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Peptency Academy · Testing

The four questions a peptide CoA must answer

Which four questions does a certificate of analysis have to answer, and which test answers each?

A certificate of analysis (CoA) for a peptide lot answers four separate questions: is it the right molecule (identity), what else shows up beside it (purity), how much peptide the material holds (content), and whether endotoxin or microbes are present (contaminants). Source sentenceTheir quality is evaluated using a series of analytical tests, many of which depend on well-characterized reference standards to determine identity, purity, and strength.1 Pharm Res 20232 ICH Q4B Annex 14, Bacterial Endotoxins Test, 20123 ICH Q4B Annex 8(R1), Sterility Test, 2010 No single test answers all four, so each needs its own method and its own CoA line. Source sentenceMethods described include NMR, mass spectrometry, and chromatography techniques for identity testing and HPLC and GC methods for assessing peptide content and impurities.1 Pharm Res 20234 J Med Internet Res 2024

Version 3Edited 9 sources

Regulatory content shown for: EU and UK.

Known, not known, contested

Known

  • Mass spectrometry answers identity: it compares the measured mass with the mass expected from the sequence. Source sentenceElectrospray LC-MS/MS was used to confirm the mass of the peptide and to verify the amino acid sequence. (Methods, Mass Spectrometry; dash normalised)1 Pharm Res 2023
  • Chromatographic purity and counterion content are separate numbers: replacing the TFA counterion of synthetic peptides with chloride left their LC-UV purity unchanged. Source sentenceNo impact on peptide purity was observed at all HCl concentrations. (Abstract, Results) The purity of the peptides determined by LC-UV. (Methods 3.2)5 Pharmaceuticals (Basel) 2025
  • A counterion can be a large share of a peptide powder: in a candidate reference material of angiotensin II, TFA made up nearly 25 percent of the mass. Source sentencea validated 19F-qNMR method to measure the trifluoroacetic acid (TFA) counter-ion, considered an impurity in this case at nearly 25% by mass6 Anal Bioanal Chem 2018
  • Endotoxin and sterility are two different pharmacopoeial tests, each with its own harmonised chapter. Source sentencePh.Eur. 2.6.14. Bacterial Endotoxins, JP 4.01 Bacterial Endotoxins Test, and USP General Chapter <85> Bacterial Endotoxins Test, can be used as interchangeable in the ICH regions (section 2.1)2 ICH Q4B Annex 14, Bacterial Endotoxins Test, 20123 ICH Q4B Annex 8(R1), Sterility Test, 2010

Not known

  • Whether the pattern in the 2024 test purchase study holds for other peptides or other sellers is not known from that study: it analysed three vials of one peptide. Source sentenceAbstract, Results: test purchases from 6 sellers; 3 vial purchases were delivered and analysed4 J Med Internet Res 2024
  • The same study reports the sellers' 99 percent purity figure only as a label or website claim; how the sellers measured it is not reported. Source sentenceDocumentation, available on vendors' websites or shipped with the product, claims a purity of at least 99%. (Results, purity analysis)4 J Med Internet Res 2024

Contested

  • Sources use the word purity for two different numbers: the share of the chromatographic signal that is the main peptide, and the peptide share of the whole material by mass. The 2024 test purchase study reported a measured purity of 7.7 to 14.37 percent as semaglutide mass in the powder, while its chromatograms showed a single semaglutide signal. Source sentencethe chromatogram only displayed 1 signal for each delivered sample, indicating the exclusive presence of semaglutide ... revealed significantly lower polypeptide concentrations of 14.37%, 8.97%, and 7.7% (Results)4 J Med Internet Res 20246 Anal Bioanal Chem 20185 Pharmaceuticals (Basel) 2025

Which test answers which question

Illustrative layout, not a Peptency lot

  • Identity
    Method
    Mass spectrometry (LC-MS), ideally with a second method such as NMR or amino acid analysis
    Reports
    Measured mass beside the mass expected from the sequence, with the spectrum
  • Purity
    Method
    HPLC with UV detection
    Reports
    Main peak as a share of all UV peaks, with the chromatogram and the wavelength
  • Content
    Method
    Mass balance, amino acid analysis, qNMR, or HPLC assay against a reference standard
    Reports
    Peptide mass in the vial, or the peptide share of the powder by mass, with counterion and water stated separately
  • Counterion
    Method
    19F NMR, HPLC with ELSD, or FT-IR (for TFA)
    Reports
    Salt form and its share of the powder mass
  • Endotoxin
    Method
    Bacterial endotoxin test, technique named
    Reports
    Result with its unit, such as EU per mg, and the pharmacopoeia chapter
  • Sterility
    Method
    Pharmacopoeial sterility test
    Reports
    Growth or no growth of microorganisms
One line per question. Illustrative layout, not a Peptency lot.

