Peptency Academy · Testing
Purity is not content: what a peptide powder is made of
Why is peptide purity not the same as peptide content?
Full text, Purity Determination of Bulk Material by Mass Balance: "The 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."; Value Assignment: "All 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 (USP and MHRA authors)
Regulatory content shown for: EU and UK.
Known, not known, contested
Known
- Peptides made by solid phase synthesis are cleaved and purified with trifluoroacetic acid, so they are obtained as TFA salts. Source sentence
Abstract: "The 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."
2 Pharmaceuticals 2025 (ETH Zurich) - In the USP mass balance method, HPLC impurities are measured as a percent of total detected area and counterion, residual solvents and inorganic residue as percent by weight, and the two are combined into one mass fraction: the share of the material's mass that is peptide. Source sentence
Full text, Table VII and text: "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"; full text, mass balance worked example (Table VII and text): purity 0.93, peptide as free base per unit mass of material, anhydrous basis
1 Pharm Res 2023 (USP and MHRA authors) - In a candidate certified reference material of angiotensin II, the TFA counterion was nearly 25 percent of the mass and the assigned purity was a mass fraction of 69.1 ± 0.9 percent angiotensin II. Source sentence
Abstract: "a validated 19F-qNMR method to measure the trifluoroacetic acid (TFA) counter-ion, considered an impurity in this case at nearly 25% by mass"; abstract: final purity value assignment of the candidate CRM, 691 ± 9 per thousand by mass (k = 2) (69.1 ± 0.9 percent, unit conversion)
3 Anal Bioanal Chem 2018 (National Research Council Canada) - Two commercial lots of the same 21 residue peptide, MOG35-55, with similar purity had peptide fractions of 45 and 72 percent. Source sentence
Abstract: "a 21-residue peptide"; "two commercially available MOG35-55 peptides of similar purity but different peptide fraction (MOG-A being 45%; MOG-B being 72%)"
4 J Neurochem 2014
Not known
- No peer reviewed survey of net peptide content across sellers of research peptides was found in a PubMed title and abstract search on 13 Oct 2026. Source sentence
Query in the source record; 14 hits, none measures net peptide content across research peptide sellers
5 PubMed search, 13 Oct 2026 - The authors of the 2024 test purchase study did not identify the material that made up the rest of the powder mass. Source sentence
Full text, Results after Table 4: "These findings suggest the presence of low-mass, weakly ionizing impurities, although further studies using additional analytical techniques are necessary to conclusively identify contaminants."
6 J Med Internet Res 2024
Contested
- The word purity names two different measurements. USP and the National Research Council Canada use it for a mass fraction, the share of the material's mass that is peptide, while product labels in the 2024 test purchase study used purity for a 99 percent claim that the authors' mass based measurement did not match. Source sentence
PMID 36949371 full text, mass balance worked example (Table VII and text): purity 0.93, peptide as free base per unit mass of material, anhydrous basis; PMID 30143839 abstract: final purity value assignment of the candidate CRM, 691 ± 9 per thousand by mass (k = 2); PMID 39509151 abstract: "measured semaglutide purity was significantly low, ranging between 7.7% and 14.37% and deviating from the 99% claimed on product labels"
1 Pharm Res 2023 (USP and MHRA authors)3 Anal Bioanal Chem 2018 (National Research Council Canada)6 J Med Internet Res 2024
Does 99 percent purity mean 99 percent of the powder is peptide?
