Methodological standard · §6

CEI-MS-02 — worked example

A worked calculation, and an illustrative mass balance showing what a purity figure alone does not establish.

Document identifier: CEI-MS-02/6
Series: Methodological standard
Version: 1.4
Published: 02 Feb 2023
Last reviewed: 02 Jun 2023
Next review: 02 Jun 2026
Identifier: 10.71829/cei.meth.2
Certainty: High
Cycle: 2023 Q1
Category: Identity

§6Worked example

§6.1Worked example — the one-dalton problem

Bremelanotide has an average mass of 1025.16 Da; melanotan-II, its C-terminally amidated analogue, 1024.18 Da. The difference is 0.98 Da at a nominal mass near 1025.
Resolution required to separate two peaks differing by 0.98 Da at m/z 1025.5, taking resolution as m/Δm at half height: R = 1025.5 ÷ 0.98 = 1047.
That figure is deceptively low. It describes the resolution needed to see two adjacent peaks as distinct, not the resolution needed to assign a single peak correctly against a background of the ¹³C isotopologue of the lighter species, which sits 1.003 Da above the monoisotopic peak and therefore almost exactly on top of the heavier compound.
Discriminating the +1 isotopologue of melanotan-II (1024.54 + 1.003 = 1025.55 monoisotopic) from the monoisotopic peak of bremelanotide (1025.52) requires separating 0.03 Da at m/z 1025.5, that is R = 1025.5 ÷ 0.03 ≈ 34 200.
The Institute therefore sets 30 000 as the threshold for near-isobaric discrimination rather than the naive 1 047. A nominal-mass instrument reporting "1025" has established nothing about which of the two compounds is present, and one of them is an approved medicine while the other is unlicensed in every jurisdiction.

§6.2Illustrative mass balance

Illustrative — not a published result

The figures below are constructed by the Institute to make the arithmetic concrete. They describe no real batch.

Table 2. Illustrative mass balance for a labelled vial.

Quantity	Value	Note
Label claim, mg	7.1	The figure on the vial.
Chromatographic purity, % area	96.10	Relative area of the main peak under a stated method.
Peptide content, % w/w	76.2	Mass fraction that is peptide, against a named reference standard.
Water, % w/w	6.1	By Karl Fischer titration.
Counter-ion, % w/w	16.3	Trifluoroacetate, by ion chromatography.
Mass balance, %	98.6	Sum of the three preceding rows. A certificate that does not permit this sum to be formed is incomplete.
Net peptide in vial, mg	5.410	Label claim multiplied by peptide content.
Target-sequence mass, mg	5.199	Net peptide multiplied by chromatographic purity.
Shortfall against label, mg	1.901	A shortfall of 26.8 % against the labelled mass, none of which is visible from the purity figure alone.

The same calculation can be run on any set of values in the purity-against-content calculator.

References cited on this page

References are numbered in order of first citation in this document. Each superscript in the text links to its entry below.

International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. ICH Q2(R2) Validation of Analytical Procedures. ICH Harmonised Guideline 2023;Step 4 version, 1 November 2023. identifier not held by the Institute
Kang L, Weng N, Jian W. LC–MS bioanalysis of intact proteins and peptides. Biomedical Chromatography 2020;34(1):e4633. doi:10.1002/bmc.4633 · PMID 31215680

Identifiers are reproduced only where the Institute holds them. Where a digital object identifier or PubMed identifier is not shown, the Institute has recorded the journal and year and has not constructed an identifier.

Compound Evidence Institute · CEI-MS-02/6 · https://compoundevidence.com/methods/lcmsms-identity/worked-example/ · retrieved 30 July 2026