Peptide Impurities Explained: Deletions, Oxidation & Other Related Substances

For in-vitro laboratory research use only. Not for human or veterinary use.

Our guide to HPLC purity vs net peptide content explained what a purity percentage measures. This article looks at the other side of that number: what the remaining fraction of peaks usually is, where it comes from, and how laboratories tell the different types apart.

1. What counts as an “impurity”?

In peptide chemistry, an impurity is anything in the sample that is not the exact target molecule. The most important group is peptide-related impurities, sometimes called related substances. These are close cousins of the target: the same chain with one amino acid missing, one extra, or one chemically altered.

They matter because they are hard to separate and can behave a little like the target in an assay. A review by D’Hondt et al. (2014) noted that such impurities can influence early functionality studies and lead to wrong conclusions. Water and counter-ions are also present in a peptide powder, but they are not peptides and are covered in our counter-ions guide.

2. Impurities that come from synthesis

Most research peptides are made by solid-phase peptide synthesis (SPPS): the chain is built one amino acid at a time on a tiny resin bead (see our Science page). In the common Fmoc method, every amino acid arrives with temporary chemical “caps”, called protecting groups, that stop it reacting in the wrong place. Each cycle removes one cap, adds the next amino acid, then repeats (Behrendt et al., 2016). Every step works very well, but not perfectly, and over dozens of cycles small slips add up.

• Deletion sequences: the chain is missing one amino acid because a cap was not fully removed or a coupling step did not complete. In a study of research-grade peptides, Verbeke et al. (2015) found that most of the related impurities they detected were missing one or more amino acids.
• Insertion sequences: an extra copy of an amino acid, linked to excess reagent (D’Hondt et al., 2014).
• Truncated sequences: chains that stopped growing part-way. Many protocols deliberately “cap” these with an acetyl group so they are easier to remove during purification.
• Protecting-group adducts: a side-chain cap that was never removed, leaving a heavier molecule (for example, about +56 Da for a leftover tert-butyl group). A dalton (Da) is the unit used for molecular mass.
• Racemisation: amino acids are “left-handed” (L-form) in nature. During synthesis a residue can flip to its mirror image (D-form). The result has exactly the same mass, so it is invisible to a simple mass check.
• Aspartimide formation: aspartic acid can fold back on itself to form a ring, especially next to glycine. This side reaction is a well-known challenge in Fmoc chemistry (Behrendt et al., 2016).

3. Impurities that form over time

Even a well-made peptide slowly changes if it meets water, oxygen, heat or light. These degradation products are one reason peptides are supplied freeze-dried and stored cold and dry (see our reconstitution and storage guide).

• Oxidation: the sulphur atom in methionine (and, in other ways, cysteine and tryptophan) picks up oxygen. One extra oxygen adds about +16 Da.
• Deamidation: the side chain of asparagine (or, more slowly, glutamine) loses its amide group and becomes an acid, adding about +1 Da. Geiger and Clarke (1987) showed that in the test peptide Val-Tyr-Pro-Asn-Gly-Ala, held at 37 °C and pH 7.4, this happened with a half-life of only 1.4 days. It went through a ring-shaped intermediate called a succinimide and produced a mixture of normal and “iso” forms, plus mirror-image versions.
• Sequence matters: in the same study, swapping the glycine after asparagine for a bulkier leucine or proline slowed degradation 33- to 50-fold, and using aspartic acid instead of asparagine slowed ring formation 34-fold.
• Dimers and aggregates: two or more chains can stick or bond together, producing larger species (D’Hondt et al., 2014).

4. How laboratories detect them

HPLC: separating the peaks

High-performance liquid chromatography (HPLC) pushes the sample through a packed column. Slightly different molecules travel at slightly different speeds and reach the detector at different times. A UV detector set near 214 nm, where the peptide bond absorbs light, draws each one as a peak. Purity is the main peak’s area as a share of all peak areas. If an impurity travels at the same speed as the target (it co-elutes), it hides inside the main peak.

