Accepted answer
96.4 per cent purity with no content figure leaves the milligram number unmeasured. Purity says 96.4 of every 100 units of detected area is oral semaglutide and 3.6 is something else. Content says how many milligrams are in the glass. The two do not constrain each other: a vial can be 96.4 per cent pure and still be under label, because water and counter-ion are part of the gross mass and neither shows up as an impurity peak. If you buy one test, buy the one that changes your arithmetic.
To be exact about it, understanding purity requires separating the chemistry from the method from the reporting convention, and the three are not independent.
The fraction of your main peak that is actually your target versus isomers, fragments or related sequences is invisible without complementary identity testing.
What each test answers
| Test | Answers | Does NOT answer |
|---|
| RP-HPLC, area % | What fraction of detected material is the target | How much target is present |
| Quantified content | Milligrams of peptide per vial | What the impurities are |
| ESI-MS identity | Whether the molecular weight matches | Purity, or isomeric substitution |
| Peptide mapping | Sequence, localised to a fragment | Quantity |
| Karl Fischer | Water content of the solid | Solvent content |
| LAL endotoxin | Pyrogen load in EU/mg | Sterility |
| Sterility test | Growth in defined media over 14 days | Endotoxin, or bioburden count |
Worth being precise here: column pore size affects mass transfer — a 100 Angstrom packing on a 5 kDa peptide restricts diffusion, broadening the peak and potentially hiding small impurities in the shoulders.
The Arrhenius relationship for peptide degradation is the basis of accelerated stability testing and also governs how quickly methods drift with temperature.
Compare purity within a single laboratory on the same method, never across laboratories.