Methodological standard · §6

CEI-MS-01 — worked example

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

Document identifier: CEI-MS-01/6
Series: Methodological standard
Version: 2.1
Published: 01 Feb 2023
Last reviewed: 01 Oct 2023
Next review: 01 Oct 2026
Identifier: 10.71829/cei.meth.1
Certainty: High
Cycle: 2023 Q1
Category: Chromatographic purity

§6Worked example

§6.1Worked example — reporting purity with an explicit integration window

A tirzepatide sample is analysed under the conditions of CEI-MS-01 with the gradient specified in the tirzepatide monograph. Integration from 1.20 min (end of void disturbance) to 32.0 min gives a total area of 4 812 640 µV·s with a main peak of 4 668 900 µV·s.
Purity = 4 668 900 ÷ 4 812 640 × 100 = 97.0 %.
Repeating the integration from 0.60 min, that is including the void disturbance, gives a total area of 5 104 210 µV·s and an apparent purity of 4 668 900 ÷ 5 104 210 × 100 = 91.5 %.
The two figures differ by 5.5 percentage points for the same chromatogram. Neither is wrong arithmetically; only the first is a purity determination. This is why the Institute requires the integration start time to be stated, and why a purity figure supplied without it cannot be compared against any other.

§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	4.6	The figure on the vial.
Chromatographic purity, % area	99.40	Relative area of the main peak under a stated method.
Peptide content, % w/w	78.2	Mass fraction that is peptide, against a named reference standard.
Water, % w/w	4.1	By Karl Fischer titration.
Counter-ion, % w/w	14.4	Trifluoroacetate, by ion chromatography.
Mass balance, %	96.7	Sum of the three preceding rows. A certificate that does not permit this sum to be formed is incomplete.
Net peptide in vial, mg	3.597	Label claim multiplied by peptide content.
Target-sequence mass, mg	3.575	Net peptide multiplied by chromatographic purity.
Shortfall against label, mg	1.025	A shortfall of 22.3 % 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.

United States Pharmacopeial Convention. General Chapter ⟨1225⟩ Validation of Compendial Procedures. United States Pharmacopeia — National Formulary (USP–NF) 2024;USP 2024 Issue 1. identifier not held by the Institute
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

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-01/6 · https://compoundevidence.com/methods/rp-hplc-purity/worked-example/ · retrieved 30 July 2026