On Dec 15, 2024, the China Chamber of Commerce for Import and Export of Medicines and Health Products (CCCMHPIE) released the raw material L-Ergothioneine Quality Standard and Detection Method. The CCCMHPIE follows national standardization procedures by China’s “Standardization Law” and “Group Standards Management Regulations” and has also announced group standards for “Tribulus Terrestris Extract,” “Solanum Lycopersicum (Tomato) Extract,” and “L-Ergometrine.”
The document has been prepared following GB/T 1.1—2020 guidelines, which dictate the structure and drafting rules for standardization documents. It’s important to be aware that the document may reference patented content; however, the CCCMHPIE is not responsible for identifying such patents. The standards were proposed by the CCCMHPIE.
1 Scope
The scope of this document outlines the standards for ergometrine, covering technical specifications, testing procedures, inspection guidelines, labeling, packaging, transport, storage, and shelf life. It applies to ergometrine manufactured through microbial fermentation and subsequent processes like separation, purification, and crystallization.
2 Normative References
This document mandates adherence to specific normative references that form its integral parts. For documents with specified dates, only those editions are applicable here; for undated documents, their latest versions, including amendments, are to be used. Key references include:
- GB 5009.3 National Food Safety Standard – Determination of Moisture in Food
- GB 5009.12 National Food Safety Standard – Determination of Lead in Food
- GB 5009.11 National Food Safety Standard – Determination of Total Arsenic and Inorganic Arsenic in Food
- GB 5009.15 National Food Safety Standard – Determination of Cadmium in Food
- GB 5009.17 National Food Safety Standard – Determination of Total Mercury and Organic Mercury in Food
- Pharmacopoeia of the People’s Republic of China (2020 Edition), Part 4, General Rule 0621: Optical Rotation Determination Method
- General Rule 0612 of Part 4 of the Pharmacopoeia of the People’s Republic of China (2020 Edition) for Determination of Melting Point
- Pharmacopoeia of the People’s Republic of China (2020 Edition) Part 4 General Rule 0512 High-Performance Liquid Chromatography
- Chapter 4 pH value of the Safety Technical Specification for Cosmetics (2015 edition)
- Chapter 4 of the Safety Technical Specifications for Cosmetics (2015 Edition), which includes 37 components such as ethanol
- Chapter 5 of the Safety Technical Specification for Cosmetics (2015 Edition), Methods for Total Colony Count Inspection, Chapter 5 of the Safety Technical Specification for Cosmetics (2015 Edition), Methods for Testing Heat Resistant Escherichia coli Groups
- Chapter 5 of the Safety Technical Specifications for Cosmetics (2015 Edition) – Testing Methods for Pseudomonas aeruginosa
- Chapter 5 of the Safety Technical Specifications for Cosmetics (2015 Edition) – Testing Methods for Staphylococcus aureus
- Chapter 5 of the Safety Technical Specifications for Cosmetics (2015 Edition): Methods for Testing Fungi and Yeasts
3 Terms and Definitions
No terms or definitions need to be defined in this document.
4 Ergothioneine Features
| Attribute | Detail |
| Chemical Name | Ergothioneine (mercaptohistidine trimethyl betaine, EGT) |
| Molecular Formula | C9H15N3O2S |
| Relative Molecular Weight | 229.3 (according to the 2018 international atomic masses) |
| CAS Number | 497-30-3 |
| Structural formula |  |
5 Technical Requirements
Sensory Requirements
The ergothioneine powder must meet the following sensory technical specifications:
| Item | Requirement |
| Color | White, uniformly colored |
| Odor | Odorless |
| Appearance | Uniform powder or crystals, no visible foreign matter |
Physical and chemical requirements of ergothioneine
| Item | Standard |
| Identification | There should be a chromatographic peak in the test sample spectrum consistent with the retention time of ergothioneine in the control sample spectrum. |
| Content of EGT (calculated as dry product)/% | ≥98.0 |
| Purity (area normalization method) /% | ≥99.0 |
| Specific Rotation [α]D25（c=1,H2O） | ≥(+)122° |
| pH（50mg/mL，25℃） | 6.0-7.5 |
| Melting Point /℃ | 275-277 |
| Moisture /% | ≤0.3 |
| Pb /（mg/kg） | ≤10 |
| Cd/（mg/kg） | ≤5 |
| As/（mg/kg） | ≤2 |
| Hg/（mg/kg） | ≤1 |
| Residual solvent ethanol/(mg/kg) | ≤300 |
Microbial requirements
| Item | Standard |
| Total colony count/(CFU/g) | ≤100 |
| Mold and yeast/(CFU/g) | ≤10 |
| Heat-resistant coliform bacteria/g | not detectable |
| Staphylococcus aureus/g | not detectable |
| Pseudomonas aeruginosa/g | not detectable |
6 Inspection methods
Sensory testing
Place 5g of the product on a clean white porcelain plate. Under good natural light, observe its color and shape with the naked eye and smell the odor with the nose.
