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Peptency Academy · Testing

HPLC purity explained

What does an HPLC purity figure on a peptide CoA measure?

HPLC purity is the area of the main peak as a percentage of the total peak area in a UV chromatogram of the sample. Source sentenceThe peptide purity was quantified through normalization by calculating the area (UV) of the most abundant peak as a percentage of total peak areas.1 J Pharm Anal 2015 Detection is generally at 210 to 220 nm, where the peptide bond absorbs strongly. Source sentencePeptide bonds absorb light strongly in the far ultraviolet (UV) (<220 nm), providing a convenient means of detection (generally at 210 to 220 nm).2 Methods Mol Biol 2007 The figure describes how clean the peptide signal is, not how much of the powder is peptide: counterion, water and solvents are measured by separate tests, by mass. Source sentenceThe equation used to calculate purity separates HPLC impurities, which are measured as a % of the total detected area by chromatography, and counter ion / impurities measured on a weight/weight basis.3 Pharm Res 2023

Version 3Edited 16 sources

Regulatory content shown for: EU and UK.

Known, not known, contested

Known

  • A 2023 USP paper on peptide reference standards reports HPLC impurities as a percent of total detected area, and acetic acid, TFA, residual solvents and water as percent by mass. Source sentenceTable VII: Sum of HPLC impurities (I1)/ Monograph, % Total Detected Area; Acetic acid Content (I2)/ USP <503>, %w/w; Trifluoroacetic acid Content (I3)/ USP <503.1>, %w/w; Residual solvents Content (I4)/ USP <467>, %w/w; Water Content was determined by Karl Fischer titration as described in USP general chapter <921> Water Determination.3 Pharm Res 2023
  • In a 2015 Belgian study, all 98 synthetic research peptides ordered at a requested purity of at least 95.0 percent came with a certificate stating at least 95.0 percent; the lab's own UV chromatography found that 43 of the 98 met it. Source sentenceDespite a requested purity of at least 95.0%, only 43 out of 98 peptides met these purity specifications (Fig. 2). However, all the 98 peptides were accompanied by a certificate of analysis stating at least 95.0% purity.1 J Pharm Anal 2015
  • In the same study, most related impurities were peptides missing one or more amino acids of the intended sequence. Source sentenceWe also found that the majority of the related impurities were lacking amino acid(s) in the desired peptide sequence.1 J Pharm Anal 2015
  • In the same study, the main compound of one sample had a different structure from the peptide that was ordered. Source sentenceThe main compound of one sample was even found to have a different structure compared to the desired peptide.1 J Pharm Anal 2015
  • In a 2024 test purchase study of three lyophilised semaglutide vials sold online, the chromatogram showed a single signal for each sample, while semaglutide made up 7.7 to 14.37 percent of the powder against 99 percent purity claimed on the labels. Source sentencethe chromatogram only displayed 1 signal for each delivered sample, indicating the exclusive presence of semaglutide; Table 4: Biotech Peptides vial, total powder 50.1, measured semaglutide 3.86, measured purity 7.70 percent; abstract: the measured semaglutide purity was significantly low, ranging between 7.7% and 14.37% and deviating from the 99% claimed on product labels by manufacturers.4 J Med Internet Res 2024
  • In a candidate angiotensin II reference material, the TFA counterion was nearly 25 percent of the mass, and the final assigned purity, combining mass balance, qNMR and LC-MS/MS amino acid analysis, was a mass fraction of 69.1 ± 0.9 percent (2018). Source sentenceAbstract: "a validated 19F-qNMR method to measure the trifluoroacetic acid (TFA) counter-ion, considered an impurity in this case at nearly 25% by mass"; "Ideally, results from these three orthogonal methods can be combined" (mass balance, 1H qNMR and LC-MS/MS amino acid analysis); final purity value assignment of the candidate CRM, 691 ± 9 per thousand by mass (k = 2) (69.1 ± 0.9 percent, unit conversion)5 Anal Bioanal Chem 2018
  • In a 2025 ETH Zurich study, exchanging the TFA counterion for chloride left HPLC-UV purity unchanged: one test peptide stayed above 96 percent over three rounds of exchange. Source sentenceThe purity of AT1 for all concentrations of HCl over three [exchange rounds] was constantly above 96% as shown in Figure 3 D. No degradation products were observed HPLC-UV. ... No impact on peptide purity was observed at all HCl concentrations.6 Pharmaceuticals (Basel) 2025

