A white lyophilised powder always looks the same. Whether it is a pure peptide, a mixture with synthesis by-products or a completely different molecule, you cannot tell by eye. Only laboratory testing shows what is really in the vial. In this guide we go through a certificate of analysis step by step.
What a certificate of analysis is and why it matters
A certificate of analysis (CoA) is a report with the test results for one specific batch of a substance. It states which methods were used, what was measured and whether the results met predefined limits.
This matters in practice for research. Synthetic peptides are made in batches, and synthesis produces by-products: peptides with a missing or an extra amino acid, oxidised forms, residual protecting groups or unwanted counter-ions. A review in the Journal of Pharmaceutical and Biomedical Analysis points out that such impurities can distort the results of early laboratory studies and lead to wrong conclusions. If you want an experiment to be reproducible, you need to know which substance you worked with and in what quality.
What a proper CoA should contain
The format varies between manufacturers and laboratories. A trustworthy certificate should, however, contain at least the following:
- the name of the substance, ideally with its sequence or molecular formula,
- a batch or lot number or a production date, so you can match the certificate to a specific package,
- the date of analysis,
- the name of the laboratory that carried out the tests,
- the methods used, for example RP-HPLC with UV detection and mass spectrometry,
- results together with acceptance criteria, i.e. what was measured and which limit the sample had to meet (for example "purity ≥ 98%"),
- a signature or another means of verification, such as the name of the responsible person or a verification code.
Raw outputs attached to the certificate, i.e. the chromatogram and the mass spectrum, are a big advantage. A number without a graph is much harder to check.
The European Medicines Agency (EMA) draft guideline on synthetic peptides lists an even broader set of tests for medicinal substances: besides identity and purity, also peptide content, counter-ions, water, endotoxins and microbiological purity. A CoA for a research substance does not have to cover everything, but the list shows what to ask about.
HPLC purity: what "area %" means
The most common figure on a CoA is purity determined by high-performance liquid chromatography (HPLC), usually in reversed-phase mode (RP-HPLC). The sample passes through a column that separates its components, and a detector at the outlet measures the absorption of ultraviolet (UV) light. The result is a chromatogram: a curve with peaks, where each peak corresponds to one or more substances.
For peptides, UV detection is usually set to a wavelength of around 214 nm. At this wavelength the peptide bond, the bond between amino acids that every peptide has, absorbs strongly. The detector therefore picks up both the main peptide and most peptide impurities.
Purity is calculated as area percent (area %): the area of the target peptide's peak divided by the sum of the areas of all recorded peaks. A purity of 98.5% thus means that the main peak accounts for 98.5% of the total peak area at that wavelength.
It is a useful number, but it has limits. HPLC purity:
- does not prove identity. It only shows that one component dominates the sample, not that it is the right molecule.
- may miss impurities with the same retention time as the main peak. A Thermo Fisher Scientific application note states explicitly that such co-eluting impurities cannot be ruled out on the basis of the UV trace alone.
- is not an exact mass balance. Different peptides do not have the same UV response, so peak area does not always correspond exactly to mass.
- says nothing about quantity. Purity expresses the share of the target peptide among the peptide components. Water and salts are not included.
Mass spectrometry: is it really the right molecule?
Identity, meaning that the vial contains the declared peptide, is confirmed by mass spectrometry (MS). For peptides, electrospray ionisation (ESI-MS) is commonly used, often coupled directly to chromatography (LC-MS). The instrument does not weigh molecules directly. It measures the mass-to-charge ratio, written m/z.
During ionisation a peptide picks up one or more protons. A single molecule can therefore appear in the spectrum as several peaks: with one charge ([M+H]+), with two ([M+2H]2+) and so on. The mass of the molecule can be calculated back from each peak using M = z × (m/z − 1.007), where z is the number of charges and 1.007 is the mass of a proton.
