Two tests, two questions
Every lot gets two tests because each answers a different question. Neither is enough alone.
HPLC: how clean is it?
- Separates everything in the sample over time
- Reports the main peak as a share of all peaks
- Sees anything that absorbs UV at 220 nm, which includes every peptide
- Cannot tell you which peptide the main peak is
- Does not see water or most salts
Mass spec: is it the right molecule?
- Weighs the molecules after giving them a charge
- Compares the found mass with the expected mass
- Distinguishes molecules that HPLC might not, such as CJC-1295 with and without DAC
- Says little about how much impurity is present
- Needs the right expected mass to compare with
How HPLC works, in four lines
The dissolved sample is pumped through a steel column packed with fine, water-repelling particles. A solvent mixture flows through, slowly getting less watery. Each substance comes off the column at its own time, called its retention time, and a UV detector at the outlet records how much is passing.
The result is the chromatogram: a flat baseline with a peak for each substance. Purity is the area of the main peak divided by the area of all peaks.
purity (%) = area of main peak ÷ total area of all peaks × 100
Why big peptides show several peaks in mass spec
Electrospray gives a molecule one or more extra protons, each adding a positive charge. The spectrometer measures mass divided by charge, so one molecule appears at several places. Small peptides mostly carry one or two charges; larger ones carry several. Software works backwards from the series to a single mass.
| Compound | Avg mass | Main ions expected, m/z |
|---|---|---|
| KPV | 342.4 | 1+ at 343.4 |
| BPC-157 | 1419.5 | 1+ at 1420.5 · 2+ at 710.8 |
| MOTS-c | 2174.6 | 2+ at 1088.3 · 3+ at 725.9 · 4+ at 544.7 |
| CJC-1295 (DAC) | 3647.3 | 3+ at 1216.8 · 4+ at 912.8 · 5+ at 730.5 |
| Tesamorelin | 5135.9 | 4+ at 1285.0 · 5+ at 1028.2 · 6+ at 857.0 |
Calculated from average mass plus one proton (1.007) per charge. Real spectra also show isotope spread and adducts such as sodium.
What neither test covers
- Water and salt content. That is net peptide content, a separate measurement.
- Sterility and endotoxin. These are separate tests, not part of a standard identity and purity certificate.
- The exact structure of each impurity. HPLC shows that impurities exist; identifying them takes further work.
Common questions
Why 220 nm?
The peptide bond absorbs ultraviolet light strongly around that wavelength, so every peptide shows up whatever its sequence.
What does "LC-MS" mean?
Liquid chromatography joined directly to mass spectrometry: the HPLC separates, and the mass spectrometer weighs each peak as it leaves.
Can two different peptides give the same purity result?
Yes. HPLC purity only says the sample is mostly one substance. Mass spectrometry says which substance.
Why do you show the expected mass on the certificate?
So you can compare it with the found mass yourself instead of trusting a pass mark.