GB 5009.198-2016National food safety standard - Shellfishes - Test method of domoic acid in amnesic shellfish poisoning (English PDF)

3.2.5 Eluent: PIPETTE 0.5 mL of tween-20; USE PBS solution (pH7.4) to dilute

to 1000 mL.

3.2.6 Sulfuric acid solution (6 mol/L): PIPETTE 319.2 mL of sulfuric acid; ADD

slowly it to 600 mL of water; USE water to dilute to 1000 mL.

3.3 Standard

Domoic acid (DA, C15H21NO6, CAS No. 14277-97-5) standard solution.

3.4 Preparation of standard solutions

DA standard series working solutions: PIPETTE accurately appropriate amount of DA standard solution; USE PBS solution to dissolve and dilute, to prepare the DA standard series working solutions with mass concentrations of 0 ng/mL,

0.5 ng/mL, 1.0 ng/mL, 2.0 ng/mL, 5.0 ng/mL, and 10.0 ng/mL, respectively.

They are used right after they are prepared.

3.5 Materials

3.5.1 Microporous plate coated with DA capture antibody.

Note: Commercialized kits, if the technical parameters for evaluation meet the requirements of this Standard, are also suitable for this Standard. See Appendix A.

3.5.2 Aqueous-phase microporous filter membrane: 0.45 µm.

5.1 Sample collection

COLLECT at least 10 shellfish samples and make the shellfish meat more than

200 g. The frozen sample is placed in a heat preservation box for freezing inspection; or it is guaranteed to be in a low temperature state (0 °C~10 °C) for inspection. For a shelled sample, it shall be opened, and after removing the water, frozen for inspection.

microporous rack and labeled, including blank control pore, standard solution pore, and sample solution pore. MAKE parallel pores respectively. ADD 150 µL of PBS solution (pH7.4) to the blank control pore. ADD 50 µL of amnesic shellfish poisoning standard series working solution to the standard solution pore. ADD 50 µL of the sample solution to the sample solution pore. ADD 100

µL of DA enzyme marker working solution to all the above micropores and mix gently; ADD 100 µL of DA antibody working solution and rapidly mix well for 1 min. USE viscose paper to seal the micropores to prevent evaporation of the solutions; incubate at 4 °C for 2 h. After the incubation, pour out the liquid from the pores; inject 300 µL of eluent into each micropore to rinse; overturn the microporous plate; pour out the liquid from the pores; repeat the above plate- washing operation twice and pat dry on the absorbent paper. ADD 150 µL of hydrogen peroxide and TMB to each pore and mix well; incubate at room temperature for 30 min in the dark. ADD 50 µL of sulfuric acid solution (6 mol/L) to each pore and mix rapidly; terminate the reaction; measure in 30 min and record the absorbance value at a wavelength of 450 nm. If the sample diluent is measured beyond the linear range of the standard curve, the dilution factor may be expanded and the determination repeated.

5.5 Making of standard curve

USE the base 10 logarithm of the mass concentration of amnesic shellfish poisoning standard series working solution as the abscissa. USE the percentage absorbance value of standard solution calculated according to formula (1) as the ordinate. DRAW the standard curve.

The percentage absorbance value of amnesic shellfish poisoning standard solution (or sample solution) is calculated according to formula (1):

Where:

A - Percent absorbance value;

S - Average absorbance value of amnesic shellfish poisoning standard working solution or sample solution;

S1 - Average absorbance value of blank control pores;

S0 - Average absorbance value of 0 µg/L amnesic shellfish poisoning standard working solution.

9.1 Reagents

9.1.1 Acetonitrile (CH3CN).

9.1.2 Methanol (CH3OH).

9.1.3 Formic acid (HCOOH).

9.1.4 Glacial acetic acid (CH3COOH).

9.2 Reagent preparation

9.2.1 Acetonitrile solution (10%): MEASURE and take 10 mL of acetonitrile;

USE water to dilute to 100 mL.

