How to read a peptide certificate of analysis

Key points

• HPLC purity is an area percentage of the ultraviolet signal. It describes the impurities that can be seen, not the mass of peptide in the powder [1][5].
• Mass spectrometry confirms identity by comparing theoretical with observed mass. The EMA recommends at least two orthogonal identification methods [5].
• In a candidate angiotensin II reference material, the TFA counter-ion made up nearly 25% of the mass and the assigned content was 691 mg/g [7].
• Sterile does not mean endotoxin-free: semaglutide vials with no viable microorganisms contained 2.16 to 8.95 EU/mg [13].
• In an independent check, only 43 of 98 peptides with a ≥95% purity certificate met that specification [1].

US status (October 2026): A certificate of analysis does not make a compound lawful to compound or prescribe. Check the compound first in the peptide status tracker.

A certificate describes one batch on one date

The certificate of analysis (COA) summarizes the tests performed on a specific batch. It is the first quality filter between a supplier and your patient: peptide-related impurities can change biological effects and lead to wrong conclusions about what a product does [2]. The EMA guideline, in effect since June 1, 2026, lists the expected specification, from identification and purity to counter-ion, residual TFA, water, residual solvents, bacterial endotoxins and microbiological purity [5]. That list is a good template for reading any certificate a pharmacy or supplier sends you. Reading it is one part of peptide supplier diligence.

HPLC purity measures the ultraviolet signal

The most visible number usually comes from reversed-phase liquid chromatography with an ultraviolet detector, the routine quality-control method for peptides [3]. The calculation is area normalization: the area of the main peak against the area of all detected peaks. In a quality-control study from Ghent University, detection was at 210 and 215 nm, with a reporting threshold of 0.1% of the main-peak area [1]. So “≥98%” means that at least 98% of the integrated ultraviolet signal belongs to the main peak.

That definition has limits. Water and counter-ions are measured by separate tests and are not part of that percentage [5][7]. An impurity that elutes with the main peak is hidden inside it. The EMA asks manufacturers to consider the risk of co-eluting impurities and, when they appear as one peak, to apply the 1.0% qualification threshold [5]. A good certificate states the method and attaches the chromatogram with the integrated peaks. FDA rules require laboratory records to keep those data [15].

The remaining percentage is mostly impurities typical of solid-phase synthesis: sequences missing an amino acid (deletions) or carrying an extra one (insertions), diastereomers from racemization, protecting-group adducts, oxidations and dimers [2]. Regulators set precise thresholds. Under the European Pharmacopoeia, peptide-related impurities are reported above 0.1%, identified above 0.5% and qualified above 1.0% [5]. FDA, in its guidance on synthetic generics of glucagon, liraglutide, nesiritide, teriparatide and teduglutide (summarized in an official 2022 presentation), asks that any new impurity between 0.10% and 0.5% be identified and justified, and states that a new impurity “> 0.5% is not acceptable” [6].

Mass spectrometry confirms identity

Purity tells how much of the material is a single component. It does not say which component. That is what mass spectrometry is for. Electrospray ionization (ESI) produces multiply charged ions, and the instrument software reconstructs the mass from that series of peaks [4]. The EMA asks for tables of theoretical and observed mass values [5]. Check that the observed mass matches the theoretical mass of the stated sequence, that it is clear whether monoisotopic or average mass is reported, and that the spectrum is attached.

Mass has a blind spot: two diastereomers weigh exactly the same, and racemization is a known synthesis impurity [2]. The EMA recommends at least two orthogonal identification methods (for example, mass together with relative retention time, peptide mapping, amino acid analysis or NMR) and notes that controlling diastereomers may require specific methods [5]. In the Ghent study, the main compound of one sample had a different structure from the one ordered [1], an error that purity alone would not have revealed.

Purity is not content

A lyophilized powder with 99% HPLC purity is not 99% peptide by weight. It also contains counter-ions, water and sometimes solvents. The best-documented case is a candidate angiotensin II reference material from the National Research Council Canada: the trifluoroacetate counter-ion accounted for nearly 25% of the mass and the final assigned content was 691 ± 9 mg/g [7]. About 69% of the powder was peptide. That value is the net peptide content. The EMA states that a chromatographic assay is expressed on the anhydrous, counter-ion-free substance [5].

• 43 of 98: peptides with a ≥95% certificate that met that purity in an independent check [1]
• About 25%: of the mass of an angiotensin II standard was TFA counter-ion [7]
• 0.10 to 0.5%: range in which FDA asks for new impurities to be identified and justified [6]

Peptides are cleaved from the resin and purified with trifluoroacetic acid, so cationic peptides are obtained as trifluoroacetate salts. Most approved peptides, by contrast, are marketed as acetates [8]. The difference goes beyond weight. In osteoblast cultures, TFA at 10⁻⁸ to 10⁻⁷ M reduced cell numbers, and the TFA salts of amylin and calcitonin masked a proliferative effect or suggested a nonexistent antiproliferative one [9]. Salt forms also matter for GLP-1s, as explained in Compounded GLP-1s: salts, impurities and antibodies.

Water is the third component. Residual moisture is a critical quality attribute of lyophilized products. Karl Fischer titration is the usual method [11]. The EMA includes it in the specification and expects it in stability studies when the powder is hygroscopic [5]. The practical consequence: know whether the milligrams on the label refer to powder or to net peptide. For example, 10 mg of powder with 70% peptide content provide 7 mg of peptide. Storage of the powder and the Reconstituted vial is covered in Peptide stability.

