairing reagent, which can make identifying unknown impurities harder. They also note that the current USP monograph does not list oligomeric species as potential impurities. The team used the peptide as a model and states that the approach can be applied to other oligopeptides.

Why it matters for peptide research

The study is a reminder that a single purity number reflects the method behind it. Impurities that co-elute in one chromatographic mode, or that are larger aggregates, can be missed unless a second or third orthogonal method and mass-spectrometry identification are used. For labs reading analytical data on research peptides, it is useful context for what a purity figure on a certificate of analysis can and cannot show.

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