Isotope patterns and why the monoisotopic peak is not the tallest

Isotope patterns and why the monoisotopic peak is not the tallest — setting out what I have, and where I think it stops being reliable.

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.

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.

Worth separating two things that the opening post runs together.

Resolution: "high resolution" commonly means <5 ppm across the mass range. Unit-resolution instruments achieve ±1 Da at best and cannot distinguish two species differing by less than 1 Da in total mass.

post #3 is right about the mechanism and I think understates the practical bit.

Thank you for the correction. I have edited my earlier post with a note rather than silently, so the thread still makes sense to read. The error was mine and it was the kind that comes from remembering a figure instead of looking it up.

Electrospray ionisation produces multiply charged ions. For a 4 kDa peptide you expect mostly 2+, 3+, and 4+ charge states. Reading an electrospray spectrum means recognizing the envelope, not looking for one peak.

Picking up post #3: that is the part I would want checked first.

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).

On post #3 — agreed on the reasoning, with one qualification.

Sample matrix effects: if a sample is dissolved in a complex matrix, other compounds in the matrix can suppress the peptide signal. Clean samples give higher sensitivity than dirty samples.

Quantitation by MS: most quantitation is done by LC-UV detection at 214 nm, not by MS, because extinction coefficients are better known. MS can quantify if an internal standard is used but that requires preparation.

I read post #7 twice before replying, because I had assumed the opposite.

Common adducts: sodium adds ≈22, potassium adds ≈38 compared to hydrogen. A [M+Na]+ peak is common and its mass is predictable from the base mass.

For anyone arriving from a search: the marked solution above is the direct answer, and the replies underneath it add the caveats that make it safe to use.

Mass accuracy is expressed in parts per million. It is the difference between observed and theoretical mass divided by theoretical mass, multiplied by a million. A high-resolution instrument in good calibration achieves low single-digit ppm on a peptide of this size.

post #12 is right about the mechanism and I think understates the practical bit.

Charge states observed: for semaglutide (4113.6 Da) the doubly charged ion appears at m/z ≈ 2057, triply charged at ≈ 1371, quadruply at ≈ 1029. Those are the positions to look for; the heights depend on the ionization efficiency.

Worth separating two things that post #10 runs together.

Calibration matters: a high-resolution instrument out of calibration can report mass with ppm error large enough to be uninformative. Check when the instrument was last calibrated before trusting the reported accuracy.