How to Read a Peptide Certificate of Analysis: Purity, HPLC and Mass Spec Explained

A certificate of analysis (CoA) is the one document that tells you what is actually in a vial of research peptide. It is also the document most often skimmed, misread or taken on trust. A single number such as "99% purity" can look reassuring while leaving the most important questions unanswered.

This guide walks through a typical peptide CoA line by line. It covers what each test measures, what a good result looks like, and the gaps that a headline number can hide. It is written for laboratory buyers who want to check the material in front of them before it goes anywhere near an experiment.

What a certificate of analysis is (and is not)

A CoA is a report from an analytical laboratory. It states which tests were run on a specific batch, or lot, of material and what the results were, compared against a specification.

Three things make a CoA worth trusting:

1. It names a specific lot. A CoA that doesn't tie to a lot number printed on your vial describes some other material, not yours.
2. It names the laboratory and the test date. An anonymous report with no date cannot be checked.
3. It shows the evidence. A chromatogram and a mass spectrum, not just a typed number.

A CoA is not a statement that the material is safe or suitable for any use outside the laboratory. It documents identity and quality attributes of a research material. Nothing more.

Section 1: The header — lot, compound, date, laboratory

Start at the top. Before you read any result, check:

• Compound name and sequence. Does the name match what you ordered? For well-characterized peptides, a CoA often lists the amino acid sequence and molecular formula. If you want a refresher on how peptide sequences are written, see our guide to what a peptide is.
• Lot or batch number. It should match the number printed on your vial label exactly.
• Test date and laboratory name. Look for a named lab, ideally with an accreditation reference such as ISO/IEC 17025.
• Specification vs. result. A good CoA shows both the target (for example "≥ 98%") and the measured value (for example "99.2%"). A report that shows only the specification has told you what was hoped for, not what was found.

Section 2: HPLC purity — the number everyone quotes

High-performance liquid chromatography (HPLC) separates the components of a sample as they pass through a packed column. Each component leaves the column (elutes) at a characteristic time and shows up as a peak on a UV detector trace, the chromatogram.

"Purity by HPLC" is usually calculated as the area of the main peak divided by the total area of all peaks detected. A result of 99% means that, under those conditions, 99% of the UV signal came from the main peak.

What to look for on the chromatogram

• One dominant peak at the expected retention time.
• Small, well-separated minor peaks. These are impurities, usually peptide-related ones such as sequences missing a residue (deletion sequences), truncated chains, or chains with leftover protecting groups from synthesis.
• Detection wavelength. Peptide backbones absorb around 210–220 nm, so this is the usual range. The method should be stated.
• An actual trace image. A typed "99.1%" without a chromatogram is a claim, not evidence.

What HPLC purity does not tell you

This is where headline numbers mislead:

• It does not confirm identity. HPLC shows that one component dominates. It does not show that the component is the right molecule. A wrong peptide can be very pure.
• It does not measure how much peptide is in the vial. A 5 mg vial at 99% HPLC purity may still contain less than 5 mg of peptide, because the powder also contains water and counterions (see Section 4).
• It only sees what the detector sees. Contaminants that don't absorb UV at the chosen wavelength, such as inorganic salts or some metals, don't appear.

Published analyses of peptide products bought online show why this matters. Researchers at Belgium's Scientific Institute of Public Health screened the ten most frequently encountered falsified peptide products on the Belgian market, bought from suspected illegal internet pharmacies. They reported high variation in the amount of peptide per unit, purities as low as 5–75% for cysteine-containing peptides, and elemental impurities including arsenic and lead (Janvier et al., Talanta, 2018; DOI: 10.1016/j.talanta.2018.06.023). HPLC purity alone would not have caught the metals.

Section 3: Mass spectrometry — confirming identity

Mass spectrometry (MS), usually coupled to liquid chromatography as LC-MS, measures the mass-to-charge ratio of ionized molecules. For a peptide, it answers the question HPLC can't: is the main component the molecule it's supposed to be?

What to look for

• Theoretical vs. observed mass. The CoA should state the expected molecular weight (calculated from the sequence) and the observed mass. They should agree closely. High-resolution instruments agree to within a few parts per million. Lower-resolution instruments agree to within a fraction of a dalton.
• Charge states. Peptides often ionize with more than one proton attached, so you may see peaks at [M+2H]²⁺ or [M+3H]³⁺. For example, [M+2H]²⁺ appears at roughly half the molecular mass plus one. A good report labels these.
• The spectrum itself. Again, an image beats a typed number.

Forensic and control laboratories use exactly this pairing. A 2015 method from the Belgian Official Medicines Control Laboratory used LC-MS/MS to identify 25 different peptides in suspected counterfeit and illegal preparations, then UHPLC with diode-array detection to quantify them (Vanhee et al., Talanta, 2015; DOI: 10.1016/j.talanta.2015.04.022). Identity and quantity were two separate measurements, which is the point.

A Danish study of melanotan II vials bought from three online shops makes the same case. Researchers confirmed identity by retention time and five fragment ions, then quantified the peptide by LC-UV. Every shop claimed 10 mg per vial. The measured content ranged from 4.32 mg to 8.84 mg (Breindahl et al., Drug Testing and Analysis, 2015; DOI: 10.1002/dta.1655). The right molecule was present. The label amount was not.

Section 4: Peptide content, water and counterions

This is the section most CoAs leave out, and the one that explains the gap between "5 mg" on the label and what is actually there.

Lyophilized (freeze-dried) peptide powder is never 100% peptide. It also contains:

• Residual water. Freeze-dried material still holds some moisture, often measured by Karl Fischer titration.
• Counterions. Peptides made by solid-phase synthesis are usually cleaved from the resin and purified using trifluoroacetic acid (TFA), so they are commonly isolated as TFA salts. Acetate and chloride are other common counterions.