Not known

Contested

  • Sources use the word purity for two different measurements: the chromatographic area percent, and the share of the material that is the peptide by mass. Source sentenceTable II: Purity, Chromatographic purity by HPLC, Determine quantity of peptide impurities; Value Assignment: the theoretical example described in Table VII "would have a purity of 0.93" of peptide (free base) relative to material on the anhydrous basis.3 Pharm Res 20235 Anal Bioanal Chem 20184 J Med Internet Res 2024

How an HPLC purity test works

  1. The dissolved sample runs through a column packed with silica carrying C8 or C18 chains, the packing favoured for most peptide separations. Source sentenceFavored RP-HPLC packings for the vast majority of peptide separations continue to be silica-based supports containing covalently bound octyl (C8) or octadecyl (C18) functionalities2 Methods Mol Biol 2007
  2. A gradient of water and acetonitrile, each with 0.1 percent TFA, carries the peptides off the column in order of increasing hydrophobicity. Source sentencea gradient mobile phase consisting of 0.1% trifluoroacetic acid (TFA) in water (A) and 0.1% TFA in acetonitrile (B); Mant 2007: with peptides being eluted from these hydrophobic stationary phases in order of increasing overall peptide hydrophobicity10 Biomed Chromatogr 20152 Methods Mol Biol 2007
  3. A UV detector records absorbance as each compound leaves the column, generally at 210 to 220 nm. Source sentencePeptide bonds absorb light strongly in the far ultraviolet (UV) (<220 nm), providing a convenient means of detection (generally at 210 to 220 nm).2 Methods Mol Biol 2007
  4. Peaks that also appear in a blank run of the solvent alone are excluded. Source sentenceSolvent and system peaks, observed in the blank (i.e. the solvent used to dissolve a certain peptide, Table 1), were excluded.1 J Pharm Anal 2015
  5. Each remaining peak is integrated, and the main peak area is divided by the total peak area (area normalisation). Source sentenceThe peptide purity was quantified through normalization by calculating the area (UV) of the most abundant peak as a percentage of total peak areas.1 J Pharm Anal 2015
  6. A reporting threshold sets which small peaks count: the 2015 study used 0.1 percent of the main peak area for reporting and 0.5 percent for identification, as stated in the European Pharmacopoeia. Source sentenceA reporting threshold (i.e. 0.1% of main peak AUC in the UV chromatogram) and identification threshold (i.e. 0.5% of main peak AUC in the UV chromatogram) as stated in the European Pharmacopoeia were applied.1 J Pharm Anal 2015
  7. To learn what the main peak and the impurities are, the same samples were run by LC-MS with fragmentation (MS/MS). Source sentence2.3.2 Identification of main LC peak and related impurities: Mass spectroscopic characterization was performed with a Thermo HPLC system ... a Finnigan LCQ ion trap mass spectrometer with electrospray ionization (ESI); MS2 fragmentation patterns1 J Pharm Anal 2015

What does an HPLC purity result measure?

It measures how much of the UV signal at the detection wavelength belongs to the main peak, after blank peaks are removed and peaks below the reporting threshold are set aside. Source sentenceThe peptide purity was quantified through normalization by calculating the area (UV) of the most abundant peak as a percentage of total peak areas. Solvent and system peaks, observed in the blank (i.e. the solvent used to dissolve a certain peptide, Table 1), were excluded. A reporting threshold (i.e. 0.1% of main peak AUC in the UV chromatogram) and identification threshold (i.e. 0.5% of main peak AUC in the UV chromatogram) as stated in the European Pharmacopoeia were applied.1 J Pharm Anal 20152 Methods Mol Biol 2007 Counterion, water and residual solvents are not part of it; reference standard work measures them by separate tests and reports them by mass. Source sentenceThe equation used to calculate purity separates HPLC impurities, which are measured as a % of the total detected area by chromatography, and counter ion / impurities measured on a weight/weight basis.3 Pharm Res 2023

What an HPLC purity line on a CoA looks like

Illustrative layout, not a Peptency lot

  • Purity (HPLC)
    Method
    UV chromatography, 210 to 220 nm, area normalisation, chromatogram attached
    Reports
    Main peak area percent
  • Identity
    Method
    Mass spectrometry (LC-MS)
    Reports
    Observed mass against the calculated mass
  • Peptide content
    Method
    Amino acid analysis, or LC assay against a reference standard
    Reports
    Peptide share of the powder, by mass
  • Counterion
    Method
    LC or ion chromatography (acetate, TFA)
    Reports
    Counterion percent by mass
  • Water
    Method
    Karl Fischer titration
    Reports
    Water percent by mass
Illustrative layout, not a Peptency lot. The purity line and the tests that sit beside it.