1. Identity: is it the right molecule?

  • Reference standard work confirms identity with more than one method: NMR for structural features, LC-MS/MS for mass and sequence, and HPLC retention compared with a standard. Source sentenceTable II, Identity: NMR, confirm structural features; LC-MS/MS, peptide mass and amino acid sequence; HPLC, compare to standard (dashes normalised)1 Pharm Res 2023
  • Mass alone cannot tell leucine from isoleucine, which have the same mass; the USP work used amino acid analysis to distinguish them. Source sentenceAmino acid analysis was used as an identity method to distinguish between leucine and isoleucine in selected peptides, like bivalirudin, that contain both amino acids.1 Pharm Res 2023
  • An identity result says which molecule is present, not how much of it there is or what else the material contains. Source sentencequality is evaluated using a series of analytical tests ... to determine identity, purity, and strength (Abstract, Purpose)1 Pharm Res 2023

2. Purity: what else shows up beside the peptide?

  • Chromatographic purity comes from HPLC with UV detection: the peptide and its impurities are separated, read at a UV wavelength (220 nm in the USP example), and impurities are counted as a percent of the total detected peak area. Source sentencePeptides and impurities were monitored at 220 nm. (Methods, Chromatography) The equation used to calculate purity separates HPLC impurities, which are measured as a % of the total detected area by chromatography (Purity Determination of Bulk Material by Mass Balance)1 Pharm Res 2023
  • Counterion, water and other non peptide material are measured separately, by weight, so an HPLC purity figure describes the peptide signal only. Source sentenceseparates HPLC impurities, which are measured as a % of the total detected area by chromatography, and counter ion / impurities measured on a weight/weight basis (Purity Determination of Bulk Material by Mass Balance)1 Pharm Res 20235 Pharmaceuticals (Basel) 2025

3. Content: how much peptide is in the material?

  • USP assigns content to a peptide reference standard by mass balance: peptide related impurities, counterion, water, residual solvents and non combustible residue are each measured and subtracted from 100 percent. Source sentenceAll detectable impurities (e.g., peptide related impurities, counter ion, water, residual solvents, non-combustible residues) are measured and subtracted from 100% to assign purity.1 Pharm Res 2023
  • Content can also be measured directly, by amino acid analysis with isotope dilution LC-MS/MS after hydrolysis or by quantitative NMR (qNMR) of the intact peptide. Source sentenceConventionally, amino acid analysis by isotope dilution tandem mass spectrometry (LC-MS/MS) following peptide hydrolysis is employed as a reference method. By contrast, quantitative nuclear magnetic resonance (qNMR) spectroscopy allows for quantitation of intact peptides6 Anal Bioanal Chem 2018
  • For a certified reference material, the National Research Council Canada combined mass balance, qNMR and amino acid analysis results into one value. Source sentenceIdeally, results from these three orthogonal methods can be combined for final purity assignment of peptide reference standards.6 Anal Bioanal Chem 2018

4. Contaminants: endotoxin and microbes

  • The harmonised endotoxin text allows any of three techniques, and the gel clot limit test decides when a result is in doubt or dispute. Source sentenceAny of the three techniques can be used for the test. In the event of doubt or dispute, the gel-clot limit test should be used to make the final decision on compliance for the product being tested. (2.1.1)2 ICH Q4B Annex 14, Bacterial Endotoxins Test, 2012
  • A sterility pass does not rule out endotoxin: in the 2024 test purchase study, vials with no viable microorganisms all carried endotoxin. Source sentenceThe lyophilized peptide samples were devoid of viable microorganisms at the time of testing; however, endotoxin was detected in all samples4 J Med Internet Res 2024
  • Endotoxin results come with a unit and a method: the 2024 study reported endotoxin units (EU) per mg, measured by kinetic turbidimetric testing with Limulus amebocyte lysate (LAL) reagent. Source sentencebacterial endotoxin content measurement was performed via kinetic turbidimetry technique according to the European Pharmacopoeia ... 2.6.14 and US Pharmacopeia ... Chapter 85 ... Limulus Amebocyte Lysate reagent and Escherichia coli O55:B5 Control Standard Endotoxin were used. (Methods, Microbiological Testing)4 J Med Internet Res 2024