Full text, Purity Determination of Bulk Material by Mass Balance: "HPLC impurities, which are measured as a % of the total detected area by chromatography, and counter ion / impurities measured on a weight/weight basis"1 Pharm Res 2023 (USP and MHRA authors) In a 2024 test purchase study of three lyophilised semaglutide vials bought from online sellers without a prescription, the labels claimed 99 percent purity and no peptide-like impurities were identified, yet the measured purity was 7.7 to 14.37 percent. Source sentence
Abstract: "Test purchases were completed from 6 illegal online pharmacies"; full text Table 4 lists three lyophilised powder vials; "no peptide-like impurities were identified"; "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"6 J Med Internet Res 2024 The study's full text calculates that figure as measured semaglutide mass against the total powder mass in each vial. Source sentence
Full text, Table 4: columns Total weight of powder in vial (mg), Measured semaglutide content (mg) and Measured purity (%); for example 50.1 mg powder, 3.86 mg semaglutide, 7.70 percent6 J Med Internet Res 2024
What is in a peptide powder, and how each part is measured
- Target peptide
- Measured as content or assay by liquid chromatography against a reference standard, amino acid analysis, nitrogen analysis by Kjeldahl, elemental analysis or quantitative NMR, and reported as a mass fraction. Source sentence
Section 3.2.S.4 Specification list: "assay/content; e.g. by LC, elemental analysis, amino acid analysis, nitrogen analysis by Kjeldahl, or qNMR"
7 EMA guideline on synthetic peptides, EMA/CHMP/CVMP/QWP/367182/20253 Anal Bioanal Chem 2018 (National Research Council Canada) - Peptide related impurities
- Peptide related impurities are measured by RP-HPLC and reported as a percent of the total detected peak area. Source sentence
Full text, Chromatography: "RP-HPLC was used to determine the lot homogeneity, stability, identity, content, and purity"; Table VII: "Sum of HPLC impurities (I1) ... % Total Detected Area"
1 Pharm Res 2023 (USP and MHRA authors) - Counterion: acetate
- The acetate counterion is measured by HPLC under USP general chapter 503 and reported as percent by weight. Source sentence
Full text, Methods: "Acetic Acid content was analyzed by HPLC method according to the method described in USP general chapter < 503 > Acetic Acid in Peptides"; Table VII: "Acetic acid Content (I2)/ USP < 503 > %w/w"
1 Pharm Res 2023 (USP and MHRA authors) - Counterion: TFA
- Residual TFA is measured by HPLC under USP general chapter 503.1, and also by 19F NMR, FT-IR and HPLC with an evaporative light scattering detector, and reported as percent by weight. Source sentence
PMID 36949371 full text: "Trifluoroacetic acid content was evaluated by HPLC using the method in USP general chapter < 503.1 >"; PMID 40872554 abstract: "Detection and quantification of residual TFA- were carried out via FT-IR, 19F-NMR, and HPLC using an evaporative light-scattering detector (ELSD)."
1 Pharm Res 2023 (USP and MHRA authors)2 Pharmaceuticals 2025 (ETH Zurich) - Water
- Water is measured by Karl Fischer titration under USP general chapter 921 and reported as percent by weight. Source sentence
Full text, Methods: "Water Content was determined by Karl Fischer titration as described in USP general chapter < 921 > Water Determination"
1 Pharm Res 2023 (USP and MHRA authors) - Residual solvents
- Residual solvents are measured by gas chromatography under USP general chapter 467 and reported as percent by weight. Source sentence
Full text, Methods: "Residual solvents were analyzed by gas chromatography as described in USP general chapter < 467 > Residual Solvents"
1 Pharm Res 2023 (USP and MHRA authors) - Inorganic residue
- Inorganic residue is measured as residue on ignition under USP general chapter 281 and reported as percent by weight. Source sentence
Full text, Methods: "Inorganic impurities/thermal ash were determined by residue on ignition according to USP general chapter < 281 >"
1 Pharm Res 2023 (USP and MHRA authors)
A worked example: how USP turns these numbers into content
- The USP authors give a theoretical example for a peptide with acetic acid as the counterion; the figures in these steps are theirs, not measurements of any product. Source sentence
Full text: "Table VII shows a theoretical example for a peptide with acetic acid as the counter ion and trifluoracetic acid as a reagent during synthesis."
