Every synthetic peptide sample contains small amounts of molecules that are almost, but not quite, the target. These are called peptide-related impurities. They are the small peaks around the main peak on an HPLC chromatogram, and they are the reason a purity figure exists at all. This guide explains where they come from, what they look like and how laboratories find them.
Why They Matter in Research
A peptide-related impurity is not inert filler. Because it closely resembles the target, it may bind the same targets, interfere with an assay or behave differently in a cell model. A widely cited review of impurities in peptide medicines makes the point that such impurities can strongly influence early functional studies and lead to wrong conclusions (D'Hondt et al. 2014). For a research lab, knowing what might be in the vial is part of interpreting the result.
Where Synthetic Peptides Come From
Most peptides are made by solid-phase peptide synthesis (SPPS). The method was introduced by R. Bruce Merrifield at the Rockefeller University in the early 1960s; he received the 1984 Nobel Prize in Chemistry for developing chemical synthesis on a solid matrix (Nobel Prize 1984). In SPPS the growing chain is anchored to an insoluble resin bead. Each cycle removes a temporary protecting group from the end of the chain, couples the next protected amino acid, and washes away excess reagents. Merrifield's 1969 review describes how this approach made the synthesis of biologically active peptides and small proteins practical (Merrifield 1969).
Today the dominant chemistry is Fmoc/tBu: the temporary Fmoc group is removed with a base, and at the end a strong acid, trifluoroacetic acid (TFA), cleaves the finished peptide from the resin and removes the permanent side-chain protecting groups (Sikora et al. 2018). Each of these steps is very efficient, but a peptide of 15 residues passes through dozens of them, and small inefficiencies add up.
Family 1: Synthesis-Related Impurities
| Impurity type | How it forms | What it looks like analytically |
|---|---|---|
| Deletion sequence | A deprotection or coupling step is incomplete, so one residue is skipped | Mass lower than target by one residue mass |
| Insertion sequence | Excess activated amino acid adds a residue twice | Mass higher by one residue mass |
| Truncated sequence | Chain growth stops early, often after deliberate capping of unreacted chains | Shorter peptide, much lower mass |
| Diastereomer (racemized form) | A residue flips from the L- to the D-form during activation or deprotection | Same mass; separated only by chromatography |
| Protection adduct | A side-chain protecting group is not fully removed | Mass higher by the protecting group |
| Side-chain reaction product | An unprotected side chain reacts with a synthesis reagent | Mass shift depends on the reagent |
D'Hondt and colleagues link deletions to inefficient Fmoc removal and insertions to excess amino acid reagent, and note that Fmoc removal conditions can also cause racemization (D'Hondt et al. 2014).
Aspartimide: a sequence-specific side reaction
Aspartic acid residues are a known weak point in Fmoc chemistry. Under the basic conditions used to remove Fmoc, the side chain of aspartic acid can cyclize with the backbone to form an aspartimide. That ring can reopen in more than one way, giving the normal peptide, a β-linked (isoaspartyl) form, or racemized forms, all with very similar properties. A 2025 review describes it as a persistent problem and surveys protecting-group and backbone strategies to suppress it (Kong et al. 2025). Sequences with Asp, especially Asp followed by a small residue such as glycine, are watched closely.
Family 2: Degradation-Related Impurities
These form after synthesis, during purification, lyophilization or storage.
- Oxidation. Methionine is readily oxidized to methionine sulfoxide (a gain of 16 Da). Tryptophan, tyrosine, histidine and cysteine are also sensitive, especially under light; singlet oxygen reaches Trp, Tyr, His, Cys and Met (Pattison et al. 2012).
- Deamidation and isomerization. Asparagine, and more slowly aspartic acid, can form a cyclic succinimide that reopens to aspartyl and isoaspartyl forms and can racemize. In a model peptide, an Asn-Gly sequence deamidated with a half-life of about 1.4 days at 37 °C and pH 7.4, while the Asp version formed the succinimide about 34 times more slowly; replacing the glycine with a bulky residue slowed degradation 33- to 50-fold (Geiger & Clarke 1987). Deamidation adds about 1 Da, which high-resolution MS can resolve.
- Pyroglutamate. An N-terminal glutamine or glutamic acid can cyclize, losing ammonia or water.
- Diketopiperazine. The first two residues at the N-terminus can cyclize and cleave off as a small ring, leaving a shortened peptide.
- β-elimination and dimers. Some side chains can eliminate, and peptides can form dimers or larger aggregates.
D'Hondt and colleagues group β-elimination, diketopiperazine, pyroglutamate and succinimide formation as the main degradation mechanisms in peptide products (D'Hondt et al. 2014). Stability reviews of protein and peptide products describe the same chemistry and show that it runs fastest in solution and at higher temperature (Manning et al. 2010).
Other Things in the Powder That Are Not "Peptide-Related"
- Counter-ions such as trifluoroacetate, which come from cleavage and from HPLC mobile phases (Sikora et al. 2018). Excess TFA can remain in the lyophilizate.
- Residual solvents such as acetonitrile, dimethylformamide and dichloromethane, all classed as Class 2 solvents in ICH Q3C (ICH Q3C(R9)).
- Water retained after freeze-drying.
- Unrelated peptides, which D'Hondt and colleagues found in some products and attributed to poor manufacturing practice (D'Hondt et al. 2014).
These do not appear as peptide-related peaks in an HPLC purity figure, which is why content and contaminant tests exist alongside purity.
