RB/T 033-2020Guidance on verification and validation of microbiological testing methods (English PDF)

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.

5.8 Nitrogen blowing instrument

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.

8.1 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%.

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