Worked example: three vials, four answers

  1. Ashraf and colleagues (J Med Internet Res 2024) bought semaglutide (PubChem CID 56843331) from six online sellers without a prescription; three vials arrived and were tested for content and purity by LC-MS, for sterility and for endotoxin. Source sentenceAbstract, Methods and Results: targeted product test purchases; microbiological sterility and endotoxin contamination evaluation; quantitative sample analysis using liquid chromatography coupled with mass spectrometry; 3 vial purchases delivered4 J Med Internet Res 20247 PubChem CID 56843331 (semaglutide)
  2. Identity: the chromatogram of each vial showed one signal, semaglutide, and no peptide-like impurities were identified. Source sentencethe chromatogram only displayed 1 signal for each delivered sample, indicating the exclusive presence of semaglutide (Results)4 J Med Internet Res 2024
  3. Content: the measured semaglutide per vial was 28.56 to 38.69 percent above the label. Source sentenceThe semaglutide content of samples substantially exceeded labeled amounts by 28.56%-38.69%4 J Med Internet Res 2024
  4. Purity by mass: semaglutide made up 7.7 to 14.37 percent of the powder, against at least 99 percent purity claimed by the sellers. Source sentencerevealed significantly lower polypeptide concentrations of 14.37%, 8.97%, and 7.7% (Results); Table 4 lists total powder in the vial beside measured semaglutide content4 J Med Internet Res 2024
  5. Contaminants: no viable microorganisms were found, and endotoxin was detected in all three vials, reported in the abstract as 2.1645 to 8.9511 EU/mg. Source sentenceendotoxin was detected in all samples with levels ranging between 2.1645 EU/mg and 8.9511 EU/mg4 J Med Internet Res 2024
  6. In the same three vials the four questions got four different answers, which is why a CoA needs a separate line for each. Source sentenceAbstract, Results: content above label, purity far below the label claim, no viable microorganisms, endotoxin in all samples4 J Med Internet Res 2024

What is the most accurate lab test for peptides?

No single test is the most accurate for all four questions. For identity the core test is mass spectrometry, read beside the sequence; for content, reference laboratories combine orthogonal methods, for example mass balance, qNMR and amino acid analysis for one content value. Source sentenceIdeally, results from these three orthogonal methods can be combined for final purity assignment of peptide reference standards.6 Anal Bioanal Chem 20181 Pharm Res 2023

Does a sterile result mean no endotoxin?

No. A sterility test looks for living microorganisms, while the endotoxin test detects cell wall residue of Gram-negative bacteria, and the 2024 test purchase vials passed one and failed the other. Source sentenceThe presence of endotoxin indicates residuals and remnants from the cell walls of Gram-negative bacteria. Despite the presence of endotoxin, all 3 lyophilized peptide samples ... (Results, Microbiological Testing)4 J Med Internet Res 20242 ICH Q4B Annex 14, Bacterial Endotoxins Test, 20123 ICH Q4B Annex 8(R1), Sterility Test, 2010

Terms on this page

Orthogonal methods
Independent methods that measure the same property by different principles, so their results can be cross checked. Source sentenceIdeally, results from these three orthogonal methods can be combined for final purity assignment6 Anal Bioanal Chem 2018
Mass balance
A content value found by measuring everything that is not the peptide and subtracting it from 100 percent. Source sentenceThe mass balance approach has also been employed for peptide purity measurements, whereby the purity is defined by the sum of the mass fraction of all impurities identified.6 Anal Bioanal Chem 20181 Pharm Res 2023
Counterion
The ion paired with a charged peptide in the solid. Peptides made by solid phase synthesis and purified with TFA usually end up as TFA salts. Source sentenceThe synthesis and purification of peptides via solid-phase peptide synthesis (SPPS) strongly rely on trifluoroacetic acid (TFA) as a cleavage agent and ion-pairing reagent, respectively, resulting in peptides being obtained as TFA salts.5 Pharmaceuticals (Basel) 2025
Endotoxin
Residue from the cell walls of Gram-negative bacteria, reported in endotoxin units (EU). Source sentenceThe presence of endotoxin indicates residuals and remnants from the cell walls of Gram-negative bacteria.4 J Med Internet Res 2024

