Peptide quality: a complete guide to COA, HPLC and purity

What peptide quality is and why it matters

When a physician or researcher evaluates a peptide, the first question is chemical rather than pharmacological: does the vial contain the declared molecule, in the declared amount and without relevant contaminants? The European Medicines Agency (EMA), in its guideline on the development and manufacture of synthetic peptides, in effect since 1 June 2026, puts it plainly: purity is one of the most important critical quality attributes of this class of molecules¹. Impurities are not a technical footnote: a Ghent University review shows they can influence early functional studies and lead to erroneous conclusions².

The World Health Organization (WHO) separates two problems. Substandard products are those that do not meet their quality standards and specifications, often because of poor manufacturing practices or inadequate quality control; falsified products deliberately misrepresent their identity, composition or source³. According to the same fact sheet, at least 1 in 10 medicines in low- and middle-income countries is substandard or falsified³. This guide covers both sides: how quality is measured (certificate of analysis, HPLC, mass, content, endotoxin), how it is preserved (stability) and what labs have found when they test products from the market.

Where impurities come from: solid-phase synthesis

Most peptides today are made by solid-phase synthesis: the chain is built on a resin through repeated cycles of deprotection, washing and amino acid coupling, and is cleaved from the resin at the end¹². Every step can leave a trace. Incomplete coupling produces deletion sequences (an amino acid is missing); an amino acid coupled twice produces insertion sequences; epimerization creates diastereomers; protecting groups that are not fully removed leave adducts; and cleavage releases reactive species that can attack tryptophan, tyrosine or methionine¹². Oxidation products, dimers and oligomers add to the list².

There are also impurities that are not peptides: process reagents, residual solvents, elemental impurities and counter-ions such as trifluoroacetate (TFA), which comes from the synthesis itself or from purification¹². The EMA notes that synthetic peptides usually carry acetate as the counter-ion, although trifluoroacetate or chloride salts also exist, and asks for the counter-ion type to be defined and its amount limited¹. In the Ghent study, most of the related impurities found were precisely sequences missing amino acids⁴.

HPLC purity: what it measures and what it does not

The figure on almost every certificate (“purity ≥98%”) comes from reversed-phase high-performance liquid chromatography (HPLC) with an ultraviolet detector. It is area normalization: the area of the main peak against the sum of all detected peaks. It means that at least 98% of the integrated ultraviolet signal belongs to the main peak; it does not mean that 98% of the powder is peptide. A detailed reading of that number, with its wavelengths and reporting thresholds, is in how to read a peptide certificate of analysis.

The method has limits worth knowing. An impurity that elutes with the main peak is hidden inside it; the EMA asks manufacturers to account for that co-elution risk and, when impurities appear as one peak, to apply the 1.0% qualification threshold¹. For synthetic peptides, the European Pharmacopoeia states that related impurities are reported above 0.1%, identified above 0.5% and qualified above 1.0%¹. A good certificate states the method and attaches the chromatogram with the integrated peaks; the EMA asks for copies of the relevant chromatograms¹.

Identity: mass spectrometry and orthogonal methods

Purity tells you how much of the material is a single component; it does not tell you which one. That is the job of mass spectrometry. Electrospray ionization, described for large biomolecules by Fenn and colleagues in 1989, produces multiply charged ions from which the mass of the molecule is reconstructed⁵. The EMA asks for representative spectra and tables of theoretical and observed masses¹.

Mass has blind spots: two diastereomers weigh exactly the same. That is why, to identify a peptide in the specification and at batch release, the EMA recommends at least two orthogonal methods: mass, relative retention time, LC-MS, peptide mapping, bioactivity, amino acid analysis or nuclear magnetic resonance¹. The most-cited practical example comes from the Ghent group: among peptides ordered at ≥95% purity, the main compound of one sample had a different structure from the one requested⁴, an error a purity figure would never have revealed.

Purity is not content: counter-ions, water and net peptide

This is the point that causes the most confusion. A lyophilized powder with 99% HPLC purity also contains counter-ions, water and sometimes solvents. The best-documented case is a candidate certified reference material of angiotensin II: the TFA counter-ion made up nearly 25% of the mass, and the assigned purity value was 691 ± 9 mg/g⁶. In other words, about 69% of the powder was peptide. The EMA, for its part, states that limits for an assay by chromatography are expressed on the anhydrous, counter-ion-free substance¹.

The counter-ion does not just add weight; it can also act. In osteoblast cultures, TFA at 10⁻⁸–10⁻⁷ M reduced cell numbers, and the TFA salts of amylin and calcitonin masked a proliferative effect or suggested a nonexistent antiproliferative one; the authors recommended converting peptides to the hydrochloride before testing their biological effect⁷. Salt forms have their own consequences for GLP-1 drugs, reviewed in compounded GLP-1s: salts, impurities and antibodies.

Water is the third component. The EMA warns that peptides are often very hygroscopic powders, asks for hygroscopicity to be stated and expects water content to be part of stability protocols¹. The practical consequence is knowing whether the milligrams on a label refer to the powder or to net peptide.

