CEI-MS-02 — Confirmation of peptide identity by liquid chromatography–tandem mass spectrometry

§1Scope

§1.1Scope

Applies to synthetic peptides where an expected molecular formula or average mass is available. Sets the minimum resolution required to distinguish the near-isobaric compound pairs recorded in the Institute’s monograph series.

§2Principle

The analyte is separated chromatographically, ionised by electrospray, and its charge-state envelope acquired. Deconvolution of the envelope yields an average or monoisotopic mass which is compared against the expected value. Fragmentation of a selected precursor yields b- and y-ion series from which sequence coverage is established. Mass agreement alone confirms composition; sequence coverage confirms order.^[1,2]

§3Apparatus and reagents

§3.1Apparatus

• LC–MS system with electrospray ionisation, positive and negative mode capability
• Mass analyser with resolving power ≥ 30 000 at m/z 1000 for near-isobaric discrimination; ≥ 5 000 is the minimum for any determination under this standard
• Collision cell or higher-energy dissociation capability for fragmentation
• Deconvolution software with a stated algorithm and charge-state range

§3.2Reagents and reference materials

• Water and acetonitrile, LC-MS grade
• Formic acid 0.1 % — trifluoroacetic acid suppresses electrospray ionisation and must not be used for the identity determination even where it is used for the purity determination
• Calibration standard appropriate to the mass range
• Reference standard of the analyte, where available

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. 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.