Everything you need to know
A purity result and an endotoxin result answer different questions, and no amount of purity testing will tell you anything about endotoxin. If a peptide is going to be injected, these are two separate tests and you need both.
Does HPLC purity testing detect endotoxin?
No. Purity by HPLC and endotoxin are different measurements and one tells you nothing about the other. HPLC purity separates peptide-related species and measures UV absorbance, typically at 220 nm where the peptide bond absorbs. Bacterial endotoxin is a lipopolysaccharide from the outer membrane of gram-negative bacteria. It does not chromatograph with the peptide, it does not absorb meaningfully at the wavelength being measured, and it is present at concentrations orders of magnitude below anything a purity method would register. A vial can be 99.5% pure and carry enough endotoxin to cause a febrile response.
What is an endotoxin test and how does it work?
Endotoxin is measured by a lysate assay. The classical method is LAL, Limulus amebocyte lysate, which clots or develops colour in the presence of endotoxin; recombinant factor C (rFC) is a synthetic alternative with the same readout. The result is reported in endotoxin units per millilitre or per milligram, EU/mL or EU/mg. The test is compendial, and the relevant limits depend on the route and the dose rather than on the peptide itself. The measurement is quantitative and quite sensitive, which is why sample handling matters: endotoxin contamination introduced during sampling is indistinguishable from endotoxin in the product.
When does endotoxin testing actually matter?
It matters whenever a preparation is intended to be injected or otherwise bypasses the barriers that normally keep bacterial material out. It does not matter for a peptide that will only ever be used in a bench assay that is not cell-based. The honest framing is about route, not about product category: endotoxin risk follows what will be done with the material. For anything parenteral, endotoxin and sterility are separate tests again, because a preparation can be sterile and still carry endotoxin from bacteria killed earlier in the process.
Is sterility the same as endotoxin testing?
No. Sterility asks whether viable organisms are present. Endotoxin asks whether the breakdown products of gram-negative bacteria are present. Killing the organisms does not remove the endotoxin; it is a heat-stable molecule that survives conditions that destroy the bacteria that produced it. A preparation can pass sterility and fail endotoxin. Both are ordinary requirements for injectables and neither substitutes for the other.
Can one vial be split for purity, content and endotoxin?
Usually yes, if the lab tells you up front how much material each test needs and the vial arrives unopened. Splitting has to be planned rather than improvised, because the endotoxin aliquot must be handled with endotoxin-free consumables from the first transfer or the result is meaningless. For a typical 50 mg research vial there is normally enough material for chromatographic testing and a separate endotoxin aliquot. Ask the lab for its material requirement per test before shipping rather than after, and send the vial sealed so content can be reported as mg per vial.
What should an endotoxin result look like on a certificate?
It should name the method, state the result with units, and state the limit it was compared against. A bare "pass" is not a result. If the certificate does not say what limit was applied, the pass is unverifiable, because the limit depends on the intended dose and route rather than on the material alone. More on endotoxin testing.