CEI-MS-13 — worked example

§6Worked example

§6.1Worked example — mass balance on an oxidative challenge

1. A peptide containing two methionine residues is stressed with 0.3 % hydrogen peroxide for 4 hours at 25 °C. Unstressed total area 5 002 100 µV·s with the main peak at 4 921 000 µV·s (98.4 %).
2. After stress: main peak 4 016 400 µV·s; a new peak at shorter retention 612 300 µV·s, identified by LC–MS as the mono-sulfoxide (+16 Da); a second new peak 214 800 µV·s identified as the bis-sulfoxide (+32 Da).
3. Degradation achieved = (4 921 000 − 4 016 400) ÷ 4 921 000 = 18.4 %, within the 5–20 % window.
4. Mass balance = (4 016 400 + 612 300 + 214 800 + other unchanged impurities 81 100) ÷ 5 002 100 = 4 924 600 ÷ 5 002 100 = 98.4 %.
5. The balance closes at 98.4 %, within the 95–105 % criterion, and both degradants are resolved from the analyte at R > 1.5. The method sees the degradation chemistry this peptide actually undergoes and can therefore support a purity claim over time.
6. Had the same method been validated against acid and base stress only, it would have been declared stability-indicating without ever having been shown to resolve the sulfoxides — which are, for this sequence, the only degradants that matter.

§6.2Illustrative mass balance

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.

1. 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
2. International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use. ICH Q1A(R2) Stability Testing of New Drug Substances and Products. ICH Harmonised Tripartite Guideline 2003;Step 4 version. identifier not held by the Institute

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