1 Pharm Res 2023 (USP and MHRA authors) - HPLC impurities sum to 0.92 percent of the total detected area, so the chromatographic factor is 99.08 percent. Source sentence
Full text, Table VII: "Sum of HPLC impurities (I1)/ Monograph % Total Detected Area 0.92"; "[(99.08) ÷ 100]"
1 Pharm Res 2023 (USP and MHRA authors) - Acetic acid is 5.58 percent by weight, trifluoroacetic acid 0.003 percent, residual solvents 0.003 percent and residue on ignition 0.00 percent, so the mass factor is 94.41 percent. Source sentence
Full text, Table VII: "Acetic acid Content (I2)/ USP < 503 > %w/w 5.58 Trifluoroacetic acid Content (I3)/ USP < 503.1 > %w/w 0.003 Residual solvents Content (I4)/ USP < 467 > %w/w 0.003 Residue on Ignition (I5)/ USP < 281 > %w/w 0.00"; "[(94.41) ÷ 100]"
1 Pharm Res 2023 (USP and MHRA authors) - Multiplying the two factors gives a mass fraction of 0.93: on the anhydrous basis, 93 percent of the material's mass is peptide, as free base. Source sentence
Full text, after Table VII: the theoretical example "would have a purity of 0.93" of peptide (free base) relative to material on the anhydrous basis
1 Pharm Res 2023 (USP and MHRA authors) - Correcting for 1.5 percent water, measured at the time of the HPLC analysis, gives a mass fraction of 0.92 (92 percent) on the as is basis. Source sentence
Full text, Table VIII: "L6 0.93 1.5 0.92"; "the purity is further adjusted to the “as is” basis by applying the water content of the individual lab’s material"
1 Pharm Res 2023 (USP and MHRA authors) - Read from these figures: the HPLC impurities are under 1 percent of the area, yet peptide is 92 percent of the material's mass, because the acetic acid and the water are measured by mass and subtracted separately. Source sentence
Computed from Table VII and Table VIII values above (0.92 percent area; 5.58 percent acetic acid; 1.5 percent water; mass fraction 0.92)
1 Pharm Res 2023 (USP and MHRA authors)
Published cases where purity and content diverged
- Two commercial lots of MOG35-55, a 21 residue peptide, had similar purity in the authors' words and peptide fractions of 45 percent and 72 percent. Source sentence
Abstract: "two commercially available MOG35-55 peptides of similar purity but different peptide fraction (MOG-A being 45%; MOG-B being 72%)"
4 J Neurochem 2014 - The authors write that the peptide fraction of purified material can vary from less than 50 percent to over 90 percent, with the remainder of the mass primarily the counterion used for purification, and that knowledge of peptide fraction is as critical as knowledge of peptide purity when peptides come from different sources. Source sentence
Abstract: "can vary from less than 50% to over 90%, with the remainder of mass primarily comprised of the counter ion used for peptide purification"; "These findings underscore the fact that a knowledge of peptide fraction is as critical as knowledge of peptide purity when using peptides from different sources."
4 J Neurochem 2014 - For an angiotensin II candidate certified reference material, the National Research Council Canada assigned purity as a mass fraction of 69.1 ± 0.9 percent (k = 2) by combining mass balance, 1H qNMR and LC-MS/MS amino acid analysis; the TFA counterion was nearly 25 percent of the mass. Source sentence
Abstract: "Ideally, results from these three orthogonal methods can be combined"; "TFA counter-ion, considered an impurity in this case at nearly 25% by mass"; abstract: final purity value assignment of the candidate CRM, 691 ± 9 per thousand by mass (k = 2)
3 Anal Bioanal Chem 2018 (National Research Council Canada) - Three lyophilised semaglutide vials bought from online sellers were analysed by liquid chromatography with mass spectrometry: labels claimed 99 percent purity, no peptide-like impurities were identified, and semaglutide was 7.7 to 14.37 percent of the powder. Source sentence
Abstract: "quantitative sample analysis using liquid chromatography coupled with mass spectrometry"; "no peptide-like impurities were identified"; "ranging between 7.7% and 14.37% and deviating from the 99% claimed on product labels"; full text Table 4 for the powder mass basis
6 J Med Internet Res 2024 - The same vials held more semaglutide per vial than the label stated, by 28.56 to 38.69 percent, so content per vial, label amount and mass fraction are three separate numbers. Source sentence
Abstract: "The semaglutide content of samples substantially exceeded labeled amounts by 28.56%-38.69%"
6 J Med Internet Res 2024
What does acetate or TFA in a peptide name mean?
