CEI-MS-08 — Related-substances profiling and the detection of isobaric impurities

§1Scope

§1.1Scope

Applies to synthetic peptides where the impurity profile is to be characterised rather than merely quantified in aggregate. Covers the isobaric impurity classes that CEI-MS-01 and CEI-MS-02 cannot detect: enantiomeric substitutions, sequence scrambling and regioisomerism.

§2Principle

Isobaric impurities have the same mass as the target and are therefore invisible to mass spectrometry. Detecting them requires either a chromatographic system capable of resolving diastereomers or regioisomers, or a chemical method that reads configuration directly. Chiral amino-acid analysis after complete hydrolysis and derivatisation determines the D-to-L ratio at each residue position in aggregate; enantioselective chromatography of the intact peptide resolves diastereomeric peptides where the separation is achievable.^[1,2]

§3Apparatus and reagents

§3.1Apparatus

• HPLC system with a polysaccharide-based or crown-ether chiral stationary phase for intact-peptide separation
• Gas chromatograph with a chiral capillary column, or HPLC with a chiral phase, for derivatised amino-acid analysis
• Hydrolysis vessels for vapour-phase acid hydrolysis at 110 °C
• Derivatisation reagent, typically an o-phthalaldehyde and chiral thiol combination

§3.2Reagents and reference materials

• Hydrochloric acid 6 M, constant boiling
• Derivatisation reagents of stated enantiomeric purity
• D- and L-amino-acid standards for each residue of interest
• Control peptide of known configuration

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. 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

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