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GB 5009.274-2016: National Food Safety Standard -- Determination of Ciguatoxin in Aquatic Products
GB 5009.274-2016
NATIONAL STANDARD OF THE
PEOPLE’S REPUBLIC OF CHINA
National Food Safety Standard -
Determination of Ciguatoxin in Aquatic Products
ISSUED ON: DECEMBER 23, 2016
IMPLEMENTED ON: JUNE 23, 2017
Issued by: National Health and Family Planning Commission of the PRC;
State Food and Drug Administration.
Table of Contents
Foreword ... 3
1 Scope ... 4
2 Principle ... 4
3 Reagents and materials ... 4
4 Instruments and apparatuses ... 5
5 Analysis steps ... 6
6 Description of the analysis result ... 7
7 Others ... 8
8 Principle ... 8
9 Reagents and materials ... 9
10 Instruments and apparatuses ... 10
11 Analysis steps ... 10
12 Description of the analysis result ... 13
13 Precision ... 14
14 Others ... 14
Appendix A The relationship of time of death that is caused by ciguatoxin - mouse unit
... 15
Appendix B Table of the correction coefficient of mouse weight ... 16
Appendix C Multiple reaction monitoring chromatogram of ciguatoxin (P-CTX-1, P-
CTX-2, P-CTX-3) mixed standard solution ... 17
National Food Safety Standard -
Determination of Ciguatoxin in Aquatic Products
1 Scope
This Standard specifies methods for the determination of ciguatoxin in aquatic
products.
This Standard applies to the determination of ciguatoxin in edible parts of aquatic
products.
Method 1 -- Mouse bioassay
2 Principle
Use acetone to extract ciguatoxin in the aquatic product; after the extract is evaporated
to dryness under reduced pressure, use 1% Tween-60 physiological saline as the
dispersion medium to prepare a ciguatoxin-1% Tween-60 physiological saline
suspension; inject the suspension into the peritoneal cavity of the mouse; observe the
survival of the mouse. According to the correlation chart between the death time of the
mouse and the mouse unit, find out the corresponding mouse unit; correct the mouse
unit according to the mouse weight; calculate and confirm the content of ciguatoxin
(mouse unit virulence).
3 Reagents and materials
Unless otherwise specified, all the reagents are analytical reagents, the water is grade-
1 water which is specified by GB/T 6682.
3.1 Reagents
3.1.1 Tween-60 (C64H126O26).
3.1.2 Picric acid [HO-C6H2(NO2)3].
3.1.3 Acetone (CH3COCH3).
3.1.4 Methanol (CH3OH).
4.5 Nitrogen concentrator.
5 Analysis steps
5.1 Sample preparation
Take the edible portion of the aquatic product (boning); cut it into small pieces to make
meat emulsion. Store it below -18°C.
5.2 Sample extraction
Take 200 g of sample in a plastic bag; place it in a water bath at 70°C for 15 min. Take
it out for cooling; add 400 mL of acetone; homogenize for 10 min; use a Buchner funnel
to extract and collect the filtrate. Extract the meat residue for twice more. Combine the
filtrate; place it in a rotary evaporator at 55°C to concentrate to near dryness. Add 100
mL of methanol solution (90%) to dissolve; transfer to 500 mL separatory funnel; add
100 mL of n-hexane; shake well to extract; let stand for about 30 min. After layering is
complete, discard the upper layer of n-hexane and collect the lower layer of methanol
solution. Add 100 mL of n-hexane to repeat the extraction once more. Rotary
evaporate the lower layer of methanol solution to dryness at 55°C; use 100 mL of
ethanol solution (25%) to dissolve; transfer to a separatory funnel; add 100 mL of
diethyl ether and shake well; let stand for 10 min or more. After the layering, collect
the upper layer of ether. Use diethyl ether to extract the lower solution twice more.
