Glutathione — analytical characterisation
Chromatographic conditions, identity, related substances, presentation, reconstitution and in-use stability.
§6Analytical characterisation
§6.1Chromatographic conditions
- Column
- C18 with an aqueous-compatible phase, or ion-pairing; glutathione is very polar and poorly retained
- Mobile phase and gradient
- A: 0.1 % trifluoroacetic acid or 25 mM phosphate pH 2.5 in water; B: methanol. Gradient 0–20 % B over 12 min
- Detection
- UV 214 nm; the compound has no useful chromophore above 230 nm. Electrochemical detection or derivatisation with a thiol-specific reagent gives far better sensitivity and, importantly, distinguishes the reduced thiol from the oxidised disulfide
- Retention
- Very early
§6.2Identity by mass spectrometry
[M+H]⁺ at m/z 308.1; [M−H]⁻ at m/z 306.1. Oxidised glutathione disulfide gives [M+H]⁺ at m/z 613.2.[3]
§6.3Related substances and degradation
Table 7. Related substances recorded for Glutathione, with the process or storage route that generates each and its analytical signature.
| Related substance | Origin | Analytical signature |
|---|---|---|
| Glutathione disulfide (GSSG) | Oxidation | The dominant impurity and degradant. It forms spontaneously in air and in solution. The reduced-to-oxidised ratio is the single most important quality attribute for this compound and a purity figure that does not distinguish the two forms is uninformative |
| Cysteinylglycine and free amino acids | Hydrolysis | Detected in the same method |
| S-conjugates | Reaction with electrophiles present in processing | Variable mass additions |
| Heavy metals | Thiols chelate metals avidly | Elemental impurity determination is appropriate and is not generally performed |
| Endotoxin | If intended for parenteral use | Essential and not reported in this channel |
Degradation routes
- Oxidation of the thiol to the disulfide is rapid in air and in neutral aqueous solution, and is catalysed by trace metals
- Hydrolysis of the glycine amide bond
- Racemisation is not a significant route
§7Presentation, reconstitution and storage
§7.1Presentation and reconstitution
- Presentation
- Crystalline or lyophilised powder; authorised injectable presentations in some jurisdictions
- Reconstitution
- A 600 mg vial with 6 mL gives 100 mg/mL. Solutions oxidise measurably within hours; preparation immediately before use is required and the addition of a chelator such as EDTA slows metal-catalysed oxidation.
- Storage, lyophilised
- 2–8 °C, desiccated, protected from air. The solid is more stable than the solution but still oxidises slowly
- Storage, reconstituted
- Use immediately. The Institute publishes no in-use claim
- In-use period
- Immediate use only
Glutathione makes a point that applies across this whole series in an unusually visible way: for a compound whose activity resides in a single reactive functional group, the relevant specification is the state of that group, not a chromatographic area percentage.
§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.