Key takeaways
- HPLC separates a sample into its components. It's used to calculate purity (%) and, with a reference standard, quantity (mg).
- Mass spectrometry weighs molecules. It confirms identity: that the peptide is the one on the label.
- The two are complementary. A pure sample of the wrong peptide would pass HPLC, which is why identity testing matters too.
In this guide
Browse all BioAscend COAs →Why testing needs two methods
A peptide test has to answer three separate questions: is it the right molecule, how clean is it, and how much is there. No single technique answers all three well, so labs combine methods. If you've read our guide on how to read a certificate of analysis, these are the methods behind the numbers on that report.
HPLC: measuring purity and quantity
High-performance liquid chromatography (HPLC) separates the components of a sample so each can be measured individually. Here's what happens, simplified:
- The sample is dissolved and injected into a stream of liquid (the "mobile phase") pumped at high pressure.
- It passes through a column packed with fine particles (the "stationary phase"). For peptides, this is usually reversed-phase HPLC, where molecules are separated by how strongly they stick to the column.
- Different molecules exit at different times. The target peptide and each impurity come out separately, at their own "retention time".
- A detector records each one as it leaves, usually by UV absorbance, producing a graph called a chromatogram with a peak for each component.
How purity is calculated
The size (area) of each peak reflects how much of that component is present. Purity is the target peptide's peak area as a percentage of all the peak areas combined. A result of 99.5% means the main peak makes up 99.5% of everything the detector saw.
How quantity is calculated
To get an absolute amount in milligrams, the lab compares the sample's peak against a reference standard of known concentration. That's how a report can say a vial contains, for example, 11.53 mg of peptide.
What HPLC purity doesn't show: impurities that the detector can't see or that come out at the same time as the main peak, and non-peptide content such as water or salts. It also can't confirm on its own that the main peak is the right peptide. That's where mass spectrometry comes in.
Mass spectrometry: confirming identity
Mass spectrometry (MS) measures the mass of molecules with great precision. The sample is turned into charged particles (ions), and the instrument measures their mass-to-charge ratio.
Every peptide has a known molecular weight based on its amino-acid sequence. BPC-157, for example, is 1,419.56 g/mol. If the mass the instrument detects matches the expected value, that's strong evidence the sample contains the right molecule. A different or truncated peptide would show up at a different mass.
Labs often run the two methods together as LC-MS (liquid chromatography–mass spectrometry), separating the sample first and then weighing what comes out. This can also help identify what the impurity peaks are.
Other tests you might see
HPLC and MS cover identity, purity and quantity. Other properties need their own tests, and they only appear on a report if they were specifically requested:
- Endotoxin testing (for example, the LAL test) measures bacterial endotoxins.
- Sterility testing checks for live microorganisms.
- Heavy-metal testing screens for metals such as lead or mercury.
- Peptide content analysis measures how much of the powder's total weight is peptide, as opposed to water, salts or counter-ions.
Reading results with this in mind
- Look for both purity and quantity. Purity alone doesn't tell you how much is in the vial.
- Check the "tests requested" line to see which methods the results cover. For multi-peptide blends, labs often report identity and amount for each component rather than one purity figure.
- Prefer independent, verifiable reports. A third-party lab with a public verification system gives you the most confidence that the numbers are real.
Frequently asked questions
What does HPLC stand for?
High-performance liquid chromatography. It separates the components of a sample so each can be measured, and it's the standard method for calculating peptide purity.
How is peptide purity calculated?
Purity is the target peptide's HPLC peak area as a percentage of the total area of all detected peaks. For example, 99.5% purity means the main peak accounts for 99.5% of what the detector measured.
What does mass spectrometry show on a peptide test?
Mass spectrometry measures the molecular mass of what's in the sample. If it matches the expected mass for that peptide's sequence, it confirms the identity of the compound.
Is a high HPLC purity enough on its own?
Not entirely. HPLC shows how clean a sample is, but it doesn't by itself confirm the molecule's identity or, without a reference standard, how much is in the vial. Identity and quantity results complete the picture.