Pharmacopoeia chapters for endotoxin and sterility

European Union: the EMA guideline on synthetic peptides

EU

  • EMA's guideline on synthetic peptides (EMA/CHMP/CVMP/QWP/367182/2025, in effect from 1 June 2026) lists specification tests that include identification, purity, assay or content, counterion and residual TFA, water, residual solvents, elemental impurities, bacterial endotoxins and microbiological purity. Source sentenceSpecification tests ... may include the following (non-exhaustive list): appearance; identification; purity; ... assay/content; counter-ion identity and content; residual ion content (TFA); water content; residual solvents; elemental impurities; bacterial endotoxins; microbiological purity. (4.4.1)8 EMA, Guideline on the development and manufacture of synthetic peptides, 2025
  • For identification it recommends at least two orthogonal methods, such as mass, relative retention time, LC-MS, peptide mapping, amino acid analysis or NMR. Source sentenceFor the identification of the peptide as part of the specification and release, use of at least two orthogonal methods is recommended. Identification by mass, relative retention time (RRT), LC-MS, peptide mapping, bioactivity, amino acid analysis or NMR are considered appropriate. (4.4.2)8 EMA, Guideline on the development and manufacture of synthetic peptides, 2025
  • It states assay limits by LC on the counterion free, anhydrous substance unless otherwise justified. Source sentenceLimits of assays determined by LC are expressed in terms of the counter-ion free, anhydrous substance, unless otherwise justified. (4.4.5)8 EMA, Guideline on the development and manufacture of synthetic peptides, 2025
  • The guideline sets thresholds for peptide related impurities: reported above 0.1 percent, identified above 0.5 percent and qualified above 1.0 percent. Source sentencepeptide-related impurities should be reported above 0.1%, identified above 0.5% and qualified above 1.0%. (4.4.1)8 EMA, Guideline on the development and manufacture of synthetic peptides, 2025
  • For peptides, the EMA guideline also points to Ph. Eur. general chapters 2.2.55 peptide mapping, 2.2.56 amino acid analysis, 2.5.34 acetic acid in synthetic peptides and 2.2.64 peptide identification by NMR. Source sentencePh. Eur. general chapters applicable to peptides (e.g. 2.2.55 Peptide mapping, 2.2.56. Amino acid analysis, 2.5.34 Acetic acid in synthetic peptides, 2.2.64 Peptide identification by NMR) (4.4.2)8 EMA, Guideline on the development and manufacture of synthetic peptides, 2025
  • ICH lists the EU as having implemented the interchangeable endotoxin chapters (Q4B Annex 14) on 1 May 2013 and the sterility chapters (Q4B Annex 8) on 1 September 2010. Source sentenceQ4B Annex 14, EC, Europe: Implemented; Date: 1 May 2013. Q4B Annex 8(R1), EC, Europe: Implemented; Date: 1 September 2010.9 ICH quality guidelines, Q4B implementation status, 13 Oct 2026

Studies cited

  1. Analytical only (no biological model)J Med Internet Res 2024

    LC-MS content and purity, sterility and endotoxin of semaglutide vials bought online

    Design
    Market surveillance with test purchases and laboratory analysis of delivered vials
    Population or model
    Lyophilised semaglutide vials bought online, 3 delivered
    Molecule
    semaglutide exact
    n
    3
    Comparator
    Label claims; an originator product as reference for content

    Two authors are employees of S-3 Research LLC; one author co-owns it (COI statement).

    PubMed 39509151DOI

  2. Analytical only (no biological model)Pharm Res 2023

    Identity, purity and content testing and value assignment for peptide reference standards

    Design
    Methods report with case studies on establishing peptide reference standards
    Population or model
    Synthetic peptide reference standards, with bivalirudin among the case studies
    Molecule
    synthetic peptide reference standards different molecule

    Authors are employees or collaborators of USP, which develops and sells peptide reference standards (COI statement).

    PubMed 36949371DOI

  3. Analytical only (no biological model)Anal Bioanal Chem 2018

    Peptide mass fraction by mass balance, qNMR and amino acid analysis, including TFA counterion

    Design
    Value assignment of a candidate certified reference material by three methods
    Population or model
    Angiotensin II candidate certified reference material
    Molecule
    angiotensin II different molecule

    PubMed 30143839DOI

  4. Analytical only (no biological model)Pharmaceuticals (Basel) 2025

    Residual TFA by 19F NMR, HPLC with ELSD and FT-IR, and LC-UV purity before and after exchange to chloride

    Design
    Analytical method validation and counterion exchange study
    Population or model
    Synthetic model peptides and cell penetrating peptides
    Molecule
    synthetic model peptides different molecule
    Comparator
    Before and after counterion exchange

    Authors declare no conflicts of interest (COI statement).

    PubMed 40872554DOI

Sources

  1. 1Pharm Res 2023 accessed 13 Oct 2026
  2. 2ICH Q4B Annex 14, Bacterial Endotoxins Test, 2012 accessed 13 Oct 2026
  3. 3ICH Q4B Annex 8(R1), Sterility Test, 2010 accessed 13 Oct 2026
  4. 4J Med Internet Res 2024 accessed 13 Oct 2026
  5. 5Pharmaceuticals (Basel) 2025 accessed 13 Oct 2026
  6. 6Anal Bioanal Chem 2018 accessed 13 Oct 2026
  7. 7PubChem CID 56843331 (semaglutide) accessed 13 Oct 2026
  8. 8EMA, Guideline on the development and manufacture of synthetic peptides, 2025 accessed 13 Oct 2026
  9. 9ICH quality guidelines, Q4B implementation status, 13 Oct 2026 accessed 13 Oct 2026

About this page

Research-use information. This library summarises published literature and regulator records. It is not guidance for use, not medical advice, and not a statement that any material is suitable for use in people or animals.

Reviewed by the Peptency editorial process. Version 3, last edited 13 October 2026, 9 sources.

Cite this page: Peptency Academy. The four questions a peptide CoA must answer. Version 3./eu/academy/testing/four-questions-a-coa-must-answer/