Mass spectrometry: weighing the molecules

Mass spectrometry (MS) measures molecular mass very precisely. Because each impurity type shifts the mass in a typical way (−57 Da for a missing glycine, +16 Da for oxidation), MS paired with HPLC (LC-MS) can suggest what each small peak is. De Spiegeleer et al. (2008) used this approach to compare samples of the same research peptide from five manufacturers. One product was a different peptide entirely, and two-thirds of the others fell below 95% purity and/or had individual impurities above 1%. For a walkthrough of both methods, see Laboratory Testing Explained.

5. What this means for your records

• Read the purity figure for what it is: the main peak’s share at the stated wavelength, not a full list of contents.
• Check which tests were run: most certificates in the Corex Vitality™ COA directory report HPLC purity; the MOTS-c 10mg certificate also includes LC-MS data. Our COA verification guide explains how to confirm a report with the laboratory.
• Match batch numbers: record the batch or report number with every experiment, so any unexpected result can be traced.
• Store with degradation in mind: keep vials cold, dry, sealed and away from light, as these conditions slow oxidation and deamidation.

Key takeaways

• Peptide-related impurities are near-copies of the target peptide, not random contaminants.
• Synthesis produces deletions, insertions, truncations, leftover protecting groups and racemised residues.
• Storage conditions drive slow changes such as oxidation, deamidation and aggregation.
• HPLC separates and measures peaks; mass spectrometry helps work out what each one is, except for same-mass changes.
• Browse our research peptides and check each batch’s certificate before you start. More topics are on the Learning Hub, starting with What Are Peptides?

Published Research

The peer-reviewed papers below were used for the chemistry in this article. Each entry was checked against PubMed. Summaries describe laboratory and analytical findings only. Corex Vitality™ is not affiliated with the cited authors, journals or institutions.

1. Related impurities in peptide medicines. D'Hondt M, Bracke N, Taevernier L, Gevaert B, Verbeke F, Wynendaele E, De Spiegeleer B. J Pharm Biomed Anal. 2014;101:2-30. PMID: 25044089 · DOI: 10.1016/j.jpba.2014.06.012
   Structured review of peptide-related impurities from solid-phase synthesis and degradation (deletions, insertions, racemisation, protecting-group adducts, oxidation, aggregates and counter-ions) and their effect on early in-vitro functionality studies.
2. Advances in Fmoc solid-phase peptide synthesis. Behrendt R, White P, Offer J. J Pept Sci. 2016;22(1):4-27. PMID: 26785684 · DOI: 10.1002/psc.2836
   Review of Fmoc solid-phase peptide synthesis chemistry, including protecting-group strategy and side reactions such as aspartimide formation that generate synthesis-related impurities.
3. Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. Succinimide-linked reactions that contribute to protein degradation. Geiger T, Clarke S. J Biol Chem. 1987;262(2):785-94. PMID: 3805008
   In-vitro kinetic study of synthetic model peptides showing succinimide-mediated deamidation, isomerisation and racemisation at asparagine and aspartic acid residues, and how neighbouring residues change the rate.
4. Impurity profiling quality control testing of synthetic peptides using liquid chromatography-photodiode array-fluorescence and liquid chromatography-electrospray ionization-mass spectrometry: the obestatin case. De Spiegeleer B, Vergote V, Pezeshki A, Peremans K, Burvenich C. Anal Biochem. 2008;376(2):229-34. PMID: 18342612 · DOI: 10.1016/j.ab.2008.02.014
   Analytical study using LC with photodiode-array/fluorescence detection and LC-ESI-MS to profile identity and related impurities in research samples of one synthetic peptide from five manufacturers.
5. Quality evaluation of synthetic quorum sensing peptides used in R&D. Verbeke F, Wynendaele E, Braet S, D'Hondt M, De Spiegeleer B. J Pharm Anal. 2015;5(3):169-181. PMID: 29403929 · DOI: 10.1016/j.jpha.2014.12.002
   Analytical quality-control study of synthetic quorum sensing peptides used in research, characterising purity and the nature of related impurities, most of which lacked one or more amino acids.

All Corex Vitality™ products are supplied strictly for in-vitro laboratory research use only. Not for human or veterinary use. No information here describes use in humans or animals.