What is a good purity level for peptides?

For peptide medicines there is no single total figure: limits are set per product from batch data, inside Ph. Eur. thresholds per impurity of 0.1 percent (report), 0.5 percent (identify) and 1.0 percent (qualify). Source sentenceSynthetic peptides are excluded from the scope of ICH Guideline Q3A/VICH GL10, 'Impurities in New Drug Substances', and consequently the limits laid down in this guideline are not applicable. Specific thresholds for peptide-related impurities are defined in the Ph. Eur. According to the Ph. Eur. general monograph 'Substances for Pharmaceutical Use', peptide-related impurities should be reported above 0.1%, identified above 0.5% and qualified above 1.0%. ... the acceptance criteria for the peptide-related impurities and total impurities should not only be based on the qualified limit but on data obtained from the batches used to support the quality of the active substance.11 EMA, Guideline on the development and manufacture of synthetic peptides, EMA/CHMP/CVMP/QWP/367182/2025 In the 2015 research peptide study, the requested figure was at least 95.0 percent, and 43 of 98 peptides met it when retested. Source sentenceDespite a requested purity of at least 95.0%, only 43 out of 98 peptides met these purity specifications (Fig. 2). However, all the 98 peptides were accompanied by a certificate of analysis stating at least 95.0% purity.1 J Pharm Anal 2015 A purity figure carries its method with it: the column, the detection wavelength, blank subtraction, peak integration and the reporting threshold each decide which peaks are seen and counted. Source sentenceA reporting threshold (i.e. 0.1% of main peak AUC in the UV chromatogram) and identification threshold (i.e. 0.5% of main peak AUC in the UV chromatogram) as stated in the European Pharmacopoeia were applied. Solvent and system peaks, observed in the blank (i.e. the solvent used to dissolve a certain peptide, Table 1), were excluded.; D'Hondt 2016: lower limits of detection and higher resolution separations, which resulted in more detected impurities1 J Pharm Anal 201512 J Pharm Anal 2016

Does 99 percent HPLC purity mean the vial is 99 percent peptide?

No. HPLC purity is a share of UV peak area; how much of the powder is peptide is a separate measurement by mass. Source sentenceThe equation used to calculate purity separates HPLC impurities, which are measured as a % of the total detected area by chromatography, and counter ion / impurities measured on a weight/weight basis.3 Pharm Res 2023 In the 2024 semaglutide test purchases, each chromatogram showed a single signal, yet semaglutide was 7.7 to 14.37 percent of the powder; TFA alone was nearly 25 percent of the mass of one angiotensin II reference material. Source sentencethe chromatogram only displayed 1 signal for each delivered sample, indicating the exclusive presence of semaglutide; Table 4: Biotech Peptides vial, total powder 50.1, measured semaglutide 3.86, measured purity 7.70 percent; abstract: the measured semaglutide purity was significantly low, ranging between 7.7% and 14.37% and deviating from the 99% claimed on product labels by manufacturers.4 J Med Internet Res 20245 Anal Bioanal Chem 2018

Can HPLC confirm that a peptide is the right molecule?

Not on its own. In the USP reference standard programme, the HPLC identity test compares the sample with a well characterised standard, while NMR, LC-MS/MS and amino acid analysis confirm structure, mass and sequence. Source sentenceIdentity: NMR, Confirm structural features; LC-MS/MS, Peptide mass and Amino acid sequence; HPLC, Compare to standard (previous lot or other well characterized material); AAA, Distinguish Ile vs. Leu3 Pharm Res 2023 A clean main peak can still be the wrong molecule: in the 2015 Belgian study, the main compound of one sample had a different structure from the peptide that was ordered. Source sentenceThe main compound of one sample was even found to have a different structure compared to the desired peptide.1 J Pharm Anal 2015

How is an HPLC chromatogram read?

