MOTS-c — 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
- Mobile phase and gradient
- A: 0.1 % trifluoroacetic acid in water; B: acetonitrile. Gradient 15–50 % B over 30 min
- Detection
- UV 214 nm; 280 nm strong (Trp3, Tyr8, Tyr11) — this is one of the few peptides in the series for which 280 nm quantification is genuinely reliable, and the 280/214 ratio is a robust identity indicator
- Retention
- Intermediate to late
§6.2Identity by mass spectrometry
[M+2H]²⁺ at m/z ≈ 1088.3; [M+3H]³⁺ at m/z ≈ 725.9. Average mass 2174.6 ± 1.5 Da.[3]
§6.3Related substances and degradation
Table 7. Related substances recorded for MOTS-c, with the process or storage route that generates each and its analytical signature.
| Related substance | Origin | Analytical signature |
|---|---|---|
| Met1 and Met6 sulfoxides | Oxidation | +16 Da each and +32 Da for the bis-sulfoxide. Two methionines plus a tryptophan make this the most oxidation-prone sequence in the series and a mono-oxidised species is almost always present |
| Trp3 oxidation products | Oxidation and photolysis | +16 and +32 Da with characteristic spectral change |
| Des-Met1 | Aminopeptidase cleavage or incomplete coupling | −131 Da |
| Deamidated Gln4 | Storage | +1 Da |
| Trifluoroacetate counter-ion | Purification | Three arginines and a lysine; content is substantial and should be quantified |
Degradation routes
- Methionine oxidation at two sites is the dominant route and proceeds in air
- Tryptophan oxidation and photolysis
- Deamidation of Gln4
- Tyrosine nitration in the presence of nitrite and oxidants
§7Presentation, reconstitution and storage
§7.1Presentation and reconstitution
- Presentation
- Lyophilised powder in vial (research supply only)
- 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 or below, desiccated, protected from light and air
- Storage, reconstituted
- 2–8 °C protected from light, and the Institute notes that with two methionines and a tryptophan this peptide should be regarded as having a short aqueous life regardless of refrigeration
- In-use period
- No supported claim
MOTS-c is the best illustration in this series of why an oxidation-state determination belongs on a certificate. A preparation that is 97 % pure with 3 % mono-sulfoxide and one that is 97 % pure with 3 % truncated chains are chemically and biologically different, and a single purity number cannot distinguish them.
§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.