How to read a certificate of analysis (COA)

The certificate of analysis summarizes the tests performed on a specific batch. The EMA's list for the specification of a synthetic peptide works as a template for reading any of them¹. Each test answers a different question:

• Appearance and identification (mass plus a second method): is it the declared molecule?¹⁵
• Purity (total and individual impurities): how much of the signal belongs to the main peak?¹
• High-molecular-weight impurities (aggregates, dimers, oligomers): has the peptide aggregated?¹
• Assay or content (for example by LC, amino acid analysis or qNMR): how much peptide is in the powder?¹⁶
• Counter-ion and residual TFA: which salt is it, and how much does it weigh?¹⁷
• Water and residual solvents: what else is in the powder?¹
• Bacterial endotoxins and microbiological purity: is it microbiologically acceptable?¹⁸

Endotoxins deserve a separate note. They are measured with the Limulus amebocyte lysate (LAL) assay, which has been accepted for more than 30 years in place of the rabbit pyrogen test and which is described in United States Pharmacopeia chapter <85>⁸. Sterile and endotoxin-free are not the same thing: the semaglutide vials tested in a market-surveillance study had no viable microorganisms, yet all contained endotoxin, between 2.16 and 8.95 EU/mg¹⁸. A properly reported result carries a number, units and a limit.

Finally, a certificate is valid only for the batch it describes: lot number, test dates, method, raw data and signature. The EMA expects material traceability from synthesis through to the final active substance¹. For newcomers, reading the label and the certificate of analysis, without fear walks through the same ground step by step.

Stability: from lyophilized powder to reconstituted vial

A high-purity peptide can still degrade later. The EMA lists the usual degradation pathways of synthetic peptides: oxidation, hydrolysis, isomerization, deamidation, cyclic imide (aspartimide) formation, diketopiperazine and pyroglutamic acid formation, β-elimination, disulfide exchange and acetylation, among others¹. Several are well quantified. Deamidation of asparagine and glutamine depends strongly on neighboring amino acids: a model covering 648 sequence combinations included very long half-times, 2–50 years for some sequences⁹. Aspartic acid residues hydrolyze at very acidic pH and isomerize above pH 6¹⁰. Photooxidation mainly affects tryptophan, tyrosine, phenylalanine and cysteine/cystine¹².

Lyophilized powder is not inert. In a lyophilized model hexapeptide, residual moisture, temperature and, above all, the type of bulking agent significantly influenced solid-state degradation¹¹. The EMA states that, to prevent or minimize degradation, peptides are generally stored refrigerated (5 °C ± 3 °C) or frozen (−20 °C ± 5 °C)¹. Physical pathways matter just as much: aggregation depends on sequence, concentration, pH, charge, surfaces and interfaces, temperature, agitation and lyophilization¹³, and adsorption to glass or plastic tubes can make the real concentration differ from the nominal one¹⁴.

Reconstitution starts the clock. Bacteriostatic water is sterile water with 0.9% or 1.1% benzyl alcohol in a multiple-dose container; its label warns against use in neonates and says prepared solutions should not be stored unless the drug manufacturer directs otherwise¹⁵. Approved peptides set specific in-use periods derived from their own studies: reconstituted tesamorelin is swirled, not shaken, kept at 20–25 °C and discarded after 7 days, with its bacteriostatic water bottle discarded after 28 days¹⁶; the semaglutide pen can be stored for 56 days after first use and must not be frozen or used if it was frozen¹⁷. Those periods do not transfer to other peptides. Each pathway is covered in detail in peptide stability: from lyophilized powder to reconstituted vial, and an example applied to one peptide in GHK-Cu: a complete guide to the copper peptide.

Counterfeit and substandard products: what the labs found

Market studies show how far the declared can be from the measured. In a surveillance study of semaglutide sold by illegal online pharmacies, all three lyophilized vials received were judged probably substandard or falsified: the semaglutide amount exceeded the label by 28.6–38.7%, measured purity was 7.7–14.4% against 99% claimed, and all contained endotoxin¹⁸. In a systematic analysis of the ten falsified peptides most often found on a European market, bought from illegal online pharmacies, purity of the cysteine-containing peptides was 5–75%, several samples carried arsenic up to ten times the toxicity limit for parenteral drugs, and one contained lead¹⁹. Another report found haemolytic Bacillus cereus in two illegal injectable peptides²⁰.

The clinical consequences can be serious. A case series described two people without diabetes or obesity who injected semaglutide obtained without a prescription and arrived in a coma with severe hypoglycemia, hypokalemia and hypothermia: the cause was synthetic insulin²¹. In 2024 the WHO issued an alert on falsified Ozempic batches detected in three countries, warning of incorrect dosing, contamination or substituted ingredients, and advised checking the lot number and serial number²². Problems exist outside falsification too: in compounded products combining tirzepatide with vitamin B12 analogs, a new and widespread impurity formed by a reaction between the two molecules was identified, with unknown clinical effects²³. The lab findings are reviewed in What is really in the vial? and the clinical background of these drugs in GLP-1 drugs: a complete guide.

Open questions

Several questions remain unresolved. The first is immunogenicity: related impurities can introduce T-cell epitopes, which is why frameworks have been proposed to assess the immunogenicity risk of peptides and their impurities²⁴. The second is missing data: for many peptides outside approved drugs, little stability data in reconstituted form has been published, and the available principles come from proteins, model peptides and approved products⁹¹¹¹³. The third is methodological: market studies test few samples (three vials in the semaglutide study) and cannot estimate the average quality of a market¹⁸. The reasonable approach is to treat quality as something verified batch by batch, with orthogonal methods and independent testing of key batches¹⁴.