Related substances on a certificate of analysis

Some certificates go beyond a single purity figure and list "related substances": the individual impurities the HPLC method detected, each with its retention time and amount. Reading that table well tells you more about a lot than the purity number alone. This article explains what related substances are, how they are reported, how the numbers fit together and what the conventions mean.

What "related substances" means

Related substances are impurities that are structurally related to the peptide: molecules that come from the same synthesis or form from the peptide itself. They include deletion and truncated sequences, incompletely deprotected forms, diastereomers, oxidized, deamidated and isomerized forms, and dimers. They are distinct from non-peptide components such as water, counter-ions and residual solvents, which are reported separately if at all.

Because related substances are peptides too, they absorb at 214–220 nm much like the main peptide, which is why HPLC area percentages are a reasonable measure of their amount.

How they are reported

A related-substances table usually lists each impurity peak above a reporting threshold, by its relative retention time (its retention time divided by that of the main peak), with its area percentage. It then gives the largest single impurity and the total impurities. An illustrative example:

Related substances (illustrative)

Peak	Relative retention time	Area %	Identity (if assigned)
1	0.87	0.42	Oxidized form (+16 Da)
2	0.96	0.31	Deletion sequence (−Gly)
Main peak	1.00	98.71	Intended peptide
3	1.08	0.38	Unassigned
4	1.21	0.18	Unassigned

In this example the largest single impurity is 0.42% and the total is 1.29%. The purity, 98.71%, and the total impurities add up to 100%, because both are shares of the same integrated chromatogram.

Thresholds

Impurity reports use three kinds of threshold: a reporting threshold, above which a peak is listed; an identification threshold, above which its structure should be established; and a qualification threshold, above which its safety would be assessed for a medicine. The general ICH guideline on impurities in new drug substances (Q3A) excludes peptides. The European Pharmacopoeia sets its own thresholds for synthetic peptides in its general monograph on substances for pharmaceutical use: 0.1% for reporting, 0.5% for identification and 1.0% for qualification.

These thresholds are written for medicines, not research materials. They are still a useful reference for what a thorough impurity report looks like.

Identifying impurities

An impurity's mass, measured by LC-MS, usually points to its identity: +16 Da for oxidation, +1 for deamidation, the mass of one residue less for a deletion, the mass of a protecting group more for incomplete deprotection. Same-mass impurities, such as diastereomers and isoaspartate forms, need comparison with standards or other techniques. Mass match labels observed masses against a sequence; How synthetic peptides are made explains where each kind comes from.

Reading the table

• Check that purity and total impurities add up to about 100%; if not, ask how the figures were calculated.
• Look at the largest single impurity, not only the total: one 2% impurity is a different situation from twenty 0.1% impurities.
• Look for the reporting threshold; small peaks below it are not listed and not counted.
• Note which impurities are identified, and whether any that matter for your experiment, such as a truncated sequence that could compete in a binding assay, are present.
• Remember the method dependence: a different gradient or column can resolve more or fewer peaks. See Purity depends on the method.

Frequently asked questions

Why might a certificate show only a purity, without related substances?

A purity figure is the minimum report. A full related-substances table takes more work to prepare and is more common for well-characterized lots and reference materials.

What is a relative retention time?

An impurity's retention time divided by the main peak's. It stays more consistent between runs than the absolute time, which makes impurities easier to track across lots.

Are related substances included in net peptide content?

Largely yes, when content is measured by amino acid analysis, because related peptides hydrolyze to nearly the same amino acids. That is why content and purity are read together.

Sources & Further Reading

1. D'Hondt M, Bracke N, Taevernier L, Gevaert B, Verbeke F, Wynendaele E, et al. Related impurities in peptide medicines. Journal of Pharmaceutical and Biomedical Analysis 2014;101:2–30. doi:10.1016/j.jpba.2014.06.012
2. International Council for Harmonisation. ICH Q3A(R2): Impurities in New Drug Substances. 2006.
3. European Pharmacopoeia. General monograph 2034, Substances for pharmaceutical use.
4. Stoppacher N, Josephs RD, Daireaux A, Choteau T, Westwood SW, Wielgosz RI. Impurity identification and determination for the peptide hormone angiotensin I by liquid chromatography–high-resolution tandem mass spectrometry and the metrological impact on value assignments by amino acid analysis. Analytical and Bioanalytical Chemistry 2013;405(25):8039–8051. doi:10.1007/s00216-013-6953-7
5. Mant CT, Chen Y, Yan Z, Popa TV, Kovacs JM, Mills JB, et al. HPLC analysis and purification of peptides. In: Fields GB, ed. Peptide Characterization and Application Protocols. Methods in Molecular Biology, vol. 386. Humana Press; 2007:3–55. doi:10.1007/978-1-59745-430-8_1

Cosmic Peptides. (2026, October 25). Related substances on a certificate of analysis. Cosmic Peptides Research Library. Retrieved October 26, 2026, from https://cosmicpeptides.com/research/methods/related-substances

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  author       = {{Cosmic Peptides}},
  title        = {Related substances on a certificate of analysis},
  howpublished = {Cosmic Peptides Research Library},
  year         = {2026},
  month        = oct,
  url          = {https://cosmicpeptides.com/research/methods/related-substances},
  urldate      = {2026-10-26},
  note         = {Last reviewed 2026-10-25}
}

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