Combine the ether extracts; rotary evaporate to dryness at 40°C. Use 5 mL of
chloroform-methanol solution (3+97) to dissolve the concentrate; transfer to a
graduated test tube; use nitrogen to concentrate to dryness at 45°C; use 1% Tween
60-normal saline to fix-volume to 2 mL; shake well to make even ciguatoxin-1% Tween
60-normal saline suspension. 0.5 mL of this suspension is equal to 50 g of sample.
Use this suspension to perform the mouse experiment.
Note: In order to avoid the harm of toxins, gloves shall be used for operation. The used
equipment shall be soaked in sodium hypochlorite solution (5%) for more than 1
hour to decompose the toxin.
5.3 Mouse test
5.3.1 Breeding of experimental animals
The mouse shall be raised in a ventilated, light-transparent, clean indoor environment;
and standard rodent food and water shall be normally supplied during the experiment.
5.3.2 Mouse test
The mice are randomly divided into a test group, a solvent-control group and a blank
control group, of each group there are 3 mice and each mouse is weighed (accurate
to 0.5 g); and the body weight is recorded. Use the staining method (such as 3% ~ 5%
Where:
X -- the virulence of ciguatoxin in the sample, in MU per 50 grams (MU/50g);
MU -- the mouse unit of ciguatoxin that is contained in 0.5 mL of mouse intraperitoneal
injection, in MU per 50 grams (MU/50g);
C -- the correction coefficient of mouse weight;
f -- the dilution factor of sample extract.
1MU is defined as the virulence of death in 400 min of a specific pathogen-free (SPF)
Kunming male mouse of 20.0 g; it is equivalent to 9.10 ng of ciguatoxin.
6.2 Result judgment
If the death time of all mice in the test group is greater than 400 min, the virulence of
ciguatoxin in the sample of the report is less than 0.98 MU/50g.
If the median death time of the test group is 50 min ~ 400 min, calculate according to
Formula (1); and report the virulence of ciguatoxin in the sample as: ××× MU/50g.
If the median death time of the test group is less than 50 min, dilute the sample solution;
and choose 3 mice for the experiment until the median death time is 50 min ~ 400 min;
calculate according to Formula (1). Report the virulence of the ciguatoxin in the sample
as: ×××MU/50g.
7 Others
The detection-limit of the method is 0.98 MU/50g.
Method 2 -- Liquid chromatography - tandem mass
spectrometry
8 Principle
The ciguatoxin in the sample is extracted by methanol, purified by C18 solid phase
extraction column, determined by liquid chromatography-tandem mass spectrometry,
and quantified by external standard method.
9.5 Materials
9.5.1 C18 solid phase extraction column: 200 mg/3mL. Before use, use 6 mL of
methanol and 6 mL of water to activate it and to keep the column moist.
9.5.2 Microporous membrane: 0.22 μm, organic phase.
10 Instruments and apparatuses
10.1 Liquid chromatography - tandem mass spectrometer: equipped with electrospray
ion source (ESI).
10.2 Analytical balance: sensitivity of 0.1mg and 0.01g.
10.3 Nitrogen concentrator.
10.4 Freeze-dryer.
10.5 Pulverizer.
10.6 Centrifuge: speed ≥ 5 000 r/min.
10.7 Homogenizer: 3 400 r/min ~ 24 000 r/min.
10.8 Vortex oscillator.
10.9 Ultrasonic generator.
11 Analysis steps
11.1 Sample preparation
Take the edible portion of the aquatic product; cut it into small pieces to make meat
emulsion. Store at -18°C.
11.2 Sample extraction
Weigh 5 g (accurate to 0.01 g) of sample; freeze-dry; then, fully crush; place in a 50
mL stoppered centrifuge tube; add 10 mL of methanol and 5 mL of n-hexane;
homogenize at 5 000 r/min for 1 min; vortex and mix; use ultrasound to extract for 15
min; centrifugate at 5 000 r/min for 3 min; discard the upper layer of n-hexane phase;
transfer the methanol to a test tube. Use 10 mL of methanol to extract the residue once
again; combine the extracts; use nitrogen to concentrate ...