How to read a certificate of analysis (COA) for peptides

What a certificate of analysis is

A certificate of analysis, often called a COA, is a document that shows the results of testing performed on a specific batch, or lot, of a product.

The word ‘specific’ is important.  A COA should tell you what was found in the particular batch you received – not merely what the product is supposed to contain.

A COA is different from a Specification Sheet:

• • A Specification Sheet describes what a product is designed or expected to be like.
  • A COA reports what testing found in one particular batch.

These documents are useful for different reasons and should not be treated as interchangeable.

Identity: the top block

Compound name and sequence

The product name should be clear and specific. For peptides, the amino-acid sequence should also be listed, usually as a series of one-letter or three-letter codes.

The sequence is more reliable than the product name because different suppliers may use different names or abbreviations for similar materials. Compare the sequence on the COA with the sequence you ordered, one character or residue at a time.

If the product has a recognized reference number in a public scientific database, including that identifier can provide additional confirmation.

Molecular formula and molecular weight

The molecular formula and molecular weight are usually calculated from the product’s stated structure or sequence. These values are later compared with the results from mass spectrometry testing.

When reviewing the molecular weight, check whether the COA reports:

• • Average molecular weight, or
  • Monoisotopic molecular weight.

These are calculated differently. Comparing one type with the other can make a correct result appear to be incorrect.

Also check whether the weight refers to:

• • The main compound by itself, or
  • The compound combined with a salt or counterion.

For example, a peptide supplied in a salt form may weigh more than the same peptide by itself. The COA should make clear which form is being reported.

CAS number

A CAS number is an identification number used for many chemical substances.

Some compounds have one and some do not. This is especially common for custom or less-established peptide sequences. The absence of a CAS number is not automatically a problem. However, if a CAS number is included, it should match the material being described.

Lot number and dates

The lot number, also called the batch number, connects the COA to the actual vial or container you received.  The lot number on the COA should match the lot number on the product label. If the numbers do not match, the test results may not apply to your material.

The COA should also include relevant dates, such as:

• • Testing or analysis date
  • Retest date or expiry date, when applicable

The dates should make sense together.

Appearance and physical description

If the COA describes a white powder but the material you received looks very different – for example, it is noticeably yellow, wet, or has an unexpected texture – it is worth asking the supplier for an explanation.

A difference in appearance could have several causes, including exposure to moisture, degradation, chemical changes, or a labeling or handling mistake. Appearance alone cannot identify the cause, but it can signal that further review is needed.

Purity: the HPLC section

The purity section usually contains results from a testing method called **HPLC**. HPLC separates the main substance from other substances in the sample.

The COA may report purity as a percentage. In simple terms, this number estimates how much of the detected material appears to be the main product rather than related impurities.

A simplified calculation is:  Purity percentage = area of the main peak ÷ area of all peaks × 100

For example, a reported purity of 98% generally means that approximately 98% of the detected material in that test appeared as the main product peak.

A chromatogram is the graph produced by the HPLC test. It shows the separated components as peaks.

When available, review it rather than relying only on the reported percentage. Things to notice include:

• • A clear main peak
  • Small additional peaks, which may represent impurities
  • A reasonably flat baseline
  • A peak that is not severely stretched, uneven, or irregular

Two samples may both be reported as 98.2% pure but have very different-looking chromatograms. The graph can provide useful context that the percentage alone does not show.

Identity confirmation: the mass spectrometry section

The mass spectrometry section is usually used to confirm that the material has the expected molecular weight.

The COA should ideally show both:

• • The expected, or theoretical, molecular weight
  • The measured, or observed, molecular weight

The two values should be reasonably close, taking the test instrument’s normal tolerance into account. Make sure you are comparing the same type of molecular weight – for example, average with average or monoisotopic with monoisotopic.

A difference does not automatically mean the material is wrong, but it should be investigated. The size of the difference may provide clues about what happened. For example, a change of approximately 16 mass units can sometimes be associated with oxidation, while a difference corresponding to one amino-acid residue can suggest a shortened version of the peptide.

If the original mass spectrum is included, it provides more detail than a single summary number. It may show the different charge states and the calculated final mass.

Content: the section most often missing

Chromatographic purity describes the peptide fraction of the ultraviolet-absorbing material. It does not describe how much of the vial is peptide. The following entries close that gap.

• Water content. Determined by Karl Fischer titration. Lyophilizates commonly retain several percent.
• Counterion content. Most commonly trifluoroacetate from reversed-phase purification, and it can represent a substantial share of the total mass. Some applications are sensitive to residual TFA, in which case a salt exchange to acetate or hydrochloride may be specified.
• Net peptide content. Determined by amino acid analysis or nitrogen determination, and the figure that ties everything together:

Net peptide content:  net peptide % = (mass of peptide / total mass of lyophilizate) x 100

A lot reporting 99% chromatographic purity and 80% net peptide content is not contradicting itself. It is telling you that four fifths of the powder is peptide, and that the peptide present is 99% homogeneous. Any calculation involving quantity needs the first number. Most certificates in circulation omit it.

Additional testing where applicable

Depending on the material and how it will be used, the COA may include other testing results, such as:

• • Residual solvents: checks for leftover processing solvents
  • Endotoxin: checks for substances that can be important in cell-based or biological work
  • Bioburden: estimates the level of living microorganisms present
  • Specific rotation: measures how the material affects polarized light and may help characterize certain compounds

Not every COA needs to include every type of test. The importance of a missing test depends on how the material will be used.

For example, endotoxin information may be important for some cell culture applications but may not be relevant for a basic binding experiment.

Authorization

The bottom of the COA should identify who performed or released the testing. Look for:

• • The testing laboratory’s name
  • The name or signature of the person approving the results
  • The date the results were released
  • Any relevant laboratory accreditation information

If accreditation is claimed, the information should be possible to verify through the appropriate accrediting organization.

A document with no named laboratory, no responsible person, and no release information provides much less confidence because it is not clear who stands behind the results.

Before relying on a COA, check the following

1. The lot or batch number matches the number on the vial.
2. The sequence matches what you ordered.
3. The measured molecular weight is reasonably close to the expected value.
4. The COA explains how the purity result was measured.
5. A chromatogram is included and appears generally reasonable.
6. Net product content is provided when accurate quantity calculations are needed.
7. Water and salt or counterion content are reported when relevant.
8. The manufacturing, testing, and expiry or retest dates make sense together.
9. The testing laboratory and approving person are identified.
10. Testing laboratory is identified.

The first three checks usually take less than a minute and can catch many common problems. The remaining checks help you judge how much confidence to place in the document.

Keeping the record

Save each COA with the matching lot information and keep it even after the material has been used.

If a result is questioned later, one of the first questions will be: “What material was used?” A COA that was read once and then discarded cannot answer that question.

A good record should connect:

• • The product name and sequence
  • The lot number
  • The vial or container
  • The testing results
  • The date and purpose of use

In plain language

A good COA should answer four basic questions:

1. What is this product?
2. Which batch was tested?
3. What did the testing actually find?
4. Who performed and approved the testing?

If a COA cannot answer those questions clearly, ask the supplier for clarification before relying on it.