SC/T 3053-2019 English PDF (SCT3053-2019)

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SC/T 3053-2019: Determination of astaxanthin in fish and fishery products by high performance liquid chromatography method
SC/T 3053-2019
SC
AQUACULTURE INDUSTRY STANDARD
OF THE PEOPLE’S REPUBLIC OF CHINA
ICS 67.120.30
X 20
Determination of Astaxanthin in Fish and Fishery
Products by High Performance Liquid
Chromatography Method
ISSUED ON: AUGUST 01, 2019
IMPLEMENTED ON: NOVEMBER 01, 2019
Issued by: Ministry of Agriculture and Rural Affairs of the People’s
Republic of China
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Normative References ... 4
3 Principle ... 4
4 Reagents ... 4
5 Instruments ... 6
6 Determination Procedures ... 6
7 Calculation of Results ... 8
8 Method Quantification Limit, Recovery Rate and Precision ... 9
Appendix A (Informative) Chromatogram ... 10
Determination of Astaxanthin in Fish and Fishery
Products by High Performance Liquid
Chromatography Method
1 Scope
This Standard specifies the principle, the used reagents and instruments, the
determination procedures, the calculation method of the results, the sensitivity,
accuracy and precision of the high-performance liquid chromatography method for the
determination of astaxanthin content in fish and fishery products.
This Standard is applicable to the determination of astaxanthin content in fish,
crustaceans, shrimp meal, krill oil and other products.
2 Normative References
The following documents are essential to the application of this document. For the
dated documents, only the versions with the dates indicated are applicable to this
document; for the undated documents, only the latest version (including all the
amendments) is applicable to this document.
GB/T 6682 Water for Analytical Laboratory Use - Specification and Test Methods
GB/T 30891 Practice of Sampling Plans for Aquatic Products
3 Principle
The to-be-tested substance in the sample is extracted by the mixed solution of acetone
or dichloromethane-methanol; after alkali saponification, the astaxanthin ester is
converted into free astaxanthin; separated by liquid chromatography; measured by
ultraviolet detector; and quantified by external standard method.
4 Reagents
4.1 Unless otherwise specified, the used reagents are all analytically pure; and the
water is the Class-I water specified in GB/T 6682.
protected from light. The validity period is 1 month.
4.21 Preparation of geometric isomers of astaxanthin: Accurately pipette an
appropriate amount of all-trans astaxanthin standard stock solution (4.20); dilute with
acetone to prepare a standard solution of 10µg/mL. Pipette 2mL of standard solution
into 10mL test tube with a stopper. Add 3mL of dichloromethane; mix well; add 50µL of
0.01 g/mL iodine-dichloromethane solution (4.17); vortex thoroughly; seal and place
under natural light for reacting 15min. Then add 1mL of 0.1mol/L sodium thiosulfate
solution (4.18) to shake sufficiently and remove excessive iodine; then stand and
separate into layers to take the lower phases. After drying with nitrogen, add 1mL of
acetone to dissolve it; and it shall be prepared for immediate use.
4.22 Primary secondary amine (PSA) filler: Particle size of 40µm~60µm.
5 Instruments
5.1 High performance liquid chromatograph: Equipped with ultraviolet detector.
5.2 Analytical balance: Sensitivity of 0.01g.
5.3 Analytical balance: Sensitivity of 0.0001g.
5.4 Analytical balance: Sensitivity of 0.00001g.
5.5 Ultrasonic cleaner.
5.6 Centrifuge: Rotating speed of 8000 r/min.
5.7 Vortex mixer.
5.8 Nitrogen blowing instrument
6 Determination Procedures
6.1 Preparation of specimen
Take a representative specimen and implement it in accordance with the provisions of
GB/T 30891.
6.2 Extraction
6.2.1 Fish and crustaceans
Take 2g (accurate to 0.01g) of the specimen into a 50mL centrifuge tube; add 4g of
anhydrous MgSO4; then add 10mL of acetone; vortex fully. Perform the ultrasonic
Where:
X - astaxanthin content in the sample, in mg/kg;
1.3 – correction factor of 13-cis astaxanthin against all-trans astaxanthin;
A13-cis – peak area of 13-cis astaxanthin in the specimen solution;
Atrans – peak area of all-trans astaxanthin in the specimen solution;
1.1 – correction factor of 9-cis astaxanthin against all-trans astaxanthin;
A9-cis - peak area of 9-cis astaxanthin in the specimen solution;
Cs - all-trans astaxanthin content in the standard working solution, in µg/mL;
V – volume of specimen solution, in mL;
As – peak area of all-trans astaxanthin standard working solution;
m – mass of sample, in g;
f – dilution rate.
8 Method Quantification Limit, Recovery Rate and
Precision
8.1 Quantification limit
The quantification limit of astaxanthin in fish and crustaceans is 2.5mg/kg; the
quantification limit of astaxanthin in shrimp meal is 5mg/kg; and the quantification limit
of astaxanthin in krill oil is 10mg/kg.
8.2 Recovery rate
When the addition concentration of this method is 2.5mg/kg~100mg/kg, the recovery
rate shall be 90%~110%.
8.3 Precision
The inter-assay coefficient of variation of this method is ≤10%; and the intra-assay
coefficient of variation is ≤10%.