Sensible. I would want the same detail before I acted on it either.
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Analytics · Impurities & related substances · continued
Endotoxin and why it is a separate question from purity — what changed since posts 61–90
This is a continuation of a long topic, addressed by post number rather than by page. Start at post 1.
Picking up post #60: that is the part I would want checked first.
Truncation products are substantially lower in mass and usually well separated chromatographically, which makes them the easiest class to see and the least likely to be missed.
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VPoulsen, post #21: Post #20 is the version of this I will quote in future. One addition. That last point is the ceiling on what any purity figure can claim. A method that cannot see a species cannot exclude it, and no certificate says which species its method cannot see. Go to post
On post #62 — agreed on the reasoning, with one qualification.
Endotoxin: I would want to see the raw numbers rather than the summary before agreeing. Summaries lose exactly the information that would settle this.
Dimer and higher-order multimers: two or more peptide molecules bonded together. They appear at double the mass and higher. They may or may not separate from the monomer on HPLC depending on the method.
That holds under the stated conditions and I have stated them.
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buffer_margin, post #14: Taking post #11 at face value and following it one step further. The useful distinction on Endotoxin is between what was measured and what was inferred from it. Both end up in the same sentence and only one of them has error bars. Go to post
Adding the measurement that post #64 says would settle it.
Adding what did not work for me on Endotoxin, since the failures never get written up and they are half the useful information.
customs_ledger, post #29: On Endotoxin, I would rather understate and be corrected upward than overstate and be quoted. That is a house style here and it is a good one. Go to post
Oxidation at methionine and tryptophan: adds 16 per oxygen. Usually elutes earlier. Oxidation is common in storage, especially if the solution is exposed to light or if antioxidants are not present.
That has held every time I have looked, which is not the same as always.
Where I part company with post #66, and it is a narrow parting.
Off-target structures: if the sequence synthesis goes wrong, a completely different amino acid can be incorporated. The resulting off-target peptide is a structural isomer with the same mass but a different sequence. No chromatographic purity method detects this without a reference standard.
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i.wojcik, post #22: Where I part company with post #19, and it is a narrow parting. Posting my Endotoxin numbers with the method attached so they can be discounted properly. Uncontrolled, unblinded, and collected by someone who wanted a particular answer. Go to post
The arithmetic in post #69 is right; the assumption feeding it is the part to check.
I would be cautious about generalising from the Endotoxin example above. It is a good example. It is one example.
Clear enough that I do not think I have a follow-up, which is unusual.
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Oxidation at methionine or tryptophan adds sixteen per oxygen and typically elutes earlier. In aged material it is the modification that grows.
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l.vermeulen, post #47: Building on post #46 rather than restating it. Taking Endotoxin seriously for a moment rather than deflecting: the honest position is that the community has observations and no controlled comparison, and those two things support very different sentences. Go to post
Post #74 describes the usual case. This is about the unusual one.
That last point is the ceiling on what any purity figure can claim. A method that cannot see a species cannot exclude it, and no certificate says which species its method cannot see.
Adding it in case it saves somebody the afternoon it cost me.
d.magalhes, post #31: That is consistent with mine, for whatever one more account is worth. Go to post
Racemisation produces a diastereomer that is mass-identical and chromatographically resolvable only on a method chosen for the purpose. Standard reversed-phase frequently will not separate it.
That is the version I use. It may not be the version that is correct.
Confirming post #77 from a second method, which matters more than confirming it from a second person.
Endotoxin was covered in the wiki last year and the page has a review date on it, which is a better starting point than my memory of a thread.
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buffer_sheet, post #25: The related-substances total is only as informative as the method that generated it. Two per cent by a method that resolves everything is a different statement from two per cent by a method that resolves little. For what it is worth, the same held on the two occasions I checked. Go to post
Comparing impurity profiles across suppliers is much more informative than comparing purity figures, and almost nobody has the documents to do it.
That is the practical version. The rigorous version is longer and says the same thing.
Reading this Endotoxin thread as someone who came in with a fixed view: the third and seventh replies moved me and the confident ones did not.
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Collapsed as off-topic by two members at trust level 3 or above
Post #78 answers the question as asked. The question underneath it is different.
I would rather this thread reach "we do not know" about Endotoxin than reach a confident answer that nobody can support when asked.
Acetate content: counter-ion content. Trifluoroacetate or acetate from the salt form of the peptide. Affects mass calculations and should be stated on a complete certificate.
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g.bakken, post #79: Comparing impurity profiles across suppliers is much more informative than comparing purity figures, and almost nobody has the documents to do it. That is the practical version. The rigorous version is longer and says the same thing. Go to post
I changed my mind about Endotoxin after someone here asked me for the source and I could not produce one. That is worth saying out loud because it is the ordinary way it happens.
j.hartmann, post #15: What would change my mind on Endotoxin is a second dataset collected by someone with no stake in the first. Until then I hold it loosely and I would rather say so than pretend to more. Go to post
The honest reading of a purity figure: it is an upper bound on how much of what the method could see was the intended species, under one integration convention, on one sample.
That has been true for the cases I have seen and I have not seen many.
Coming back to post #84, because the follow-up matters more than the original answer.
A note on scope: what I am saying about Endotoxin applies to the case in the first post and I would not extend it further without checking.
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s.grimaldi, post #73: Clear enough that I do not think I have a follow-up, which is unusual. Go to post
Nothing to add, except that this is the answer I would give if asked.
Storage-related degradation and synthesis-related impurity look different on a chromatogram. A growing oxidation peak over time is not a manufacturing finding.
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Deamidation at asparagine and glutamine: adds 1 approximately. Frequently appears as a close-eluting pair. It is a chemical modification that occurs during storage.
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e.iyer, post #75: Post #74 describes the usual case. This is about the unusual one. That last point is the ceiling on what any purity figure can claim. A method that cannot see a species cannot exclude it, and no certificate says which species its method cannot see. Adding it in case it saves somebody the afternoon it cost me. Go to post
I had written a reply contradicting post #88 and deleted it. Here is what survived.
Related substances: compounds chemically related to the target peptide but not the target peptide itself. The standard method separates them and reports them as area percent. How related they can be before they exceed specification is a regulatory question.
Written from notes rather than memory, which is why the numbers are specific.