GHK-Cu — analytical characterisation
Chromatographic conditions, identity, related substances, presentation, reconstitution and in-use stability.
§6Analytical characterisation
§6.1Chromatographic conditions
- Column
- C18, 4.6 × 250 mm, 5 µm; the copper complex requires a mobile phase that does not strip the metal, so trifluoroacetic acid at 0.1 % may dissociate the chelate and an ammonium acetate system is preferable
- Mobile phase and gradient
- A: 10 mM ammonium acetate pH 6.5; B: acetonitrile. Gradient 2–20 % B over 15 min
- Detection
- UV 214 nm for the peptide; visible absorbance near 520 nm is characteristic of the copper(II) complex and its presence is the simplest confirmation that the metal is bound
- Retention
- Very early; a highly polar tripeptide
§6.2Identity by mass spectrometry
The complex gives [M+H]⁺ at m/z 402.1 with the characteristic ⁶³Cu/⁶⁵Cu isotope pattern in an approximately 69:31 ratio. That isotope signature is the single most useful identity check for this compound and its absence indicates the free peptide rather than the complex.[3]
§6.3Related substances and degradation
Table 7. Related substances recorded for GHK-Cu, with the process or storage route that generates each and its analytical signature.
| Related substance | Origin | Analytical signature |
|---|---|---|
| Free GHK peptide (uncomplexed) | Incomplete complexation | −61 Da and loss of the copper isotope pattern; the free peptide is a different substance with different activity |
| Excess free copper salt | Over-addition | Determined by inductively coupled plasma mass spectrometry; a peptide purity assay does not detect it |
| GHK 2:1 and other stoichiometries | Complexation control | Distinguishable by mass and by visible spectrum |
| Histidine racemisation | Coupling | Isobaric; histidine is among the most racemisation-prone residues in solid-phase synthesis and this sequence has only three residues, so a single racemisation event affects a third of the structure |
| Counter-ion and copper content | Both required | A conformant certificate for this compound reports peptide content, copper content by an elemental method, and complexation stoichiometry. The Institute has not assessed a research-supply certificate that reports all three |
Degradation routes
- Loss of copper on exposure to competing chelators or low pH
- Histidine oxidation in the presence of free copper and peroxide — the metal that makes the compound active also catalyses its degradation
- Peptide-bond hydrolysis
§7Presentation, reconstitution and storage
§7.1Presentation and reconstitution
- Presentation
- Lyophilised powder, characteristically deep blue; topical formulations
- Reconstitution
- A 50 mg vial with 5.0 mL gives 10 mg/mL. The solution should be visibly blue; a colourless solution indicates the free peptide rather than the copper complex.
- Storage, lyophilised
- −20 °C, desiccated, protected from light
- Storage, reconstituted
- 2–8 °C protected from light; copper-catalysed oxidation proceeds in solution
- In-use period
- No supported claim
The visible blue colour of a correctly complexed GHK-Cu preparation is a rare instance of a quality attribute that can be checked without instrumentation, and the Institute notes it for that reason.
§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.