This standard specifies the sampling, sample preparation and liquid chromatography determination methods for the amnesic shellfish toxin domoic acid in the meat, scallops, mantles and their products of marine bivalve shellfish. This standard is applicable to the determination of amnesic shellfish toxin domoic acid in the meat, scallops, mantles and their products (excluding salted products) of marine bivalve shellfish. GB/T 5009.198-2003 Determination of amnesic shellfish toxin domoic acid in shellfish GB/T5009.198-2003 Standard download decompression password: www.bzxz.net

ICS67.040
National Standard of the People's Republic of China
GB/T5009.198-2003
Determination of domoic acid in amnesic shellfish poisoningShellfishes-Test method of domoic acid in amnesicshellfish poisoning2003-08-11Published
Ministry of Health of the People's Republic of China
Standardization Administration of the People's Republic of China
2004-01-01Implementation
GB/T5009.198—2003
This standard is proposed and managed by the Ministry of Health of the People's Republic of China. This standard is drafted by the Liaoning Entry-Exit Inspection and Quarantine Bureau of the People's Republic of China and the Food Hygiene Supervision and Inspection Institute of the Ministry of Health. The main drafters of this standard are Wei Feng, Wang Zhutian, Chen Mingsheng, Wang Yuping and Tang Shouting. 564
1 Scope
GB/T5009.198—2003
Determination of amnesic shellfish toxin domoic acid in shellfishThis standard specifies the sampling, sample preparation and liquid chromatography determination methods for the amnesic shellfish toxin domoic acid in marine bivalve shellfish meat, shellfish scallops, mantle and their products. This standard is applicable to the determination of amnesic shellfish toxin domoic acid in marine bivalve shellfish meat, shellfish scallops, mantle and their products (excluding salted products).
The detection limit of this standard is 1.0μg.
The linear range of this standard is 1μg~250ug. 2 Principle
The sample is extracted with methanol/water, cleaned up by LC-SAX strong anion column solid phase extraction (SPE), and quantitatively analyzed by RP-HPLC. 3 Reagents
3.1 Methanol: chromatographic grade.
3.2 B: chromatographic grade.
3.3 Trifluoroacetic acid: high-grade pure.
3.4 ​​Citric acid monohydrate: high-grade pure.
3.5 Triammonium citrate: high-grade pure. bzxZ.net
3.6 Acetonitrile + water (1+9).
3.7 0.5mol/L citric acid solution (pH=3.2): 40.4g citric acid monohydrate and 14.0g triammonium citrate are dissolved in 400mL water, pH is adjusted to 3.2 with concentrated nitrogen solution, 50mL acetonitrile is added, and after complete dissolution, the volume is adjusted to 500mL with water. 3.8 LC-SAX column: 3mL
3.9 Standard stock solution DACS-1C (100pg/mL), or equivalent. 3.10 Domoic acid standard solution: dilute the standard stock solution with acetonitrile + water (1 + 9) to prepare standard working solutions containing 0.1, 1.0, 2.5, 10.0, and 25.0 μg/mL of domoic acid. 4 Instruments
4.1 High-performance liquid chromatograph: equipped with a diode array or UV detector. 4.2 Tissue homogenizer: maximum speed 20000r/min. 4.3 Desktop high-speed centrifuge: maximum speed 6000r/min. 4.4 Vortex homogenizer.
4.5 Ultrasonic cleaner.
5 Analysis steps
5.1 Extraction
Weigh 5.0g of the sample (accurate to 0.1g) into a 50ml covered centrifuge tube, add 10mL of methanol + water (1+1) solution, vortex mix for 1min, ultrasonically extract for 5min, centrifuge at 4000/min for 20min to completely separate the solid and liquid phases. Remove the supernatant into a 25mL volumetric flask, add 5mL of methanol + water (1+1) to the residue and repeat the extraction twice, transfer the supernatant into a 25mL volumetric flask, dilute to the mark with water, and mix.
GB/T5009.198—2003
5.2 Purification
Take 5.0mL of the above extract and transfer it to the LC-SAX column which has been treated with 6mL methanol, 3mL water, and 3mL methanol + water (1+1) in sequence. After the liquid flows out at a rate of 1 drop/s, use 5mL acetonitrile + water (1+9) and 0.5mL citric acid eluent in sequence at a rate of 1 drop/s to pass through the column, discard the effluent, and finally use 2mL citric acid eluent to elute the domoic acid adsorbed on the column for HPLC determination. Note: When the liquid passes through the column, when the lower end of the liquid concave surface is level with the upper end of the column packing, stop the liquid from flowing out to prevent the LC-SAX column packing from contacting with air. 5.3 Determination
5.3.1 Chromatographic conditions
5.3.1.1 Chromatographic column: ZorbaxSB-Ci, 150mm×4.6mm(id), 5μm, or equivalent; 5.3.1.2 Mobile phase: acetonitrile + 0.1% trifluoroacetic acid (13+87); 5.3.1.3 Flow rate: 1mL/min
Injection volume: 10pL:
5.3.1.5 Determination wavelength: 242nm
5.3.1.6 Column temperature: room temperature.
5.3.2 Chromatographic determination
Take 10μL (or the same volume) of the sample solution and the standard solution and inject them into the high performance liquid chromatograph for determination. The retention time is used for qualitative determination and the peak area is used for quantitative determination. Under the above chromatographic conditions, the retention time of domoic acid is about 6.6min. The liquid phase chromatogram of the standard solution of domoic acid is shown in Figure 1.
Figure 1 Liquid phase chromatogram of the standard sample of domoic acid 5.4 Blank test
Except for not adding the sample, the above determination steps are followed. 6 Result calculation
AXc.XVX1000
A.xmx(V./V.)
Wherein:
X—the content of domoic acid in the sample, in milligrams per kilogram (mg/kg); A—the peak area of ​​domoic acid in the sample solution;
-the peak area of ​​domoic acid in the standard solution;
c,——the concentration of domoic acid in the standard solution, in micrograms per milliliter (ug/mL); m is the mass of the sample weighed, in grams (g). V,—the total volume of the sample extract, in milliliters (mL); V—the volume of the extract for purification, in milliliters (mL); V, the volume of the eluent, in milliliters (mL). The calculation result shall retain two significant figures.
CB/T5009.198—2003
Note: The calculation result shall deduct the blank value, and the result shall be reported as the content of amnesic shellfish toxin chondroitin in the edible tissue of shellfish (mg/kg).
7 Precision
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 10% of the arithmetic mean. 567
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