Example: according to PubChem, BPC-157 has the formula C62H98N16O22 and a molecular weight of 1419.5 g/mol. The spectrum should therefore show approximately these values (calculated from the average mass and rounded):
| Ion | Charge (z) | Expected m/z |
|---|---|---|
| [M+H]+ | 1 | ≈ 1420.5 |
| [M+2H]2+ | 2 | ≈ 710.8 |
| [M+3H]3+ | 3 | ≈ 474.2 |
For MOTS-c, with a molecular weight of 2174.6 g/mol, the doubly charged ion would be at around m/z 1088.3 and the triply charged ion at around 725.9.
The CoA should state both the theoretical and the observed mass, and the two should agree. An article on the control of synthetic peptides in Pharmaceutical Technology gives as an example the requirement that the observed monoisotopic mass be within ±1.0 mass units of the theoretical value. Watch out for the two kinds of mass: the average mass (1419.5 for BPC-157) includes the natural distribution of isotopes, while the monoisotopic mass (about 1418.7) uses only the lightest isotopes. Lower-resolution instruments usually report the average mass, high-resolution instruments the monoisotopic one.
Mass alone does not reveal everything, though. A peptide with the same amino acids in a different order has the same mass. That is why fragmentation (MS/MS) is used to confirm the sequence, and the EMA draft guideline recommends at least two independent (orthogonal) methods for identifying a peptide, for example LC-MS and amino acid analysis.
Purity is not content: how much peptide is in the vial
This is where the most common misunderstanding arises. Purity tells you what share of the peptide material is the target peptide. Net peptide content tells you what share of the total powder weight is peptide material at all. The rest is counter-ions and residual water.
Counter-ions enter the peptide during manufacture. Trifluoroacetic acid (TFA) is used to cleave the finished peptide in solid-phase synthesis and also as an additive in HPLC purification. After lyophilisation the peptide remains as a trifluoroacetate salt. A 2025 study measured TFA contents of up to 35% of the total salt weight in the peptides examined. According to the EMA, acetate is the counter-ion usually used for synthetic peptides, but acetate is not peptide either. On top of that, lyophilised powder binds water.
So the weight of the powder is made up of peptide, counter-ions and water. The peptide manufacturer Bachem gives an example with a net peptide content of 85%: of 1 mg of powder, 0.85 mg is peptide material. That share also includes peptide impurities. Bachem also notes that two samples with the same 95% purity can contain different amounts of the target peptide if their content differs (for example 80% versus 95%).
This is why a vial with 99% purity can contain less than 10 mg of peptide even though it is labelled 10 mg. It depends on whether the figure refers to the weight of the powder or to the net peptide content. A good CoA either states this explicitly or includes a separate figure for peptide content or quantity.
Peptide content is determined, for example, by:
- amino acid analysis, in which the peptide is broken down into individual amino acids that are then measured,
- elemental nitrogen analysis, because neither counter-ions nor water contain nitrogen,
- quantitative HPLC against a reference standard of known content.
Counter-ion content is measured, for example, by ion chromatography. The Pharmaceutical Technology article recommends adding up peptide content, counter-ions and water. The result should approach 100%.
Other tests on a CoA
- Bacterial endotoxins. Endotoxins are components of the cell wall of Gram-negative bacteria. They are determined with the LAL test (Limulus amoebocyte lysate, from the horseshoe crab), and the procedures are described, for example, in chapter USP <85> of the United States Pharmacopeia. Neither HPLC nor MS detects endotoxins, so high purity says nothing about them.
- Sterility and microbiological purity. Mainly relevant for substances in solution. The EMA draft guideline lists microbiological purity among the typical tests.
- Appearance. For example "white lyophilised powder" or "clear colourless solution". A simple check that reveals gross deviations.
- Solubility. Which solvent the substance dissolves in to give a clear solution.
Red flags
- No batch number or production date. The certificate cannot be matched to a specific package.
- No laboratory name or date of analysis. It is unclear who carried out the tests and when.
- All batches have the same chromatogram or the same numbers. Results relate to a specific batch. Bachem states explicitly that peptide content is a lot-specific value.