The trace plots UV absorbance against time; each peak is a compound, or several unresolved compounds, leaving the column. Source sentencePeptide bonds absorb light strongly in the far ultraviolet (UV), providing a convenient means of detection; elution profiles and retention time; EMA guideline: When co-eluting impurities are observed as one peak2 Methods Mol Biol 200711 EMA, Guideline on the development and manufacture of synthetic peptides, EMA/CHMP/CVMP/QWP/367182/2025 Structurally related impurities tend to leave the column just before or just after the main peak, and full separation from it is not always achieved. Source sentenceAs complete separation of the desired peptide and structurally related pre- and post-eluting impurities is not always achieved, it is common to collect fractions and pool the main fractions with highest purity for further processing.11 EMA, Guideline on the development and manufacture of synthetic peptides, EMA/CHMP/CVMP/QWP/367182/2025 Peaks that also appear in a blank run come from the solvent or the system and are not counted. Source sentenceSolvent and system peaks, observed in the blank (i.e. the solvent used to dissolve a certain peptide, Table 1), were excluded.1 J Pharm Anal 2015

Does UPLC give a different purity figure than HPLC?

It can, and so can two labs running different HPLC methods: the figure depends on the method as well as the material. In a 2016 Ghent comparison on four peptide medicines, UPLC with sub 2 µm particles gave lower detection limits and higher resolution, which resulted in more detected impurities than HPLC-UV methods derived from the pharmacopoeia. Source sentenceThe QC and impurity profiling results obtained using the ultra-high performance liquid chromatography ultraviolet/mass spectrometry (UPLC-UV/MS) detection system were analyzed against the results obtained using traditional high performance liquid chromatography-ultraviolet detection (HPLC-UV) methods derived from pharmacopoeial methods. In general, the used stationary phases of sub-2 µm particle (UPLC) technology resulted in lower limits of detection and higher resolution separations, which resulted in more detected impurities12 J Pharm Anal 2016 Peak integration matters too: the same paper describes an HPLC-UV method whose inadequate integration led to a false low quality conclusion. Source sentenceThis is an example of how an averagely performing HPLC-UV QC method, combined with inadequate integration, can result in a fals[e] low quality conclusion.12 J Pharm Anal 2016 A single quad mass detector coupled to the UPLC run identified the main compounds and small impurities directly, without reference standards. Source sentenceonline coupling with a single quad MS detector allowed direct peak identification of the main compounds as well as small impurities, hereby increasing the information content without the need of reference standards.12 J Pharm Anal 2016

Terms on a chromatogram and a CoA

Area percent
A peak's area as a percentage of the total integrated area. It is a share of the UV signal, not a share of mass. Source sentenceThe equation used to calculate purity separates HPLC impurities, which are measured as a % of the total detected area by chromatography, and counter ion / impurities measured on a weight/weight basis.3 Pharm Res 2023
Related impurity
A peptide that differs from the intended one, for example by a missing amino acid (deletion), an extra one (insertion), a wrong stereoisomer, an oxidised side chain or a deamidated residue. Source sentencedeletion and insertion of amino acids are related to inefficient Fmoc-deprotection and excess use of amino acid reagents, respectively. Fmoc-deprotection can cause racemization of amino acid residues and thus diastereomeric impurities. ... Oxidation of amino acid side chains and dimeric-to-oligomeric impurities were also observed.; EMA guideline 4.3.2: the following pathways can contribute to the formation of degradation products of synthetic peptides: oxidation; hydrolysis; isomerisation; deamidation13 J Pharm Biomed Anal 201411 EMA, Guideline on the development and manufacture of synthetic peptides, EMA/CHMP/CVMP/QWP/367182/2025
Co-elution
Two or more compounds leaving the column together and showing as one peak. Source sentenceWhen co-eluting impurities are observed as one peak the qualification threshold of 1.0% applies unless otherwise justified.11 EMA, Guideline on the development and manufacture of synthetic peptides, EMA/CHMP/CVMP/QWP/367182/2025
Stability indicating method
A method shown to separate the peptide from its degradation products: the 2015 KPV method separated KPV from lys-pro-diketopiperazine formed under acid, alkali and peroxide stress. Source sentenceUnder stress conditions (acid, alkali and hydrogen peroxide) KPV yielded lys-pro-diketopiperazine as major degradation product, which was identified by flow [...] MS analysis. The developed HPLC method was found to be efficient in separating the active peptide from its degradation products10 Biomed Chromatogr 2015
Mass balance
Assigning purity by mass: every detectable impurity (related peptides, counterion, water, residual solvents, inorganic residue) is 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.3 Pharm Res 2023
Size exclusion chromatography (SEC)
Size exclusion chromatography separates by size and is used to look at dimers, oligomers and aggregates; EMA lists these as a separate specification test. Source sentenceassessment of oligomerization state of peptides/proteins by size-exclusion chromatography; EMA guideline 4.4.1: high molecular weight impurities (e.g., aggregates / oligomers) by SEC-LC or other techniques (if relevant)2 Methods Mol Biol 200711 EMA, Guideline on the development and manufacture of synthetic peptides, EMA/CHMP/CVMP/QWP/367182/2025