How Laboratories Find Them
- Reversed-phase HPLC with UV at about 214 nm. Separates species by hydrophobicity and sees almost every peptide species, because the peptide bond absorbs there (Aguilar & Hearn 1996).
- Mass spectrometry. Assigns masses to peaks. A deletion, an oxidation (+16 Da) or a deamidation (+1 Da) each leaves a recognizable mass signature. Electrospray made routine MS of peptides possible (Fenn et al. 1989).
- High-resolution LC-MS. Can quantify impurities that co-elute with the main peak. An FDA laboratory reported detecting impurities below 0.1% of the main compound with this approach (Zeng et al. 2015).
- Orthogonal methods. A second separation with different chemistry, so that an impurity hidden in one method is revealed by another. FDA reviewers ask for this in synthetic peptide drug submissions (FDA SBIA presentation, 2022).
Diastereomers are the hardest case: same mass, nearly the same chromatography. They usually need a method specifically developed to separate them.
How Small Is Small? A Regulatory Reference Point
Research materials are not drug products, but drug rules show how seriously small impurities are taken. For certain synthetic peptide drug products, FDA's 2021 guidance asks applicants to identify each peptide-related impurity at or above 0.10%, and regards a new impurity above 0.5% as generally not acceptable without further justification (FDA SBIA presentation, 2022; Federal Register 2021). Notably, the general ICH impurity guideline for new drug substances, Q3A(R2), excludes peptides from its scope altogether (ICH Q3A(R2)), which is one reason peptide-specific guidance exists.
Reading Impurities on a Report
- A purity figure tells you the main peak's share. The remainder is a mix of the impurity types above.
- If the chromatogram is shown, look for peaks close to the main peak; those are the near-copies.
- If an LC-MS table lists impurity masses, compare them with the target mass: −1 residue, +16, +1 and similar shifts point to specific types.
- A report that lists only a purity number answers fewer questions than one that also shows the trace and the masses.
Every Peptide Privé batch is tested by an independent lab. See also How to Read a Peptide Analytical Report and Storing Lyophilized Peptides in the Lab.
Sources
- D'Hondt M et al. Related impurities in peptide medicines. J Pharm Biomed Anal. 2014. PubMed 25044089: https://pubmed.ncbi.nlm.nih.gov/25044089/
- Merrifield RB. The synthesis of biologically active peptides and proteins. JAMA. 1969. PubMed 5394648: https://pubmed.ncbi.nlm.nih.gov/5394648/
- Nobel Prize in Chemistry 1984, summary: https://www.nobelprize.org/prizes/chemistry/1984/summary/
- Nobel Prize in Chemistry 1984, Bruce Merrifield facts (affiliation, 1960s method): https://www.nobelprize.org/prizes/chemistry/1984/merrifield/facts/
- Sikora K et al. Counter-ion effect on antistaphylococcal activity and cytotoxicity of selected antimicrobial peptides. Amino Acids. 2018. PubMed 29307075: https://pubmed.ncbi.nlm.nih.gov/29307075/
- Kong MJW, van den Braak TJHP, Neumann K. Aspartimide Formation and Its Prevention in Fmoc Chemistry Solid Phase Peptide Synthesis. ChemBioChem. 2025. PubMed 40857621: https://pubmed.ncbi.nlm.nih.gov/40857621/
- Pattison DI, Rahmanto AS, Davies MJ. Photo-oxidation of proteins. Photochem Photobiol Sci. 2012. PubMed 21858349: https://pubmed.ncbi.nlm.nih.gov/21858349/
- Geiger T, Clarke S. Deamidation, isomerization, and racemization at asparaginyl and aspartyl residues in peptides. J Biol Chem. 1987. PubMed 3805008: https://pubmed.ncbi.nlm.nih.gov/3805008/
- Manning MC et al. Stability of protein pharmaceuticals: an update. Pharm Res. 2010. PubMed 20143256: https://pubmed.ncbi.nlm.nih.gov/20143256/
- Aguilar MI, Hearn MT. High-resolution reversed-phase HPLC of peptides and proteins. Methods Enzymol. 1996. PubMed 8803962: https://pubmed.ncbi.nlm.nih.gov/8803962/
- Fenn JB et al. Electrospray ionization for mass spectrometry of large biomolecules. Science. 1989. PubMed 2675315: https://pubmed.ncbi.nlm.nih.gov/2675315/
- Zeng K et al. Liquid Chromatography-High Resolution Mass Spectrometry for Peptide Drug Quality Control. AAPS J. 2015. PubMed 25716148: https://pubmed.ncbi.nlm.nih.gov/25716148/
- U.S. FDA, SBIA 2022 presentation on peptide-related impurities in synthetic peptide ANDAs: https://www.fda.gov/media/166572/download
- Federal Register notice of FDA guidance, ANDAs for Certain Highly Purified Synthetic Peptide Drug Products (May 20, 2021): https://www.federalregister.gov/documents/2021/05/20/2021-10603/abbreviated-new-drug-applications-for-certain-highly-purified-synthetic-peptide-drug-products-that
- ICH Q3A(R2) Impurities in New Drug Substances (2006): https://database.ich.org/sites/default/files/Q3A%28R2%29%20Guideline.pdf
- ICH Q3C(R9) Guideline for Residual Solvents (2024): https://database.ich.org/sites/default/files/ICH_Q3C%28R9%29_Guideline_MinorRevision_2024_2024_Approved.pdf
For in-vitro laboratory research only. Not for human or animal use.