Physical and chemical testing
distinguish
Measure according to the method specified in A.2, and refer to B.1 for the reference chart.
- Content and purity of ergothioneine: Determine according to the method specified in A.3, and refer to B.2 for liquid chromatography and reference retention time.
- Specific rotation[ α] D25 (c=1, H2O): Determine according to the method specified in Part 4 of the Pharmacopoeia of the People’s Republic of China (2020 edition), General Rule 0621, Rotational Photometry.
- PH (50mg/mL, 25 ℃): Determine according to the method specified in Chapter 4 of the Safety Technical Specifications for Cosmetics (2015 Edition) for pH value.
- Melting point: Determine according to the method specified in the first method of the melting point determination method in Part 4 of the Pharmacopoeia of the People’s Republic of China (2020 edition), General Rule 0612.
- Moisture: Determine according to the second method specified in GB 5009.3.
- Lead (Pb): Determine according to the first method specified in GB 5009.12.
- Cadmium (Cd): Determine according to the method specified in GB 5009.15.
- Arsenic (As): Determine according to the first method specified in GB 5009.11.
- Mercury (Hg): Determine according to the first method specified in GB 5009.17.
- Residual solvent (ethanol): Determine according to the methods specified in Chapter 4 of the Safety Technical Specifications for Cosmetics (2015 Edition) for 37 components, including ethanol.
- Microbial testing and total bacterial count: Determine according to Chapter 5 of the Safety Technical Specifications for Cosmetics (2015 Edition) – Colony Count Testing Method.
- Mold and Yeast: Determine, according to Chapter 5 of the Safety Technical Specifications for Cosmetics (2015 Edition), Methods for Testing Mold and Yeast.
- Heat-resistant Escherichia coli group: Determine according to Chapter 5 of the Safety Technical Specification for Cosmetics (2015 Edition) for Heat-resistant Escherichia coli group testing methods.
- Staphylococcus aureus: Determine according to Chapter 5 of the Safety Technical Specifications for Cosmetics (2015 Edition) for Staphylococcus aureus.
- Pseudomonas aeruginosa: Determination by Chapter 5 of the Safety Technical Specifications for Cosmetics (2015 Edition) – Testing Methods for Pseudomonas aeruginosa
7 Inspection rules
Group Batch
Products manufactured from identical material varieties, grades, and batches and sharing a production date are grouped together as a single inspection lot.
Factory inspection
Each product batch must undergo a thorough inspection. Only those batches that meet the required standards and for which a certificate of conformity has been issued are permitted to be dispatched from the factory.
The factory inspection process includes a comprehensive evaluation of sensory requirements, as well as tests for identification, content, purity, optical rotation, pH value, melting point, moisture content, residual solvents, total bacterial count, molds and yeasts, heat-resistant coliforms, Staphylococcus aureus, and Pseudomonas aeruginosa.
Type inspection
The type inspection items include all the items specified in this document. During normal production, a type inspection should be conducted once a year. When one of the following situations occurs, type inspection should be carried out:
- A) When there is a significant change in the source of raw materials;
- B) After formal production, if there are significant changes in the formula and production process that may affect product quality;
- C) When there is a significant difference between the results of the factory inspection and the previous type of inspection;
- D) When the product has been discontinued for more than 6 months and production resumes;
- E) When the market supervision and management department proposes the requirement for type inspection.
Judgment rules
When all the inspection results meet the requirements of this document, the batch of products shall be deemed qualified.
When the inspection results do not meet the requirements of this document, double sampling and retesting can be conducted on the original batch of products, and the retesting results shall prevail. If one or more items still do not meet the standards after re-inspection, the batch of products shall be deemed unqualified. Microbial indicators that do not meet the standards will not be retested.
