Peptide Purity Guide: HPLC, Mass Spec and COAs

Purity in one minute

• Purity answers: of everything the test can see, how much is the right peptide?
• HPLC separates a sample into its parts and measures each one. It gives the purity percentage.
• Mass spectrometry weighs the molecules. It confirms the peptide is the right one, but it is not a purity test by itself.
• Peptide content is a different number. It is how much of the powder's weight is peptide, after water and salts are counted.
• Endotoxin and sterility need their own tests. HPLC does not show them.

Synthetic peptides can carry many small structural faults. These come from the starting materials, the way the peptide is made and how it is stored. That is why a single number never tells the whole story.

Sources: Lian Z et al, J Am Soc Mass Spectrom, 2021. Bachem, Peptide calculator, Knowledge Center.

How HPLC works

HPLC stands for high-performance liquid chromatography. A dissolved sample is pushed through a narrow column packed with fine particles. Different molecules travel through at different speeds, so they come out at different times.

A detector sits at the end. It usually measures how much ultraviolet light the liquid absorbs, and it draws a graph called a chromatogram. Each substance shows up as a peak. The tall main peak should be the target peptide. Smaller peaks are other substances.

The purity figure is the area of the main peak as a share of the total area of all the peaks. So 98 percent purity means 98 percent of the material that the detector could see was the main peak. It does not tell you:

• What the other 2 percent is
• Whether something was present that the detector cannot see
• Whether two different molecules came out at the same time and sit under one peak
• How much of the powder's weight is peptide

Some impurities are very hard to separate. One example is an epimer. This is a peptide with one amino acid in its mirror-image form. It behaves almost the same as the correct peptide. Special chiral HPLC methods combined with mass spectrometry were developed to find these.

Sources: Lian Z et al, J Am Soc Mass Spectrom, 2021. Strege MA et al, J Chromatogr B, 2023.

What mass spec adds

Mass spectrometry, often written MS, turns molecules into charged particles and measures their mass. If the main mass matches the expected weight of the peptide, that is strong evidence the right molecule is present.

When the two methods are joined, called LC-MS, each peak on the chromatogram can be weighed. That lets a lab name the impurities, not just count them. A 2022 study of the peptide thymalfasin used this approach to identify a range of closely related impurities in batches of the material.

What MS cannot do alone is tell you how pure a sample is. A mirror-image impurity has exactly the same mass as the correct peptide, so MS sees no difference. That is why a good report includes both an HPLC trace and an MS spectrum.

Sources: Lian Z et al, J Am Soc Mass Spectrom, 2021. Badgujar D et al, Enantiomeric purity of synthetic therapeutic peptides: a review, Chirality, 2024. Cheng Y et al, Identification and determination of structurally related peptide impurities in thymalfasin by liquid chromatography-high-resolution mass spectrometry, Anal Bioanal Chem, 2022.

Purity vs content

A vial labelled 10 mg is almost never 10 mg of pure peptide by weight. The powder also holds parts that are not peptide, such as salts and water. Peptides also pull moisture from the air, which lowers the peptide share of the weight further.

So two numbers can both be true at once. A sample can have high HPLC purity, meaning nearly all the peptide-like material is correct. It can still have a lower peptide content by weight, because some of the powder is water and salts. For example, two samples can both be 95 percent pure. If one has 80 percent peptide content and the other has 95 percent, the first holds less peptide. When a report gives only one number, ask which one it is.

Sources: Bachem, Handling and storage guidelines for peptides, Knowledge Center. Bachem, Peptide calculator, Knowledge Center.

Hidden impurities

These are the impurities most often discussed in the analytical literature for synthetic peptides:

• Deletion sequences: one amino acid missing from the chain
• Truncated sequences: a chain that stopped growing early
• Epimers: an amino acid in its mirror-image (D) form instead of the normal (L) form
• Chemical changes: oxidised or deamidated forms that appear during making or storage
• Leftover reagents: traces from the chemistry used to build the peptide

Why do they matter for research? One review looks at synthetic peptide drugs. It notes that regulators ask makers to assess whether impurities could trigger an immune response. This applies even when the main peptide does not. Another review explains where mirror-image impurities come from. They can form while the peptide is made, or come from the starting amino acids. For a researcher, an impurity is also simply a second molecule in the experiment that nobody planned for.

