Mass error in ppm: the arithmetic and the caveat — a second dataset

On the subject in the title: Mass error in ppm: the arithmetic and the caveat — a second dataset Working notes rather than a conclusion.

Working through the identity arithmetic and I would like it checked.

retatrutide has a monoisotopic mass close to 4731.3 Da. On an electrospray instrument I would expect to see the multiply charged series rather than the intact singly charged ion, so for the doubly charged species I calculate (4731.3 + 2 x 1.00728) / 2, and for the triply charged the analogous expression.

The observed values in the report sit within a few ppm of those. My question is what that actually establishes, because I have seen people treat a mass match as a purity result and I do not think it is one.

An acylated peptide has a mass that reflects the modification, so comparing against the mass of the unmodified backbone gives a mismatch that is not an error.

If the premise is wrong, everything after it is decoration.

Taking post #2 at face value and following it one step further.

Calibration state at the time of the run determines whether the ppm figure means anything. A report that states when the instrument was last calibrated is unusual and is worth more than one that does not.

A mass match establishes that the measured mass is consistent with the proposed composition. It does not establish purity, sequence order, or the absence of an isomer, and all three are frequently claimed from it.

Adding it because I spent an afternoon working it out and nobody should have to twice.

Sample preparation for mass spectrometry can itself introduce modifications, particularly oxidation. A finding at trace level may be telling you about the preparation rather than the material.

For what it is worth, the same held on the two occasions I checked.

Nothing to add, except that this is the answer I would give if asked.

Monoisotopic and average mass are different numbers and both appear on documents. At this molecular weight the difference is a few daltons, which is more than the tolerance being claimed.

I would not lead a decision with this, but I would not ignore it either.

Confirming post #7 from a second method, which matters more than confirming it from a second person.

Tandem mass spectrometry with fragmentation gives sequence information that intact mass cannot. It is the analysis that would actually confirm identity, and it is rarely supplied.

Tandem mass spectrometry: MS/MS fragments the molecular ion and uses fragment masses to confirm identity and detect modifications. A simple identity confirmation by LC-MS does not address post-translational modifications or impurities with the same or very close mass.

Trifluoroacetate adducts are common in material purified with TFA and are one reason a mass spectrum from a peptide can look busier than expected.

Not a strong opinion, just a consistent one.

Purity and identity are different questions: LC-MS establishes that the species at a retention time has the expected mass. It does not establish how much of the sample is that species (that is what LC-UV purity answers).

If anyone has run this properly I would rather read that than my own guess.

Picking up post #11: that is the part I would want checked first.

Resolution and mass accuracy are different specifications. An instrument can resolve two species and still assign their masses imprecisely, and the reverse is also possible.

That is the shape of it. The detail is where I would expect to be corrected.