A certificate of analysis is the main evidence a researcher has about a material before using it. It is also a document, and documents can be incomplete, mismatched, reused or simply wrong. Reading one critically does not require a laboratory: most problems are visible on the page, to anyone who knows where to look. This article sets out what a certificate should contain, how to check that it belongs to the material in hand, and the signs that it deserves a second look.
What a certificate is, and is not
A certificate of analysis reports the results of specific tests, performed on a sample, from one lot. That definition carries three limits:
- One lot. A certificate says nothing about any other lot, however similar. The lot number on the certificate must match the lot number on the container.
- Specific tests. It reports only what was tested. A purity result is not an identity result, and neither says anything about water content, counter-ions or endotoxin unless those were measured too.
- A sample. The result describes the sample that was analysed, at the time it was analysed. Storage and handling after that are outside it.
What a complete certificate contains
| Part | What it should show | What to check |
|---|---|---|
| Issuer | The testing laboratory's name and address, and its accreditation if it has one | That the laboratory exists and can be contacted independently of the seller |
| Sample identification | Product name, lot number, and the laboratory's own sample or report number | That the lot number matches the container exactly |
| Dates | Date the sample was received and date of analysis or report | That the dates are in a sensible order and fit the lot's history |
| Identity | Mass spectrometry: the technique used and the spectrum or observed mass | That the observed mass matches the calculated mass of the stated sequence |
| Purity | HPLC result as area percent, with the chromatogram | The technique and detection wavelength, and a chromatogram consistent with the reported figure |
| Other tests | Where performed: peptide content, water, counter-ion, endotoxin | The method and units for each |
| Sign-off | Name or signature of the person responsible for the report | That someone at the laboratory stands behind the result |
Matching the certificate to the material
The first check is the simplest and the most often skipped. The lot number on the certificate should match the lot number on the container, character for character. A certificate without a lot number, or one that applies to a "batch" with no link to the container, cannot be connected to the material and is of little use as evidence about it.
Where a supplier publishes certificates, it should be clear which certificate belongs to which lot, and which lot is being shipped. A certificate shown for a product, rather than for a lot, describes some lot of that product at some time.
The laboratory
A certificate is only as credible as the laboratory behind it. Useful questions:
- Can the laboratory be identified? A certificate with no laboratory name, or a name that cannot be found, cannot be checked.
- Is it independent? Testing by the seller or the manufacturer is not worthless, but it is not independent verification. The certificate should make clear who performed the work.
- Is it accredited, and for what? ISO/IEC 17025 is the international standard for the competence of testing laboratories. Accreditation applies to a defined scope: a list of the specific tests the laboratory has been assessed for. A laboratory accredited for some tests is not thereby accredited for all of them, so the useful question is whether the tests on the certificate fall within the scope.
- Can the result be verified? Some laboratories let clients or third parties confirm a report by its number. Where that is possible, it is the strongest check available.
Reading the identity result
Mass spectrometry confirms identity by comparing the observed mass with the mass calculated from the sequence and its modifications. With electrospray ionisation, a peptide usually appears as several ions carrying different numbers of protons. Each ion's mass-to-charge ratio follows a simple relation:
m/z = (M + z × 1.00728) ÷ z
where M is the peptide's monoisotopic mass and z the number of protons gained. For a peptide of monoisotopic mass 1,000.00 Da, the singly, doubly and triply charged ions appear near 1,001.01, 501.01 and 334.34. A certificate that reports an observed mass should show values consistent with the stated sequence, within the accuracy of the instrument, and ideally the spectrum itself.
Two limits are worth remembering. Mass spectrometry cannot distinguish molecules of identical mass, such as a peptide and its D-amino-acid epimer. And a correct mass says nothing about how much of the sample is that peptide: identity and purity are separate questions. The mass spectrometry article explains the method and how to work out the expected mass.
