Methodological standard · §6

CEI-MS-11 — worked example

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

Document identifier: CEI-MS-11/6
Series: Methodological standard
Version: 1.1
Published: 26 Aug 2026
Last reviewed: 26 Aug 2026
Next review: 26 Aug 2029
Identifier: 10.71829/cei.meth.10
Certainty: High
Cycle: 2026 Q3
Category: Physical quality

§6Worked example

§6.1Worked example — why the reverse-phase figure and the size-exclusion figure disagree

A long-acting amylin analogue is analysed by both methods. Reverse-phase purity by CEI-MS-01: 98.7 %. Size-exclusion analysis of the same sample: monomer 91.4 %, dimer 6.2 %, higher-order species 2.4 %.
Both determinations are correct. Under the denaturing acidified organic conditions of reverse-phase chromatography, the non-covalent dimer dissociates and its constituent monomers elute in the main peak. Under the non-denaturing isotonic conditions of size-exclusion chromatography, it survives and is counted separately.
A certificate reporting only the 98.7 % figure describes a preparation in which 8.6 % of the material is aggregated at physiological pH, and does so without saying anything untrue.
For a class of molecules whose parent hormone is amyloidogenic and whose entire engineering rationale was to prevent self-assembly, this is not a technicality. It is the quality attribute the compound was designed around, and it is invisible to the assay the channel actually performs.

§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	8.5	The figure on the vial.
Chromatographic purity, % area	98.26	Relative area of the main peak under a stated method.
Peptide content, % w/w	80.5	Mass fraction that is peptide, against a named reference standard.
Water, % w/w	5.1	By Karl Fischer titration.
Counter-ion, % w/w	14.5	Trifluoroacetate, by ion chromatography.
Mass balance, %	100.1	Sum of the three preceding rows. A certificate that does not permit this sum to be formed is incomplete.
Net peptide in vial, mg	6.843	Label claim multiplied by peptide content.
Target-sequence mass, mg	6.724	Net peptide multiplied by chromatographic purity.
Shortfall against label, mg	1.776	A shortfall of 20.9 % 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 Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. ICH Harmonised Tripartite Guideline 1999;Step 4 version. identifier not held by the Institute
United States Pharmacopeial Convention. General Chapter ⟨788⟩ Particulate Matter in Injections. United States Pharmacopeia — National Formulary (USP–NF) 2024;USP 2024 Issue 1. identifier not held by the Institute

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-11/6 · https://compoundevidence.com/methods/aggregates-sec/worked-example/ · retrieved 30 July 2026