Coming back to: Purity assay versus content assay: two different numbers posts 61–78

I read post #59 twice before replying, because I had assumed the opposite.

Peptide content and chromatographic purity are separate determinations and a certificate that reports only one has answered only one question. Content is the one that tells you how much is in the vial.

Amino acid analysis gives content: it hydrolyses the peptide and quantifies the residues. It measures the peptide content independent of chromatographic purity. Very few supplier certificates report it.

I looked this up rather than remembered it, which is the right order.

Endotoxin testing is separate: a certificate of purity says nothing about endotoxin. A chemically pure preparation can carry a clinically significant endotoxin load. If endotoxin matters, it needs to be tested and reported.

That holds under the stated conditions and I have stated them.

Taking post #63 at face value and following it one step further.

Purity assay versus content assay looks different depending on whether you are reading the primary literature or the summaries of it, and the difference is not in our favour.

Coming back to post #63, because the follow-up matters more than the original answer.

Counter-ion form is crucial: peptides are isolated as salts, most often trifluoroacetate from reversed-phase purification or acetate after salt exchange. The counter-ion is part of the mass in the vial and is not part of the peptide. Two vials of the same purity, one as TFA and one as acetate, contain different amounts of peptide per milligram.

The distinction between release testing and characterisation is one industrial documents make and retail ones usually do not. It is the difference between "we tested every lot for this" and "we established this once".

Anyone who has looked at this more carefully, please correct the record.

Two questions I would want answered before drawing anything from the Purity assay versus content assay data above: how were the cases selected, and what happened to the ones that dropped out.

An itemised related-substances table with retention times says considerably more about the synthesis than a single total. A total tells you how much is not the main peak and nothing about what it is.

That holds for the case as described. Change the assumptions and it may not.

Purity assay versus content assay: I have looked for the primary source twice and failed twice. Either it does not exist or it is somewhere I do not know to look, and I would like to know which.

For anyone finding this later: the short answer on Purity assay versus content assay is that it depends on one thing, and the rest of the thread is people identifying which thing.

I had written a reply contradicting post #68 and deleted it. Here is what survived.

Purity and content are different measurements: purity tells you what proportion of the detected material is the intended species. Content tells you how much of the intended species is present in the container. A lyophilised vial can be 99% pure and contain considerably less than the label claims because the remainder is water, counter-ion and excipient.

The part I am sure of is shorter than the part I have written.

When a document puts the specification in the result column, it has told you the release criterion, not the measurement. Those are different claims and the second is weaker.

Sterility testing is separate: chromatographic purity, mass spectrometry, and endotoxin testing all say nothing about whether a solution is sterile. If sterility matters, it needs to be tested and reported.

I have changed my mind on this once already, so take it as current rather than settled.