PMID 36949371 full text: "For identification of leuprolide acetate presented here"; "Variability of Value Assignment for Desmopressin Acetate"; PMID 40872554 full text: "stoichiometric pairing of each positive charge with a counterion"1 Pharm Res 2023 (USP and MHRA authors)2 Pharmaceuticals 2025 (ETH Zurich) TFA in a name stands for trifluoroacetate, the counterion carried over from the trifluoroacetic acid used to cleave and purify peptides made by solid phase synthesis. Source sentence
Abstract: "The 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."2 Pharmaceuticals 2025 (ETH Zurich) The EMA guideline on synthetic peptides says acetate is usually used as the counterion, that trifluoroacetate or chloride are also possible, and that the counterion type is defined and its amount controlled in the active substance specification. Source sentence
Section 3.2.S.4.1 footnote **: "For synthetic peptides, usually acetate is used as counter ion, however, other counter-ions are also possible (e.g. trifluoroacetate or chloride). The type of counter ion should be defined, and the amount of counter ions should be controlled in the active substance specification with a justified upper limit."7 EMA guideline on synthetic peptides, EMA/CHMP/CVMP/QWP/367182/2025
What is net peptide content and how is it measured?
Full text, Value Assignment: "All detectable impurities (e.g., peptide related impurities, counter ion, water, residual solvents, non-combustible residues) are measured and subtracted from 100% to assign purity"; "the peptide mass content of lyophilized vials"1 Pharm Res 2023 (USP and MHRA authors) It is measured directly by amino acid analysis after hydrolysis, by quantitative NMR of the intact peptide, by nitrogen analysis, or by liquid chromatography against a characterised reference standard, or it is assigned by mass balance, which subtracts every measured impurity from 100 percent. Source sentence
PMID 30143839 abstract: "amino acid analysis by isotope dilution tandem mass spectrometry (LC-MS/MS) following peptide hydrolysis"; "quantitative nuclear magnetic resonance (qNMR) spectroscopy allows for quantitation of intact peptides"; "The mass balance approach ... purity is defined by the sum of the mass fraction of all impurities identified"; EMA guideline, Assay: "A test for Assay (e.g., based on a chromatographic method usually the same as for purity) using a reference standard should be considered. Other tests like elemental analysis, amino acid analysis, nitrogen analysis by Kjeldahl, or qNMR may be used."3 Anal Bioanal Chem 2018 (National Research Council Canada)7 EMA guideline on synthetic peptides, EMA/CHMP/CVMP/QWP/367182/20251 Pharm Res 2023 (USP and MHRA authors) The National Research Council Canada combined amino acid analysis by LC-MS/MS, 1H qNMR and mass balance for its angiotensin II reference material and reports excellent agreement between them. Source sentence
Abstract: "Using a candidate certified reference material (CRM) for angiotensin II, excellent agreement was achieved with the three methods."3 Anal Bioanal Chem 2018 (National Research Council Canada)
Why is there water in a lyophilised peptide powder?
Full text: "Residual moisture was determined destructively by coulometric Karl Fischer titration"; "Water content from individual labs is used to account for differences in humidity during testing. Water content is determined at the time of preparation for performing the HPLC analysis"1 Pharm Res 2023 (USP and MHRA authors) The EMA guideline lists water content among the general properties to describe for a synthetic peptide and asks that hygroscopicity be indicated. Source sentence
Section 4.1.3 General Properties: "Relevant general properties of the peptide in question should be described, such as, water content ... Hygroscopicity needs to be indicated"7 EMA guideline on synthetic peptides, EMA/CHMP/CVMP/QWP/367182/2025 USP moved many peptide reference standards from powders to lyophilised vials to eliminate the need for the user to determine counterions and residual moisture before use. Source sentence
Full text, Introduction: "USP has shifted many peptide reference standards from powdered to lyophilized forms to eliminate the need for the user to determine counter-ions and residual moisture prior to use"1 Pharm Res 2023 (USP and MHRA authors)
What do Da, percent area, percent w/w, mass fraction, anhydrous basis and free base mean on a CoA?