8 Packaging, labeling, transportation, storage, and shelf life
Packaging
Packaging materials must adhere to the national food safety standard GB 4806.1 and meet the general safety requirements for food contact materials and products.
Labeling
Package labels must clearly display the product name, batch number, specifications, net content, standard number, manufacturer’s name and address, place of origin, production date, shelf life, and storage instructions.
Transportation
Products should be handled with care during transport to avoid damage. They must not be shipped with toxic, harmful, odorous, or contaminant-prone items and must be safeguarded against compression, moisture, and sunlight.
Storage
Products must be stored in a cool, dry environment and kept separate from toxic, harmful, perishable, or potentially contaminating substances.
Shelf Life
The product has a shelf life of 24 months. This is contingent upon maintaining proper storage and transport conditions, ensuring the integrity of the packaging, and keeping the seal intact.
Appendix A (Normative) Test Methods
A.1 General Provision
The testing methods are outlined in Appendix A, with general requirements specifying that reagents and water should be of analytical purity, conforming to GB/T 6682 standards, and typically prepared in aqueous form unless otherwise directed.
A.2 Identification
For identification, the ergothioneine peak in the test sample’s chromatogram must match the retention time of the reference substance’s peak according to the procedure in section A.3.
A.3 Content and Purity of Ergothioneine
Method Summary
Dissolve the sample in water and employ high-performance liquid chromatography (HPLC) to assess ergothioneine’s content and purity. Use an external standard for quantification and area normalization for purity assessment.
Instruments and Equipment
Required equipment includes an HPLC with a UV or diode array detector, an electronic balance with at least 0.1 mg sensitivity, volumetric flasks, pipettes, a 0.45 μm filter membrane, and a syringe-type filter.
Reagents and Materials
Reagents must include ergothioneine as a reference substance with a CAS number of 497-30-3 and a minimum purity of 98.0%, along with chromatographic-grade acetonitrile and purified water.
Chromatographic Conditions and System Suitability Test
The system suitability test employs specific chromatographic conditions:
- Column: Utilize a Luna Ultimate AQ-C18 chromatographic column measuring 4.6×250 mm with a particle size of 5 μm or an equivalent column.
- Mobile Phase: A mixture of acetonitrile and water at a volume ratio 2:98.
- Detection Wavelength: Set at 257 nm.
- Flow Rate: Maintain a constant flow of 0.7 mL/min.
- Injection Volume: Inject 20 μL of the sample.
- Column Temperature: Regulate the temperature to a steady 30°C.
Operation Method
Preparation of Reference Substance Solution
For the reference substance solution, precisely measure a suitable quantity of ergothioneine, dissolve it in water, and dilute it to a 0.1 mg/mL concentration. Filter this solution through a 0.45 μm filter membrane to prepare it for testing.
Preparation of Test Sample Solution
The test sample solution is prepared similarly: accurately weigh the sample, dissolve in water, and dilute to a 0.1 mg/mL concentration. Pass this solution through a 0.45 μm filter membrane before use.
Testing Method
Precisely pipette 20 μL of the reference substance solution and the test sample solution into the liquid chromatograph. Record and analyze the chromatograms. Calculate the ergothioneine content using the external standard method and determine purity using the area normalization method.
Result Calculation
The ergothioneine content in the test sample, represented as a mass fraction 𝑊 based on the dry product, is calculated and expressed as a percentage using the formula (A.1).
The following parameters are:
- W: Mass fraction of ergothioneine in the test sample, %
- A: Peak area of ergothioneine in the test sample solution chromatogram
- A0: Peak area of ergothioneine in the reference substance solution chromatogram
- m: Weight of the test sample, measured in milligrams (mg)
- m0: Weight of the reference substance, measured in milligrams (mg)
- C0: Content of the reference substance, %
- X: Dry weight loss of the test sample, %
The purity of ergothioneine in the test sample is determined as a percentage based on the peak area ratio, calculated using the formula (A.2).
In the formula:
- P: Purity of ergothioneine in the test sample, %
- A: Peak area of ergothioneine in the test sample solution chromatogram
- As: Sum of the peak areas in the test sample solution chromatogram, excluding the solvent peak.
Appendix B (Informative)
Chromatogram B.1
The chromatogram illustrating the reference substance ergothioneine is depicted in Figure B.1.
Figure B.1 presents the liquid chromatography graph of the reference substance ergothioneine.
In Figure B.2, the chromatogram of the test sample of ergothioneine is depicted.