Reading the purity result
An HPLC purity figure is the main peak's share of all UV-detected peaks, and it depends on the method. A credible result comes with the chromatogram and at least the technique and detection wavelength, such as "RP-HPLC, 214 nm". Some laboratories also report the column, gradient and run time, and a table of integrated peaks. The HPLC purity article explains what the figure includes and leaves out. When reading the chromatogram itself:
- The trace should be consistent with the reported purity: a dominant main peak, with any minor peaks small in proportion. Where a table of integrated peaks is provided, the main peak's share should equal the reported figure.
- The peaks should look like peaks: smooth, with a baseline that has some noise. Perfectly flat baselines and geometric peak shapes are not what instruments produce.
- The retention time reported for the main peak should match the trace.
Purity is not content. A 99% purity result says nothing about how much of the weighed powder is peptide; that is the net peptide content, which counts water and counter-ions as well.
Signs a certificate deserves a second look
None of these proves a problem on its own. Each is a reason to ask a question before relying on the document.
| Sign | Why it matters |
|---|---|
| No lot number, or a lot number that does not match the container | The certificate cannot be connected to the material |
| No identifiable laboratory | The result cannot be checked or attributed |
| Purity reported without identity | A pure sample of the wrong compound would pass |
| A purity figure with no chromatogram or method | The figure cannot be interpreted or compared |
| Identical chromatograms or figures across different lots | Separate analyses produce separate traces; identical ones suggest reuse |
| Results that are suspiciously round or uniformly perfect | Real measurements vary from lot to lot |
| Observed masses that do not fit the stated sequence | The identity result may not belong to this compound |
| Dates out of order, or a report older than the lot | The document may belong to different material |
| Signs of editing: mismatched fonts, misaligned text, values that do not match the image | The document may have been altered |
| Accreditation claimed for a test outside the laboratory's scope | Accreditation applies only to the tests assessed |
What no certificate can tell you
- Whether the material has been stored and shipped properly since it was tested.
- Anything about tests that were not performed.
- Anything about other lots, or about the same product from another supplier.
- Whether the material is suitable for a particular experiment. That depends on the experiment.
Frequently asked questions
- Is a certificate from the manufacturer enough?
- It is useful, but it is not independent. A certificate from an identifiable, independent laboratory, for the lot in hand, is stronger evidence.
- Does "ISO 17025 accredited" on a certificate settle the question?
- Not on its own. Accreditation covers the specific tests in the laboratory's scope. The question is whether the tests on the certificate are among them.
- Why would two lots of the same peptide have different purities?
- Because they are different syntheses, purified separately. Small differences between lots are normal; identical figures and traces across lots are more surprising than small differences.
- Can I check the mass myself?
- Yes. Calculate the monoisotopic mass of the stated sequence, including any terminal modifications, and compare it with the observed ions using the relation above.
Sources & Further Reading
- International Organization for Standardization and International Electrotechnical Commission. ISO/IEC 17025:2017, General requirements for the competence of testing and calibration laboratories. 2017.
- Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM. Electrospray ionization for mass spectrometry of large biomolecules. Science 1989;246(4926):64–71. doi:10.1126/science.2675315
- Mant CT, Chen Y, Yan Z, Popa TV, Kovacs JM, Mills JB, et al. HPLC analysis and purification of peptides. In: Fields GB, ed. Peptide Characterization and Application Protocols. Methods in Molecular Biology, vol. 386. Humana Press; 2007:3–55. doi:10.1007/978-1-59745-430-8_1
- D'Hondt M, Bracke N, Taevernier L, Gevaert B, Verbeke F, Wynendaele E, et al. Related impurities in peptide medicines. Journal of Pharmaceutical and Biomedical Analysis 2014;101:2–30. doi:10.1016/j.jpba.2014.06.012
- International Council for Harmonisation. ICH Q2(R2): Validation of Analytical Procedures. Adopted 2023.
See also
- HPLC purity analysis of peptides
- Mass spectrometry for peptide identity
- Certificate of analysis — glossary
- Mass spectrometry — glossary
- Chromatographic purity — glossary
- Net peptide content — glossary
- Lot traceability — glossary
For in vitro laboratory research only. Not for human or veterinary use. This library describes chemistry, analytical methods and published laboratory research; it gives no guidance on use in people or animals.