Full text, Table VII: "% Total Detected Area" and "%w/w"; full text, mass balance worked example (Table VII and text): purity 0.93, peptide as free base per unit mass of material, anhydrous basis; "adjusted to the “as is” basis"1 Pharm Res 2023 (USP and MHRA authors)
Analytical notation on a CoA
- Da (dalton)
- Unit of molecular mass, equal to 1/12 of the mass of a free carbon 12 atom at rest in its ground state; the dalton and the unified atomic mass unit (u) are the same unit. Source sentence
Table 8, note (i): "The dalton (symbol Da) and the unified atomic mass unit (symbol u) are alternative names (and symbols) for the same unit, equal to 1/12 of the mass of a free carbon 12 atom, at rest and in its ground state."
8 BIPM SI Brochure, 9th edition - mg
- Milligram, a mass of material, for example the total powder in a vial or the measured peptide in it. Source sentence
Full text, Table 4: Total weight of powder in vial (mg); Measured semaglutide content (mg)
6 J Med Internet Res 2024 - Percent area
- A peak's share of the chromatogram's total detected area. HPLC purity and HPLC impurities are reported this way. Source sentence
Full text, Table VII: "Sum of HPLC impurities (I1)/ Monograph % Total Detected Area"
1 Pharm Res 2023 (USP and MHRA authors) - Percent by weight (w/w)
- A part's share of the material's mass, used for counterion, residual solvents, residue on ignition and water. Source sentence
Full text, Table VII: "%w/w" for acetic acid, trifluoroacetic acid, residual solvents and residue on ignition
1 Pharm Res 2023 (USP and MHRA authors) - Mass fraction
- The share of the material's mass that is peptide, written as a fraction (0.93) or as a percent by weight (93 percent). Source sentence
PMID 36949371 full text, mass balance worked example (Table VII and text): purity 0.93, peptide as free base per unit mass of material, anhydrous basis; PMID 30143839 abstract: final purity value assignment of the candidate CRM, 691 ± 9 per thousand by mass (k = 2)
1 Pharm Res 2023 (USP and MHRA authors)3 Anal Bioanal Chem 2018 (National Research Council Canada) - Anhydrous basis
- Calculated with water excluded. Source sentence
Full text: purity 0.93 of peptide (free base) relative to material on the anhydrous basis; water is then applied separately
1 Pharm Res 2023 (USP and MHRA authors) - As is basis
- Calculated on the material as weighed, water included. Source sentence
Full text: "the purity is further adjusted to the “as is” basis by applying the water content of the individual lab’s material"
1 Pharm Res 2023 (USP and MHRA authors) - Free base, counter-ion free
- Peptide mass without the salt or counterion. Source sentence
Full text: purity 0.93 of peptide (free base) relative to material; the free base value excludes the acetic acid counterion measured separately (Table VII)
1 Pharm Res 2023 (USP and MHRA authors)
Pharmacopoeia chapters for each part of the mass balance
- USP <503>Acetic acid in peptides 1 Pharm Res 2023 (USP and MHRA authors)
- USP <503.1>Trifluoroacetic acid (TFA) in peptides 1 Pharm Res 2023 (USP and MHRA authors)
- USP <921>Water determination 1 Pharm Res 2023 (USP and MHRA authors)
- USP <467>Residual solvents 1 Pharm Res 2023 (USP and MHRA authors)
- USP <281>Residue on ignition 1 Pharm Res 2023 (USP and MHRA authors)
How the EMA guideline defines content
EU
- The EMA Guideline on the development and manufacture of synthetic peptides, EMA/CHMP/CVMP/QWP/367182/2025, was adopted by CHMP on 1 December 2025 and came into effect on 1 June 2026. Source sentence
Cover page: "Adopted by CHMP 1 December 2025"; "Date of coming into effect 1 June 2026"
7 EMA guideline on synthetic peptides, EMA/CHMP/CVMP/QWP/367182/2025 - For authorised medicines it asks for the counter ion to be indicated, with whether it is present in a stoichiometric or non-stoichiometric ratio. Source sentence
Section 4.1.3: "The counter ion needs to be indicated, if relevant, and whether it is present in a stoichiometric or non-stoichiometric ratio."