Endotoxins, sterility and residual solvents are separate tests

The endotoxin test and the sterility test measure different things. Endotoxins are determined with the Limulus amebocyte lysate (LAL) assay, which FDA has accepted for more than 30 years in place of the rabbit pyrogen test and which is described in USP chapter <85> [12]. The result should be reported with a number and units, for example in EU/mg, alongside its limit. A Hungarian study of semaglutide sold online shows why both tests matter: the three lyophilized vials purchased had no viable microorganisms at the time of testing, yet all contained endotoxins, between 2.16 and 8.95 EU/mg [13].

Residual solvents are governed by the ICH Q3C guideline, which places them in three classes, from solvents to be avoided (class 1) to solvents with low toxic potential (class 3) [14]. For class 2 it sets specific limits: 410 ppm for acetonitrile, 600 ppm for dichloromethane and 880 ppm for N,N-dimethylformamide. For class 3, up to 0.5% is considered acceptable without further justification [14].

Batch, dates and signatures make the record

A certificate is valid only for the batch it describes. FDA good manufacturing practice rules require laboratory records to identify the sample with its lot number, the date it was taken and received, the method used, all data obtained (including instrument graphs, charts and spectra identified with the lot), the calculations, the initials or signature of whoever performed each test with the date, and review by a second person [15].

The Ghent group found that although all 98 peptides came with a certificate stating ≥95% purity, only 43 met it, and recommended independent quality control rather than relying on the supplier’s certificate [1]. The strongest position combines the supplier’s certificate with independent verification, at least of identity by mass, for key batches. A licensed pharmacy should be able to give you that documentation. How 503A and 503B sources differ is covered in 503A vs 503B pharmacy sourcing.

Red flags on a certificate

• No mass spectrum or observed mass: purity alone does not confirm identity [1][5].
• Identical chromatograms (same retention times, same areas, same baseline) across different batches: each batch generates its own data [15].
• No lot number, no test dates or no signatures [15].
• Purity without peptide content, counter-ion or water when the material is weighed to prepare solutions [5][7].
• Endotoxins reported as “conforms” with no number or units [12][13].
• A 99.9% figure with no integrated chromatogram or method, or a laboratory that cannot be identified [1][15].

Even a flawless certificate has limits. It is a snapshot of the batch on the test date, which is why the EMA requires separate stability studies [5]. Market studies show how far the declared can be from the measured: in three vials of semaglutide labeled at 99%, measured purity was 7.7 to 14.4% [13], a finding reviewed in What is in the peptide vial. A clean certificate on a research use only vial also does nothing for its legal status.

What this means for you

Before a batch reaches a patient, run six checks:

1. Match the vial’s lot to the certificate’s lot.
2. Confirm observed against theoretical mass.
3. Read the method and the chromatogram, not just the number.
4. Ask for net peptide content, counter-ion and water.
5. Check endotoxins reported with a number and units.
6. File the certificate with the batch record [1][5][15].

References

1. Verbeke F, Wynendaele E, Braet S, D’Hondt M, De Spiegeleer B. Quality evaluation of synthetic quorum sensing peptides used in R&D. J Pharm Anal. 2015;5(3):169-181. PMID 29403929. Link
2. D’Hondt M, et al. Related impurities in peptide medicines. J Pharm Biomed Anal. 2014;101:2-30. PMID 25044089. Link
3. Stalmans S, et al. Quality control of cationic cell-penetrating peptides. J Pharm Biomed Anal. 2016;117:289-297. PMID 26397208. Link
4. Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM. Electrospray ionization for mass spectrometry of large biomolecules. Science. 1989;246(4926):64-71. PMID 2675315. Link
5. European Medicines Agency. Guideline on the development and manufacture of synthetic peptides (EMA/CHMP/CVMP/QWP/367182/2025). 4 December 2025; in effect 1 June 2026. Link
6. Pang E. U.S. Food and Drug Administration, SBIA 2022. Assessing immunogenicity risk of peptides: the synthetic peptide guidance (ANDAs for certain highly purified synthetic peptide drug products that refer to listed drugs of rDNA origin) and PSGs. Link
7. Melanson JE, et al. Purity assignment for peptide certified reference materials by combining qNMR and LC-MS/MS amino acid analysis results: application to angiotensin II. Anal Bioanal Chem. 2018;410(26):6719-6731. PMID 30143839. Link
8. Sikora K, Jaśkiewicz M, Neubauer D, Migoń D, Kamysz W. The role of counter-ions in peptides: an overview. Pharmaceuticals (Basel). 2020;13(12):442. PMID 33287352. Link
9. Cornish J, et al. Trifluoroacetate, a contaminant in purified proteins, inhibits proliferation of osteoblasts and chondrocytes. Am J Physiol. 1999;277(5):E779-E783. PMID 10567002. Link
10. Mrozik W, et al. Determination of counter-ions in synthetic peptides by ion chromatography, capillary isotachophoresis and capillary electrophoresis. J Pept Sci. 2012;18(3):192-198. PMID 22252914. Link
11. Clavaud M, et al. Near-infrared spectroscopy to determine residual moisture in freeze-dried products: model generation by statistical design of experiments. J Pharm Sci. 2020;109(1):719-729. PMID 31499067. Link
12. U.S. Food and Drug Administration. Pyrogen and endotoxins testing: questions and answers (Edition 2). Guidance for industry. Link
13. Ashraf AR, et al. Multifactor quality and safety analysis of semaglutide products sold by online sellers without a prescription: market surveillance, content analysis, and product purchase evaluation study. J Med Internet Res. 2024;26:e65440. PMID 39509151. Link
14. International Council for Harmonisation / European Medicines Agency. ICH Q3C (R9) guideline on impurities: guideline for residual solvents (EMA/CHMP/ICH/82260/2006). 2024. Link
15. U.S. Code of Federal Regulations. 21 CFR 211.194, Laboratory records. Link

This is general information, not medical or legal advice. Rules vary by state and change. Confirm your own facts with counsel.