What a certificate of analysis does not establish

Sterility — USP <71>

Whether the material is free of viable microorganisms. Determined by incubating samples in growth media and observing for growth over a defined period — typically fourteen days.

Chemical purity has no bearing on this. A peptide can be 99.9% pure by HPLC and carry a substantial microbial load, because bacteria are not peptide-related impurities and do not appear in a purity chromatogram.

Endotoxin — USP <85>

Endotoxins are lipopolysaccharides from the outer membrane of Gram-negative bacteria. The critical property is that they are heat-stable and survive the processes that kill the organism — so a material can be sterile and still carry a significant endotoxin burden. Sterility and endotoxin are separate questions requiring separate tests.

Measured by the Limulus amebocyte lysate assay, which USP <85> permits in three forms: gel-clot, where the lysate visibly clots above a threshold; kinetic turbidimetric, tracking the rate of turbidity development; and chromogenic, measuring colour released from a synthetic peptide-chromogen substrate. Results are reported in endotoxin units, and where two methods disagree the gel-clot limit test is the referee.

It is also an assay with known failure modes. Sample matrix can inhibit the reaction and produce false negatives, or enhance it and produce false positives — which is why method suitability testing is required rather than optional. Some formulations mask endotoxin entirely, a phenomenon called low endotoxin recovery.

Heavy metals and catalyst residues — USP <232> / <233>

Determined by inductively coupled plasma mass spectrometry. USP <232> sets the limits and <233> specifies the procedures; the pair became a requirement for pharmaceutical products in January 2018. The elements of principal concern are lead, mercury, arsenic and cadmium, which can enter from raw materials, reagents or processing equipment.

Note that this is a different mass spectrometry from the one on your certificate — ICP-MS ionises the sample in a plasma to measure elements, and shares nothing with the electrospray MS used for peptide identity.

Residual solvents — ICH Q3C

Solid-phase synthesis and purification use a great deal of organic solvent — dimethylformamide, dichloromethane, acetonitrile, piperidine, trifluoroacetic acid — and traces remain in the finished solid. Measured by headspace gas chromatography, the industry standard because it needs minimal preparation and avoids matrix interference.

ICH Q3C sorts solvents into three classes: Class 1 to be avoided outright, with benzene limited to 2 ppm; Class 2 to be limited according to permitted daily exposure; Class 3 of low toxic potential. A purity chromatogram detects none of them — the UV detector responds to the peptide bond, and these solvents do not have one.

This is worth understanding even where sterility testing has been performed, because it is a limit of the method rather than of any particular laboratory.

The sterility test is destructive. A tested vial is consumed by the test. So testing necessarily happens on a sample drawn from a lot, and the result is extrapolated to the vials that were not tested. You can never test the specific vial you are going to use — testing it would leave nothing to use.

This is well understood in pharmaceutical manufacturing, where it is managed through validated aseptic process control rather than through end-product testing alone. It is worth stating plainly here because the research-supply market sometimes presents a sterility result as though it certified the individual vial in your hand.

Describing a vial as sterile asserts something that, strictly, testing of that vial cannot have established. At minimum the claim requires a validated aseptic fill process plus lot-level testing. It cannot be inferred from chemical purity data at all.

We publish HPLC and mass spectrometry results by lot. We do not run sterility, endotoxin, elemental impurity or residual solvent testing, and we do not claim their outcomes. Where a test has not been performed, the certificate says so rather than leaving the omission to be inferred from silence. Our certificates are in the library.

We would rather be the supplier whose documentation is narrower and accurate than the one whose marketing is broader than its evidence. If you require any of the four tests above for your work, ask specifically — and treat any supplier who answers by pointing back at a purity figure as having not understood the question.

Does high HPLC purity mean a peptide is sterile?

No. They are unrelated properties measured by unrelated methods. HPLC separates peptide-related compounds by their interaction with a chromatographic column; sterility is established by incubating samples in growth media and observing for microbial growth. A chemically pure peptide can carry a microbial load, and a sterile peptide can be chemically impure.

If something is sterile, is it endotoxin-free?

Not necessarily, and this catches people out. Endotoxins are lipopolysaccharide fragments of bacterial cell walls, and they are heat-stable — they survive the sterilisation that kills the organism. Material can therefore be genuinely sterile and still carry a meaningful endotoxin burden. The two require separate tests.

Why does endotoxin matter in laboratory work?

In cell-based assays, endotoxin activates TLR4 signalling in immune cells and triggers non-specific cytokine release. That produces a response which has nothing to do with the compound under study and can invalidate an entire experimental run. It is a source of irreproducibility that is invisible unless tested for.

Is the mass spectrometry on my COA the same as ICP-MS?

No. Peptide identity uses electrospray or MALDI ionisation to measure the intact molecule. ICP-MS ionises the sample in an argon plasma at around 6,000 °C, destroying all molecular structure, to measure elements. Different instruments, different questions. A peptide identity result says nothing about elemental impurities.

Should I expect a research-grade COA to include these tests?

Generally no, and their absence is not by itself a criticism — these tests are expensive and most are aimed at material intended for administration. What matters is that the certificate does not imply results it does not contain, and that a supplier answers plainly when asked which tests were run.

• United States Pharmacopeia, general chapter <85>, Bacterial Endotoxins Test — gel-clot, turbidimetric and chromogenic techniques; gel-clot as referee method; interference and method suitability.
• United States Pharmacopeia, general chapter <71>, Sterility Tests.
• United States Pharmacopeia, general chapters <232> Elemental Impurities — Limits and <233> Elemental Impurities — Procedures.
• ICH Q3C, Impurities: Guideline for Residual Solvents — solvent classification and permitted daily exposure.
• FDA, Inspection Technical Guide: Bacterial Endotoxins/Pyrogens.