7 EMA guideline on synthetic peptides, EMA/CHMP/CVMP/QWP/367182/2025 - It lists assay or content, counter-ion identity and content, residual TFA and water content among the active substance specification tests. Source sentence
Specification list: "assay/content"; "counter-ion identity and content, e.g. acetic acid, if relevant"; "residual ion content (TFA)"; "water content"
7 EMA guideline on synthetic peptides, EMA/CHMP/CVMP/QWP/367182/2025 - It expresses liquid chromatography assay limits in terms of the counter-ion free, anhydrous substance, unless otherwise justified. Source sentence
Assay: "Limits of assays determined by LC are expressed in terms of the counter-ion free, anhydrous substance, unless otherwise justified."
7 EMA guideline on synthetic peptides, EMA/CHMP/CVMP/QWP/367182/2025 - It asks that the strength of a finished medicine be defined as the mass of peptide base, not including salt or counter-ion. Catalog research materials are not medicines; this describes how the terms are defined. Source sentence
Finished product section: "the strength of the finished product should be defined with respect to the mass of peptide base (not including salt or counter-ion)."
7 EMA guideline on synthetic peptides, EMA/CHMP/CVMP/QWP/367182/2025
What a CoA needs to say for the two numbers to be read
Illustrative layout, not a Peptency lot
- Purity
- Method
- RP-HPLC with UV detection
- Reports
- Main peak as percent of total detected area
- Content (assay)
- Method
- LC against a reference standard, amino acid analysis, nitrogen analysis or qNMR
- Reports
- Peptide mass fraction, with its basis (anhydrous, as is, free base)
- Counterion
- Method
- HPLC (USP <503>, <503.1>) or 19F NMR
- Reports
- Identity of the counterion and percent by weight
- Water
- Method
- Karl Fischer titration (USP <921>)
- Reports
- Percent by weight
- For the two numbers to be read, a CoA needs purity with its method and basis, content or assay with its method and basis, counterion identity and amount, and water content: these are the separate tests the EMA specification list and the USP mass balance use. Source sentence
EMA specification list: "purity", "assay/content", "counter-ion identity and content", "residual ion content (TFA)", "water content"; PMID 36949371 Table VII and VIII
7 EMA guideline on synthetic peptides, EMA/CHMP/CVMP/QWP/367182/20251 Pharm Res 2023 (USP and MHRA authors)
Sources
- 1Pharm Res 2023 (USP and MHRA authors) accessed 13 Oct 2026
- 2Pharmaceuticals 2025 (ETH Zurich) accessed 13 Oct 2026
- 3Anal Bioanal Chem 2018 (National Research Council Canada) accessed 13 Oct 2026
- 4J Neurochem 2014 accessed 13 Oct 2026
- 5PubMed search, 13 Oct 2026 accessed 13 Oct 2026
- 6J Med Internet Res 2024 accessed 13 Oct 2026
- 7EMA guideline on synthetic peptides, EMA/CHMP/CVMP/QWP/367182/2025 accessed 13 Oct 2026
- 8BIPM SI Brochure, 9th edition 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 2, last edited 13 October 2026, 8 sources.
Cite this page: Peptency Academy. Peptide purity vs peptide content: what each number measures. Version 2./eu/academy/testing/purity-is-not-content/