Epithalon — analytical characterisation
Chromatographic conditions, identity, related substances, presentation, reconstitution and in-use stability.
§6Analytical characterisation
§6.1Chromatographic conditions
- Column
- A conventional C18 method is inadequate; hydrophilic-interaction chromatography or ion-pairing with a strong ion-pair reagent is required for a 390 Da acidic tetrapeptide
- Mobile phase and gradient
- Hydrophilic-interaction: A: acetonitrile; B: 10 mM ammonium formate pH 3.0. Alternatively ion-exchange
- Detection
- UV 214 nm only; no aromatic residue. This peptide has among the weakest chromophores in the series and quantification at low concentration is correspondingly imprecise
- Retention
- Unretained on conventional reverse phase. The Institute regards a reverse-phase purity figure for epithalon as analytically unsupportable, and notes that such figures are nonetheless routinely supplied
§6.2Identity by mass spectrometry
[M+H]⁺ at m/z 391.1; [M−H]⁻ at m/z 389.1, with negative mode better suited to this acidic sequence.[3]
§6.3Related substances and degradation
Table 7. Related substances recorded for Epithalon, with the process or storage route that generates each and its analytical signature.
| Related substance | Origin | Analytical signature |
|---|---|---|
| Sequence-scrambled tetrapeptides | Coupling errors | Isobaric and, on an unretaining method, chromatographically unresolved. For a four-residue peptide this is the dominant unaddressed analytical problem |
| Aspartimide at Asp3 | Base-mediated side reaction | −18 Da |
| Free amino acids and dipeptides | Incomplete coupling or hydrolysis | Require a method capable of resolving the void |
| Cation counter-ion | Acidic peptide | Sodium or ammonium; a trifluoroacetate figure for this peptide is meaningless |
Degradation routes
- Aspartimide formation at Asp3
- Peptide-bond hydrolysis
- No oxidation-sensitive residues
§7Presentation, reconstitution and storage
§7.1Presentation and reconstitution
- Presentation
- Lyophilised powder in vial
- Reconstitution
- A 10 mg vial with 2.0 mL gives 5 mg/mL; 5 mg is then 1.0 mL, the full barrel of a U-100 syringe.
- Storage, lyophilised
- −20 °C, desiccated
- Storage, reconstituted
- 2–8 °C
- In-use period
- No supported claim
Epithalon is the clearest example in this series of a compound for which the analytical certificates in general circulation cannot be correct, because the method they imply does not retain the analyte. The Institute records this not as supplier misconduct but as a systematic analytical failure affecting an entire class of very short, very polar peptides.
§7.2In-use stability
Applicable standards: CEI-MS-01 · CEI-MS-02 · CEI-MS-03 · CEI-MS-04 · CEI-MS-05 · CEI-MS-06. The full series is at methodological standards.
Working calculators: reconstitution and insulin-unit conversion · purity against peptide content · certificate minimum-data checker.
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
- 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
- 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
- Manning MC, Chou DK, Murphy BM, Payne RW, Katayama DS. Stability of protein pharmaceuticals: an update. Pharmaceutical Research 2010;27(4):544–575. doi:10.1007/s11095-009-0045-6 · PMID 20143256
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.