Identity by LC-MS

Identity confirmation by mass spectrometry compares an observed mass against a theoretical mass calculated from the molecular formula. If they agree within a stated tolerance, the observed species has the elemental composition of the named compound. That is the entire claim. It is a strong claim and a narrow one.

Monoisotopic or average

The index computes theoretical masses monoisotopically — the sum of the masses of the most abundant isotope of each atom — and every ppm error on the site is calculated against a monoisotopic value. For a 4,813 Da peptide the monoisotopic and average masses differ by about 3 Da, which is 620 ppm. Reporting an observed monoisotopic mass against a theoretical average mass produces an apparent error of that size, and it is the single commonest cause of a spurious mass-accuracy flag in the index.

Charge states

Electrospray ionisation produces multiply protonated species. The measured quantity is m/z, not mass:

m/z = (M + z × 1.007276) / z

for M = 4 810.4103 Da (tirzepatide):

  z = 3   m/z = 1 604.4794
  z = 4   m/z = 1 203.6106      ← base peak
  z = 5   m/z =   963.0900
  z = 6   m/z =   802.7429

The neutral mass is recovered by deconvolution across the charge-state envelope. An error of one in the assigned charge state produces a mass error of hundreds of daltons, so a very large apparent error should always prompt a check of the assignment before any conclusion about the material.

Resolution, and the pairs where it decides the answer

A single-quadrupole instrument at unit resolution can tell a 4,813 Da species from a 4,764 Da species. It cannot tell a 1,025.16 Da species from a 1,024.18 Da species with any confidence. The index therefore records identity as confirmed only where a high-resolution instrument was used, and as consistent with otherwise, naming the alternatives not excluded.

Adducts

Sodium and potassium adducts appear at +21.982/z and +37.956/z relative to the protonated species. They are not impurities — they are the same molecule with a different cation attached in the source — and counting them as impurities inflates the impurity total. They are typically 5–17 % of the protonated peak intensity for sodium and under 7 % for potassium, and their presence is a glassware and diluent artefact.