Published examples with IDs

  1. Analytical only (no biological model)J Pharm Anal 2015

    Chromatographic purity by UV area normalisation (210 and 215 nm) against the certificate value; identity of main peaks and related impurities by LC-MS

    Design
    Analytical chemistry: in house UV chromatography purity check of supplied research peptides against their certificates, LC-MS identification of impurities
    Population or model
    98 synthetic quorum sensing peptides from suppliers
    Molecule
    synthetic peptides (various) different molecule
    n
    98

    PubMed 29403929DOI

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

    Semaglutide content and share of powder by LC-MS, chromatographic profile, sterility and endotoxin

    Design
    Market surveillance plus test purchases with laboratory analysis (LC-MS, sterility, endotoxin)
    Population or model
    3 lyophilised vials delivered from test purchases
    Molecule
    semaglutide exact
    n
    3

    Two authors are employees of S-3 Research LLC; one is its cofounder (COI statement).

    PubMed 39509151DOI

  3. Analytical only (no biological model)Biomed Chromatogr 2015

    Separation of KPV from its stress degradation products on a C18 column with a TFA water and acetonitrile gradient; degradant identity by MS

    Design
    Analytical method development and validation (stability indicating HPLC)
    Population or model
    KPV in aqueous solution and skin homogenate
    Molecule
    KPV (Lys-Pro-Val) exact

    PubMed 25298219DOI

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

    Mass fraction purity and TFA counterion content of a peptide reference material

    Design
    Metrology: purity value assignment by qNMR, LC-MS/MS amino acid analysis and mass balance
    Population or model
    Candidate angiotensin II certified reference material
    Molecule
    angiotensin II different molecule

    PubMed 30143839DOI

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

    How identity, chromatographic purity, counterion, water and content are measured and combined into one assigned value

    Design
    Methods report with case studies (reference standard value assignment)
    Population or model
    USP peptide reference standard lots
    Molecule
    synthetic peptides (various) different molecule

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

    PubMed 36949371DOI

  6. Analytical only (no biological model)J Pharm Anal 2016

    Detected impurities, resolution and peak identity by the two methods

    Design
    Analytical method comparison: UPLC-UV/MS against pharmacopoeia derived HPLC-UV
    Population or model
    Four peptide medicines (bleomycin sulfate, tyrothricin, vancomycin HCl, bacitracin)
    Molecule
    synthetic peptides (various) different molecule
    n
    4

    PubMed 29403959DOI

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

    Residual TFA after exchange for chloride, HPLC-UV purity before and after exchange, membrane permeation in a liposome assay

    Design
    Analytical method validation (FT-IR, 19F NMR, HPLC-ELSD) and counterion exchange experiments
    Population or model
    Synthetic peptides made by SPPS
    Molecule
    synthetic peptides (various) different molecule

    Authors declare no conflicts of interest (COI statement).

    PubMed 40872554DOI

Pharmacopoeia chapters

  • The EMA guideline names Ph. Eur. general monograph 2034, Substances for Pharmaceutical Use, as the basis for limits on peptide related impurities, and lists general chapters 2.5.34 (acetic acid in synthetic peptides) and 2.2.56 (amino acid analysis) among those applicable to peptides. Source sentence"The limits applied for peptide-related impurities should be based on the general monograph of the European Pharmacopoeia 'Substances for Pharmaceutical Use (2034)'." (4.4.5); "Ph. 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); reference list: "Ph. Eur. General Monograph 2034 'Substances for Pharmaceutical Use'"11 EMA, Guideline on the development and manufacture of synthetic peptides, EMA/CHMP/CVMP/QWP/367182/2025

International guidelines

  • ICH Q3A(R2), the impurity guideline for new drug substances, lists peptides among the substances it does not cover. Source sentenceThe following types of drug substances are not covered in this guideline: biological/biotechnological, peptide, oligonucleotide, radiopharmaceutical, fermentation product and semi-synthetic products derived therefrom14 ICH Q3A(R2), Impurities in new drug substances
  • ICH Q2(R2) lets a lab show that a procedure is specific by absence of interference from impurities and degradation products, or by agreement with a second procedure that ideally uses a different measurement principle. Source sentence3.1.1 The specificity or selectivity of an analytical procedure can be demonstrated through absence of interference or comparison of results to an orthogonal procedure. 3.1.1.1 ... not impacted by the presence of other substances (e.g., impurities, degradation products, related substances, matrices ...). 3.1.1.2 ... a second, well characterised analytical procedure that ideally applies a different measurement principle.15 ICH Q2(R2), Validation of analytical procedures

