Two tests show up on almost every serious certificate of analysis: HPLC and mass spectrometry. They are often listed side by side, so people assume they do the same job. They do not. One tells you how pure a sample is. The other tells you what the sample actually is. A report that has only one of them is missing half the proof.
HPLC: the purity test
High performance liquid chromatography pushes a dissolved sample through a packed column under pressure. Different components travel through the column at different speeds, so they exit at different times. A detector records each one as it comes out, producing a chart of peaks called a chromatogram.
The largest peak should be the target compound. Its share of the total peak area is reported as purity, for example 99.1 percent. The smaller peaks are impurities, such as fragments or byproducts from manufacturing.
What HPLC cannot tell you
- Identity. A clean single peak proves the sample is mostly one thing. It does not prove that one thing is the compound on the label.
- Hidden impurities. Two substances can exit the column at nearly the same time and appear as one peak.
- Anything the detector cannot see. Detectors look at a set wavelength, so some substances barely register.
Mass spectrometry: the identity test
Mass spectrometry, or MS, turns molecules into charged particles and measures their mass to charge ratio. From that, the instrument works out molecular weight. The report compares the observed weight with the expected weight calculated from the compound’s known structure. A close match is strong evidence the molecule is what it claims to be.
What mass spectrometry cannot tell you
- Purity. MS confirms the target is present. It is not designed to report what percentage of the sample it makes up.
- Quantity. It does not tell you how many milligrams are in the vial.
Why you need both
Put the two together and the gaps close. HPLC says the sample is, for example, 99 percent one substance. MS says that substance has the correct molecular weight. Many labs run them as one combined method, written as LC/MS, which separates the sample and identifies each peak in a single run.
| HPLC | Mass spec | |
|---|---|---|
| Question it answers | How pure is the sample? | Is it the right molecule? |
| What it measures | Separation of components over time | Mass of the molecules |
| Typical result | Purity as area percent, plus a chromatogram | Observed vs expected molecular weight |
| Blind spot | Cannot confirm identity by itself | Does not measure purity well |
Other tests you may see on a report
- Net content or quantity. Confirms how much compound is actually in the vial.
- Endotoxin testing. Often run with the LAL method described in USP <85>, it checks for bacterial endotoxins.
- Sterility. Checks for microbial growth.
- Heavy metals and residual solvents. Screens for contaminants left over from manufacturing.
Three questions to ask any supplier
- Does the COA include both a purity result and an identity result?
- Is the chromatogram shown, or only a number?
- Does the batch number on the COA match the batch you will receive?
If the answer to any of these is no, ask why. A supplier with nothing to hide will show you the receipts.
Want to see both tests on one report? Offline Peptides publishes an independent COA for every batch it sells. See the COAs
Sources
- United States Pharmacopeia, General Chapter <621> Chromatography.
- United States Pharmacopeia, General Chapter <85> Bacterial Endotoxins Test.
- ICH Q2(R2), Validation of Analytical Procedures. International Council for Harmonisation, 2023.
For educational purposes only. Compounds discussed on this site are for laboratory research use only and are not for human or animal use. Nothing here is medical advice.