A 2025 study from ETH Zurich describes TFA's role as both a cleavage agent and an ion-pairing reagent in peptide synthesis and purification. It notes that peptides are therefore obtained as TFA salts, and that many studies report TFA salts can interfere with biological assays. The authors validated several methods to quantify residual TFA and argued for counterion quantification and specification in assays that use synthetic peptides (Erckes et al., Pharmaceuticals, 2025; DOI: 10.3390/ph18081163). An earlier comparison of ion chromatography, capillary electrophoresis and isotachophoresis for measuring acetate, trifluoroacetate and chloride in synthetic peptides found ion chromatography performed best (Mrozik et al., Journal of Peptide Science, 2012; DOI: 10.1002/psc.1436).

What to look for

• Net peptide content (sometimes "peptide content" or "assay"), often determined by amino acid analysis or nitrogen content. It's expressed as a percentage of the powder's total mass.
• Counterion identity and amount, for example "TFA content: X%".
• Water content, for example "Water (KF): X%".

If a CoA shows only HPLC purity, you know the proportion of peptide relative to other UV-visible components. You do not know how much of the powder is peptide.

Section 5: Endotoxin (LAL)

Endotoxins are lipopolysaccharides from the outer membrane of Gram-negative bacteria. They are a common contaminant in biological materials, and they can confound cell-based and in vivo experiments even at low levels.

The standard test is the Limulus amebocyte lysate (LAL) assay, an in vitro test that uses the blood cells (amebocytes) of the horseshoe crab and detects endotoxin specifically. A review of more than 70 years of pyrogen testing traces its development: the rabbit pyrogen test in the 1940s, the LAL test in the 1970s, and the non-animal Monocyte Activation Test around 2010 (Fennrich et al., Alternatives to Laboratory Animals, 2016; DOI: 10.1177/026119291604400305).

What to look for

• A stated result in endotoxin units (EU) per mg or per vial, compared to a specification.
• The method (gel-clot, turbidimetric or chromogenic LAL; or a recombinant Factor C assay).
• "Pass" alone is weaker than a numeric result against a numeric limit.

Section 6: Appearance and solubility

Most CoAs include a simple visual check, for example "white to off-white lyophilized powder." It isn't sophisticated, but a mismatch on arrival (discoloration, a collapsed or melted-looking cake, visible moisture) is a useful early warning that the material may have been compromised in storage or transit. Our guide to lyophilized peptide storage and cold-chain basics covers why. Related reading: Lyophilized Peptide Storage and Cold-Chain Basics (2–8 °C): A Laboratory Guide.

A quick CoA checklist

Before accepting a lot into your lab, ask:

Check	Good sign	Warning sign
Lot match	CoA lot number = vial lot number	Generic CoA, no lot, or a different lot
Laboratory	Named lab, test date, accreditation reference	Anonymous, undated
HPLC	Measured result + chromatogram image	Only a spec, or a typed number with no trace
Identity	Observed mass matches theoretical, spectrum shown	No MS data at all
Content	Net peptide, water and counterion reported	Only HPLC purity
Endotoxin	Numeric EU result vs. limit	Not tested, or "pass" with no value
Traceability	Report reachable from the lot number	PDF emailed on request only

Why lot-level records matter

The single most useful habit is to tie every document back to the lot number on the vial in your hand. A generic "representative" CoA describes a batch that may have nothing to do with yours.

That is why Seratide prints a lot number on every vial and publishes a per-lot record on its Lot lookup page. The records on that page today are clearly labeled layout samples that show the format (HPLC purity, LC-MS identity and endotoxin against specification). Issued laboratory reports replace them as each lot is analyzed. The principle is the one this guide keeps coming back to: the evidence should belong to your lot, and you should be able to check it yourself.

You can browse the catalog on our research peptides shelf. Each listing shows the compound, strength, form and lot record format.

Sources

All sources below were opened and checked against their PubMed records while drafting.

1. Janvier S, et al. Impurity profiling of the most frequently encountered falsified polypeptide drugs on the Belgian market. Talanta. 2018;188:795-807. PubMed: https://pubmed.ncbi.nlm.nih.gov/30029448/ · DOI: https://doi.org/10.1016/j.talanta.2018.06.023
2. Vanhee C, et al. Analysis of illegal peptide biopharmaceuticals frequently encountered by controlling agencies. Talanta. 2015;142:1-10. PubMed: https://pubmed.ncbi.nlm.nih.gov/26003685/ · DOI: https://doi.org/10.1016/j.talanta.2015.04.022
3. Breindahl T, et al. Identification and characterization by LC-UV-MS/MS of melanotan II skin-tanning products sold illegally on the Internet. Drug Test Anal. 2015;7(2):164-72. PubMed: https://pubmed.ncbi.nlm.nih.gov/24771717/ · DOI: https://doi.org/10.1002/dta.1655
4. Erckes V, et al. Towards a Consensus for the Analysis and Exchange of TFA as a Counterion in Synthetic Peptides and Its Influence on Membrane Permeation. Pharmaceuticals (Basel). 2025;18(8):1163. PubMed: https://pubmed.ncbi.nlm.nih.gov/40872554/ · DOI: https://doi.org/10.3390/ph18081163
5. 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):192-8. PubMed: https://pubmed.ncbi.nlm.nih.gov/22252914/ · DOI: https://doi.org/10.1002/psc.1436
6. Fennrich S, et al. More than 70 years of pyrogen detection: Current state and future perspectives. Altern Lab Anim. 2016;44(3):239-53. PubMed: https://pubmed.ncbi.nlm.nih.gov/27494624/ · DOI: https://doi.org/10.1177/026119291604400305

For laboratory research use only.