Sources: Lian Z et al, J Am Soc Mass Spectrom, 2021. Badgujar D et al, Enantiomeric purity of synthetic therapeutic peptides: a review, Chirality, 2024. De Groot AS et al, Immunogenicity risk assessment of synthetic peptide drugs and their impurities, Drug Discov Today, 2023.

Endotoxin tests

Endotoxins are fragments of the outer wall of certain bacteria, released when those bacteria die. They can enter a product through water, raw materials or equipment. Even tiny amounts can cause fever-like reactions in living systems.

HPLC does not detect endotoxin. It needs its own test, with methods set out by the US Pharmacopeia, and the amount is reported in endotoxin units, or EU. Published guidance for preclinical research gives limits for different animal models, because the right level depends on the study.

Sterility is another separate question. A sterility test checks for living microbes. A clean HPLC trace says nothing about either.

These tests matter in the real world. A 2024 study ordered semaglutide from six illegal online pharmacies, and three vials arrived. Endotoxin was found in all three. The measured purity was about 8 to 14 percent, far below the 99 percent printed on the labels.

Sources: Malyala P, Singh M, Endotoxin limits in formulations for preclinical research, J Pharm Sci, 2008. Schneier M et al, Current technologies to endotoxin detection and removal for biopharmaceutical purification, Biotechnol Bioeng, 2020. Ashraf AR et al, J Med Internet Res, 2024.

Reading a COA

COAs usually cover the freeze-dried powder described in our lyophilized peptide guide. Here is a clearly fictional certificate, so you can see where each piece of information sits. Sample only, not a real test, not a Glow Labs product.

A matching sample chromatogram would show peaks like this:

Is the COA real?

A COA is only useful if it belongs to your vial. Run through these checks.

1. The lot number on the COA matches the lot number on your vial.
2. The compound name and vial strength match. A report for one size does not prove another.
3. The test date makes sense for your batch.
4. The lab is named, and it has real contact details you can check.
5. Both an HPLC chromatogram and a mass spectrum are attached, not just typed numbers.
6. For a blend, each ingredient has its own result. One purity figure cannot describe two or three peptides.
7. The document is not a blurry copy with the lot number cropped out.

If any of these fail, the COA tells you very little about the vial in your hand.

These checks apply to any supplier's certificate. Certificates of analysis available on request. See the certificates of analysis page for how to ask. Orders ship across Australia with tracked delivery in 2-3 days.

Testing in Australia

According to the TGA, unapproved peptide products have not been assessed for quality, and what is printed on them may be wrong. In July 2026 it reported intercepting unlabelled vials at the border. It said such products may contain the wrong ingredients, be contaminated, or fail basic sterility standards. The TGA also lists specific peptides by name as examples of unapproved products.

Sport Integrity Australia says counterfeit peptides are sold online illegally, often with unknown purity and concentration. It adds that some may be contaminated with substances banned in sport.

A COA from any lab shows what one batch looked like on one day. It does not make a product approved, and it does not make it suitable for any use beyond research.

Sources: Therapeutic Goods Administration, Understanding your responsibilities when importing, compounding and supplying unapproved peptide products, Safety advisory, 13 April 2026. Therapeutic Goods Administration, Unapproved peptide product promoters and suppliers are put on notice, Media release, 20 July 2026. Sport Integrity Australia, Retatrutide (Reta) explained, Substance education.

FAQ

Sources: Lian Z et al, J Am Soc Mass Spectrom, 2021. Therapeutic Goods Administration, Understanding your responsibilities when importing, compounding and supplying unapproved peptide products, Safety advisory, 13 April 2026.