European Union: the EMA synthetic peptide guideline

EU

  • EMA's Guideline on the development and manufacture of synthetic peptides (EMA/CHMP/CVMP/QWP/367182/2025) was first published on 9 Dec 2025 and took legal effect on 1 Jun 2026. Source sentenceCurrent version: Guideline on the development and manufacture of synthetic peptides, Adopted, Reference Number: EMA/CHMP/CVMP/QWP/367182/2025, Legal effective date: 01/06/2026, First published: 09/12/202516 EMA, Development and manufacture of synthetic peptides, scientific guideline page
  • It covers manufacturing, characterisation, specifications and analytical control of synthetic peptides in medicines. Source sentenceThis guideline addresses specific aspects regarding the manufacturing process, characterisation, specifications and analytical control for synthetic peptides which are not covered in the Guideline on the Chemistry of Active Substances16 EMA, Development and manufacture of synthetic peptides, scientific guideline page
  • It states that ICH Q3A limits do not apply to synthetic peptides and points to the Ph. Eur. thresholds instead: report above 0.1 percent, identify above 0.5 percent, qualify above 1.0 percent. Source sentenceSynthetic peptides are excluded from the scope of ICH Guideline Q3A/VICH GL10, 'Impurities in New Drug Substances', and consequently the limits laid down in this guideline are not applicable. Specific thresholds for peptide-related impurities are defined in the Ph. Eur. According to the Ph. Eur. general monograph 'Substances for Pharmaceutical Use', peptide-related impurities should be reported above 0.1%, identified above 0.5% and qualified above 1.0%.11 EMA, Guideline on the development and manufacture of synthetic peptides, EMA/CHMP/CVMP/QWP/367182/2025
  • If one method cannot separate all peptide related impurities, additional independent methods are expected, and co-eluting impurities seen as one peak fall under the 1.0 percent qualification threshold unless otherwise justified. Source sentenceIf one analytical method for detection and quantification of all the peptide-related impurities is not appropriate to separate all impurities, additional independent method(s) may be needed. When co-eluting impurities are observed as one peak the qualification threshold of 1.0% applies unless otherwise justified.11 EMA, Guideline on the development and manufacture of synthetic peptides, EMA/CHMP/CVMP/QWP/367182/2025
  • For identification at release, at least two orthogonal methods are recommended, for example 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.11 EMA, Guideline on the development and manufacture of synthetic peptides, EMA/CHMP/CVMP/QWP/367182/2025
  • Assay limits determined by LC are expressed on the counter-ion 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.11 EMA, Guideline on the development and manufacture of synthetic peptides, EMA/CHMP/CVMP/QWP/367182/2025

Sources

  1. 1J Pharm Anal 2015 accessed 13 Oct 2026
  2. 2Methods Mol Biol 2007 accessed 13 Oct 2026
  3. 3Pharm Res 2023 accessed 13 Oct 2026
  4. 4J Med Internet Res 2024 accessed 13 Oct 2026
  5. 5Anal Bioanal Chem 2018 accessed 13 Oct 2026
  6. 6Pharmaceuticals (Basel) 2025 accessed 13 Oct 2026
  7. 7PubMed search for interlaboratory studies of peptide HPLC purity, 13 October 2026 accessed 13 Oct 2026
  8. 8J Pharm Biomed Anal 2019 accessed 13 Oct 2026
  9. 9PubMed search for peptide purity and certificates of analysis, 13 October 2026 accessed 13 Oct 2026
  10. 10Biomed Chromatogr 2015 accessed 13 Oct 2026
  11. 11EMA, Guideline on the development and manufacture of synthetic peptides, EMA/CHMP/CVMP/QWP/367182/2025 accessed 13 Oct 2026
  12. 12J Pharm Anal 2016 accessed 13 Oct 2026
  13. 13J Pharm Biomed Anal 2014 accessed 13 Oct 2026
  14. 14ICH Q3A(R2), Impurities in new drug substances accessed 13 Oct 2026
  15. 15ICH Q2(R2), Validation of analytical procedures accessed 13 Oct 2026
  16. 16EMA, Development and manufacture of synthetic peptides, scientific guideline page 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, 16 sources.

Cite this page: Peptency Academy. HPLC purity explained: what the percent on a peptide CoA measures. Version 3./eu/academy/testing/hplc-purity/