Common impurities in synthetic peptides and how labs detect them

Prepared by: Research Desk. Sources are linked for verification.

Published 21 October 2026

Why impurities appear

Merrifield's 1963 paper introduced solid-phase peptide synthesis (SPPS). In SPPS the growing chain is attached to an insoluble resin and built through repeated deprotection and coupling steps. Excess reagents and by-products can be washed away between steps. The approach later enabled automated peptide production (Merrifield, 1963, bibliographic record).

The following is general background rather than a claim drawn from a specific source. When a step does not run to completion, or a side reaction occurs, the crude product contains the target peptide alongside closely related molecules. Purification aims to reduce these, and the analytical tests described below are how laboratories look for what remains.

The wider workflow is covered in the guide to how research peptides are made and tested.

The main impurity types

Deletion and truncated sequences. A deletion sequence lacks one or more amino acids because a coupling or deprotection step failed. A truncated sequence stopped growing early. Both are chemically very similar to the target, and UV detection alone may not distinguish them from it. A review of related impurities in peptide medicines lists the main types as deletion and insertion sequences, incomplete deprotection, oxidation, deamidation and diastereomers (D'Hondt et al., 2014, PubMed).

Racemisation and epimers. During activation or coupling, a residue can partly convert to its mirror-image form. The result is an epimer with the same nominal mass as the intended peptide. Because the mass is unchanged, MS alone cannot distinguish some epimers, and chromatographic separation is needed as evidence.

Aspartimide formation. This side reaction involves aspartic acid, and how far it occurs depends on the sequence and the reaction conditions. Subsequent products can include isoaspartate (isoAsp) variants and epimerised forms.

Oxidation. Methionine, tryptophan and cysteine are commonly affected. Oxidation can produce characteristic mass shifts, but interpreting those shifts still needs chromatographic or other orthogonal evidence.

Residual TFA, solvents and water. Trifluoroacetate (TFA) is often left over from purification as a counterion; acetate is another common counterion. Weighed material can also contain water, residual solvent and salts. These do not register in the same way as the peptide on a UV chromatogram, so a sample can show high chromatographic purity but a lower peptide content by weight. This difference is explained in HPLC purity versus peptide content.

Aggregation. Aggregation, in which peptide molecules associate into larger species, is a further quality attribute sometimes discussed for peptides. The sources used for this page do not describe how aggregates are detected or set limits for them, so it is noted here without further detail.

Which method detects what

UV detection near 214 nm is common because peptide bonds absorb strongly there; 220 nm is also used. A reported area-percent purity is the share of detected UV peak area assigned to the main peak. It is not necessarily the peptide's mass fraction or total chemical content.

In short, HPLC supports purity and MS supports identity. A report that shows only one of the two leaves a gap.

The FDA 2021 thresholds and their limited scope

The figures 0.10% and 0.5% come from a May 2021 FDA guidance on abbreviated new drug applications (ANDAs) for certain highly purified synthetic peptide drug products that refer to listed drugs of recombinant-DNA origin (FDA guidance text). Its scope is narrow: glucagon, liraglutide, nesiritide, teriparatide and teduglutide.

For those products, the guidance text states the following:

• The FDA generally recommends identifying peptide-related impurities at 0.10% or more of the drug substance.
• A new specified impurity above 0.5% is described as not acceptable for that approach.
• New impurities from 0.10% through 0.5% are to be characterised and justified, including consideration of safety, effectiveness and immunogenicity risk.
• Impurities also present in the reference listed drug should be at a level no higher than in the reference product.

The FDA has also published a summary of this guidance and related product-specific guidances (FDA summary), and an FDA presentation on common deficiencies repeats these points (FDA presentation).

These thresholds are not a universal specification for synthetic peptides, and they are not a standalone release limit. They do not automatically apply to research-use materials.

Reading a COA with impurities in mind

A more informative certificate of analysis includes:

• The HPLC method, detection wavelength and conditions, with the chromatogram attached.
• The expected and observed mass.
• The peptide content and the basis on which it was calculated.
• The identity of the counterion.
• Water and solvent results where relevant.

A COA covers only the tests it lists and only the sample tested. Whether a method can separate the relevant impurities is a matter of validation; the ICH Q2(R2) guideline on validating analytical procedures, listed on the ICH quality guidelines page, describes what that involves. Analysis of seized falsified peptide products has found impurity profiles that the accompanying paperwork did not reveal (Janvier et al., 2018, PubMed).

Sources

• Merrifield, JACS (1963): bibliographic record
• D'Hondt et al., 2014: PubMed
• Janvier et al., 2018: PubMed
• FDA, ANDAs for certain highly purified synthetic peptide drug products (2021): guidance text
• FDA summary of synthetic peptide guidance: download
• FDA presentation on common deficiencies: download
• ICH quality guidelines: ICH

Supplier documentation

Purita is a store that sells peptides for laboratory research use only. It publishes a certificates page where reports are available for some products; reports are not yet available for every item, so check the page for the specific material.

Purita certificates page

Affiliate disclosure: this is a paid link. DesignPeptide earns a commission if a purchase is made through it, at no extra cost to the buyer. A listing is not an endorsement or a statement about product quality. See the affiliate disclosure.

Related pages

• Peptide design, synthesis and testing, explained
• About DesignPeptide
• How research peptides are made and tested: from synthesis to COA
• HPLC purity vs net peptide content: two numbers that are often confused