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What are the impurities in synthetic peptides? A COA map
Synthetic peptide impurities by class, from deletion sequences to TFA counterions, and which COA line, HPLC purity or MS mass, can reveal each one.
The impurities in synthetic peptides fall into two groups. Synthesis-related impurities are deletion and insertion sequences, racemized diastereomers, protection adducts, side-chain reaction products, oxidation, dimers and oligomers, trifluoroacetate counterions and unrelated-peptide contamination. Degradation-related impurities come from beta-elimination, diketopiperazine, pyroglutamate and succinimide formation. Finished drug products can carry a third group.
This is an evergreen explainer; the classes follow from synthesis chemistry. The list follows the 2014 review by D’Hondt and colleagues at Ghent University’s DruQuaR group in the Journal of Pharmaceutical and Biomedical Analysis, whose abstract this desk read on 18 October 2026. That review concerns peptide medicines. A research-supply certificate is not a drug dossier, and nothing here implies research material is made to drug GMP.
What are the impurities in synthetic peptides, line by line on a COA
| Impurity | Origin, per D’Hondt et al. | HPLC purity figure | MS observed mass |
|---|---|---|---|
| Deletion sequence | Inefficient Fmoc deprotection | Excluded only if separated | Lighter than target |
| Insertion sequence | Excess amino acid reagent | Excluded only if separated | Heavier than target |
| Diastereomer | Racemization during Fmoc deprotection | Can co-elute under some conditions | Same mass; cannot show it |
| Protection adduct | Incomplete side-chain deprotection | Excluded only if separated | Retained group adds mass |
| Side-chain reaction product | Unprotected side chains meeting reagents | Excluded only if separated | Mass shifts where atoms are added |
| Oxidation | Side-chain oxidation | Excluded only if separated | Added oxygen adds mass |
| Dimer or oligomer | Observed in SPPS product | Excluded only if separated | Near a multiple of target mass |
| Trifluoroacetate counterion | SPPS or later purification | Outside the figure | Not this line’s job; read the salt form |
| Unrelated peptide | Contamination; the authors cite lack of appropriate GMP | Excluded only if separated | Only if the spectrum is read beyond the target |
| Degradation products | Beta-elimination, diketopiperazine, pyroglutamate, succinimide | Excluded only if separated | Each mechanism changes composition |
Why the MS line cannot see a diastereomer
A racemized residue swaps one mirror form of an amino acid for the other. The composition is unchanged, so its mass is the target’s mass. Identity by mass is silent on this class. In Mant, Hodges and colleagues’ chapter on HPLC of peptides, L- and D-peptide mixtures separate at optimum temperatures and co-elute above and below them. A diastereomer under the main peak is counted as product. The site’s note on what MS confirms that HPLC purity cannot covers the rest.
Where the counterion goes
Trifluoroacetate is on D’Hondt’s impurity list but is not part of an HPLC purity percentage. It sits in the solid as salt, which is why 99% purity is not 99% peptide by weight and why the TFA and acetate salt lines deserve their own reading.
Questions readers ask
What is a deletion sequence in peptide synthesis?
A deletion sequence is a chain missing one or more of the intended amino acids. In solid-phase synthesis, D’Hondt and colleagues tie deletions to inefficient Fmoc deprotection: an amine left protected cannot take the next coupling. Because a residue is absent, the molecule is lighter than the target, which is why the MS line can flag it.
Can HPLC purity show every peptide impurity?
No. The purity figure is the main peak’s share of detector response, so it captures only impurities the method separates from the target. Mant, Hodges and colleagues show L- and D-peptide mixtures co-eluting at some column temperatures and resolving at others. Water and counterion sit outside the figure entirely.
Is trifluoroacetate an impurity in a peptide?
D’Hondt and colleagues list unwanted counter ions such as trifluoroacetate among SPPS-related impurities, arising from the synthesis or from later purification. It is not part of the HPLC purity figure; a COA that addresses it does so on a separate line, such as the salt form. It adds mass to the powder without being peptide.
None of this concerns what any peptide does. The materials on these certificates are supplied for laboratory research use only, and this desk reads a COA for what each line documents.