- No identity test. Purity without mass spectrometry only shows that one substance dominates the sample.
- An unrealistically perfect result, such as a purity of exactly 100.0%. Peptide synthesis routinely produces by-products, and the European Pharmacopoeia sets the reporting threshold for peptide-related impurities at 0.1%. A result without a single side peak deserves questions.
- The theoretical mass does not match. Compare it with a public database such as PubChem.
- Nothing can be verified. No chromatogram, no spectrum, no laboratory contact and no verification code.
Testing by an independent laboratory with no link to the seller is stronger evidence. Some such laboratories, for example Janoshik Analytical in Prague, print a verification key on the report that lets you look up the result on the laboratory's website. This lets you check that the document matches the laboratory's record and has not been altered. Even with an independent test, everything above still applies: batch, methods, identity and content.
How it works at Arion Peptides
We publish certificates of analysis for Arion Bios products on the Certificates page. You match a certificate to a specific package by the production date printed on the packaging. When reading it, you can use the same checklist as for any other CoA.
Frequently Asked Questions
Does 99% purity mean the vial contains 99% peptide?
No. HPLC purity expresses the share of the target peptide among the peptide components. The weight of the powder also includes counter-ions and water, which are not counted in purity. How much peptide the sample really contains is shown by the net peptide content.
Why is HPLC alone not enough?
HPLC shows what percentage of the peak area belongs to the main component, but it does not confirm which molecule it is. Identity is confirmed by mass spectrometry, which compares the observed mass with the theoretical one.
How do I know the certificate belongs to my package?
Look for a batch number or production date on the certificate and compare it with the information on the packaging. For Arion Bios products, the certificate is matched by the production date on the packaging.
Why is a purity of 100.0% suspicious?
Peptide synthesis routinely produces by-products, and a sensitive method will pick up impurities at around 0.1%. A result of exactly 100.0% therefore calls for an explanation of the method and sensitivity used to obtain it.
Sources
- European Medicines Agency, "Guideline on the Development and Manufacture of Synthetic Peptides" (draft for public consultation), EMA/CHMP/CVMP/QWP/387541/2023, 2023: ema.europa.eu
- Swietlow A, Verlander M, Gregg B, Rode H, Eggen I, Szajek A, "Control Strategies for Synthetic Therapeutic Peptide APIs – Part I: Analytical Consideration", Pharmaceutical Technology, 2014: pharmtech.com
- Grosse S, Samonig M, De Pra M, Steiner F, "Impurity profiling of the synthetic peptide LL-37 using high-performance liquid chromatography with combined UV and single quadrupole mass spectrometric detection", Thermo Fisher Scientific, Application Note 72818, 2018: thermofisher.com
- D'Hondt M, Bracke N, Taevernier L, Gevaert B, Verbeke F, Wynendaele E, De Spiegeleer B, "Related impurities in peptide medicines", Journal of Pharmaceutical and Biomedical Analysis, 2014;101:2–30. DOI: 10.1016/j.jpba.2014.06.012
- Erckes V, Streuli A, Chamera Rendueles L, Krämer SD, Steuer C, "Towards a Consensus for the Analysis and Exchange of TFA as a Counterion in Synthetic Peptides and Its Influence on Membrane Permeation", Pharmaceuticals, 2025;18(8):1163. DOI: 10.3390/ph18081163
- Bachem, "What does net peptide content mean?", technical FAQ: bachem.com
- Bachem, "Peptide Calculator" (purity and peptide content), Knowledge Center: bachem.com
- U.S. Food and Drug Administration, "Pyrogen and Endotoxins Testing: Questions and Answers (Edition 2)", Guidance for Industry, 2026: fda.gov
- National Center for Biotechnology Information, "PubChem Compound Summary: BPC-157 (CID 9941957) and MOTS-c (CID 146675088)": pubchem.ncbi.nlm.nih.gov, pubchem.ncbi.nlm.nih.gov