Short answer
Mass spectrometry confirms identity by measuring the molecule's mass and matching it to the expected peptide. HPLC measures purity, the share of peak area that is the main peptide. A sample can pass one and fail the other, so a certificate of analysis needs both, with the numbers shown.
Want to see both tests on a real report? See a published certificate.
What is the difference between HPLC and mass spectrometry?
HPLC (high-performance liquid chromatography) separates a sample into its components and reports how much of the signal belongs to each. Mass spectrometry (MS) measures the mass of the molecules. One tells you how clean the sample is; the other tells you what the main molecule is.
| HPLC | Mass spectrometry | |
|---|---|---|
| Question answered | How pure is the sample? | Is it the right molecule? |
| What is measured | Retention time and detector signal (peak area) | Mass-to-charge ratio |
| Result on a COA | A percentage, plus a chromatogram | Expected mass, observed mass, a spectrum |
| Can detect | Related impurities that separate from the main peak | A wrong or modified molecule, by its mass |
| Cannot tell you | The identity of the main peak on its own | The amount of each component |
| Misses | Impurities that co-elute, and anything the detector does not see | Isomers and sequence changes with the same mass |
Many labs run the two together (LC-MS), so one run produces both a chromatogram and a mass for the main peak.
Can a peptide look pure by HPLC but be the wrong compound?
Yes. A chromatogram with one sharp peak shows that one substance dominates the signal. It does not show which substance. A different peptide, or a mislabeled one, can produce an equally clean peak.
HPLC does compare retention time to a reference run when the lab has a standard, which supports identity. But a retention time match is weaker than a mass match, and a report that gives only a purity figure leaves identity unchecked.
Read a published certificate
Our certificates are published in a public library. See our certificates of analysis.
How is identity confirmed from the mass?
The peptide's sequence fixes its theoretical mass. The lab measures the sample's mass and compares the two. If they agree within the instrument's tolerance, the sample is consistent with the expected peptide.
Larger peptides carry several charges in the instrument, so the spectrum shows a series of peaks. Software converts these into one deconvoluted mass. A good report states the expected mass, the observed mass and the difference. Labs report either monoisotopic or average mass, and the report should say which, because the two differ.
A line that says only "identity: conforms" is a conclusion without evidence. You cannot check it.
What does a clean identity result look like on a report?
You should see the peptide's name, its expected mass (calculated from the sequence), the observed mass from the run, and the difference between them, often in daltons or parts per million. You should also see the instrument type and ionization method named, and a spectrum or deconvoluted trace attached or available.
With that information a reader can check the arithmetic. Without it, the line is a claim. If the expected and observed masses differ by more than the method's stated tolerance, the report should say so rather than print "conforms".
Do I need both on a COA?
Yes, and a report should not substitute one for the other. Identity without purity can hide a sample that is mostly something else alongside the right peptide. Purity without identity can hide a clean sample of the wrong compound.
Neither test says how much peptide is in the vial. That is a third question, answered by a content measurement. See peptide purity vs content.
Why do buyers lose time when a report shows only one of the two?
Procurement and QA teams often receive a purity number with no mass data, or a mass statement with no chromatogram. They then have to ask the supplier for the missing half before they can approve a lot, which delays the order.
On our certificates the lab runs HPLC and mass spectrometry, and the report states whether identity was confirmed.
How should a buyer use both results together?
Read identity first, then purity. If the mass does not match, the purity number is irrelevant: you have a clean sample of something else. If the mass matches, the purity figure tells you how much of the signal is that molecule.
Then check the remaining gaps. Is there a content result? Is the lot on the report the lot you will receive? Was the test recent? A COA that answers identity and purity but leaves these open is a partial answer, not a rejection. Ask for the rest.
What are the limits of each method?
No single method proves everything, and a buyer should know where each one stops.
- HPLC depends on the method. A different column or gradient can separate peaks that another method merges. Purity is relative to the peaks the detector sees, so water and salts are invisible to it.
- Mass spectrometry confirms mass, not structure. Two peptides with the same composition in a different order have the same mass. MS alone does not quantify, because signal strength varies between molecules.
- Neither reports endotoxin or heavy metals. Those need separate panels.
When a decision carries more weight, buyers ask for the raw chromatogram and spectrum, or for orthogonal confirmation such as tandem MS (MS/MS) fragmentation, which supports the sequence.
We pull a vial from our own stock and send it to an accredited, independent laboratory, which issues the certificate.
Common questions
What is the difference between HPLC and mass spectrometry?
HPLC separates the components of a sample and shows the share that is the main peptide. Mass spectrometry measures the mass of the molecules and shows whether the main one matches the expected peptide.
Can a peptide look pure by HPLC but be the wrong compound?
Yes. A single clean peak only shows one dominant substance. Without a mass check, nothing says it is the peptide on the label.
How is identity confirmed from the mass?
The lab compares the observed mass with the mass calculated from the peptide's sequence. A match within the instrument's tolerance is consistent with the right molecule.
Do I need both tests on a COA?
Yes. Identity and purity answer different questions, and neither test answers how much peptide is in the vial.