9.2.2 Methanol solution (50%): MEASURE and take 50 mL of methanol; USE

water to dilute to 100 mL.

9.2.3 Formic acid solution (0.1 mol/L): PIPETTE 3.81 mL of formic acid; USE

water to dilute to 1000 mL.

9.2.4 Acetic acid solution (0.1%): PIPETTE 1 mL of glacial acetic acid; USE

water to dilute to 1000 mL.

9.3 Standard

Domoic acid standard (DA, C15H21NO6, CAS No. 14277-97-5): Purity>=90%.

9.4 Preparation of standard solutions

9.4.1 Domoic acid standard stock solution (150 µg/mL): Accurately weigh 8.3

mg (accurate to 0.01 mg) of the domoic acid standard; USE acetonitrile solution (10%) to dissolve and dilute to 50 mL. The concentration of the domoic acid standard stock solution has been converted using the purity of the standard.

STORE at 4 °C in the dark, valid for 6 months.

9.4.2 Domoic acid standard series working solutions: Separately pipette

appropriate amount of domoic acid standard stock solution (150 µg/mL). USE acetonitrile solution (10%) to dilute into standard series working solutions with a concentration of 0.3 µg/mL, 0.6 µg/mL, 3 µg/mL, 15 µg/mL, 30 µg/mL, respectively. The standard series working solutions are stored at 4 °C in the

Accurately pipette 5 mL of the extract into the activated strong-anion solid- phase extraction column. After the liquid is discharged at a flow rate of 1 mL/min, successively use 5 mL of acetonitrile solution (10%) and 0.5 mL of formic acid solution (0.1 mol/L) to rinse. KEEP pumping for 2 min and discard the effluent.

Finally, USE 3 mL of formic acid solution (0.1 mol/L) to elute; KEEP pumping for 2 min and collect the eluate (3 mL of eluate is equivalent to 1 g of sample).

After passing through a 0.22 µm aqueous-phase microporous filter membrane, it is determined by liquid chromatography.

11.5 Blank test

With the exception of no sample, USE the same operation procedures as the sample to obtain the blank solution.

11.6 Instrument reference conditions

a) Chromatographic column: C18 column. Column length is 150 mm. Inner diameter is 4.6 mm. Particle size is 3 µm. Or those with equivalent performance;

b) Mobile phase: Acetonitrile+acetic acid solution (0.1%) (13+87);

c) Flow rate: 1 mL/min;

d) Column temperature: 35 °C;

e) Injection volume: 10 µL;

f) Determination wavelength: 242 nm.

11.7 Making of standard curve

The standard series working solutions are separately injected into the liquid chromatograph, to determine the corresponding peak areas. USE the mass concentration of standard working solution as the abscissa. USE the peak area as the ordinate. DRAW a standard curve. The liquid chromatogram of domoic acid standard solution is shown in Figure B.1.

11.8 Determination of sample solution

Inject the sample solution into a liquid chromatograph to obtain a peak area.

According to the standard curve, obtain the mass concentration of domoic acid in the sample solution.

16.1 Reagents

16.1.1 Methanol (CH3OH).

16.1.2 Acetonitrile (CH3CN).

16.1.3 Formic acid (HCOOH): Analytically pure.

16.1.4 Ammonium formate (HCOONH4): Analytically pure.

16.2 Reagent preparation

16.2.1 Acetonitrile solution (10%): MEASURE and take 50 mL of acetonitrile;

USE water to dilute to 500 mL.

16.2.2 Methanol solution (50%): MEASURE and take 500 mL of methanol; USE

water to dilute to 1000 mL.

16.2.3 Ammonium formate buffer solution (2 mmol/L): WEIGH 0.126 g of

ammonium formate; ADD 200 µL of formic acid; USE water to dissolve and dilute to 1000 mL.

16.2.4 Formic acid solution (0.3%): PIPETTE 300 µL of formic acid; USE water

to dilute to 100 mL.

16.3 Standard

Domoic acid standard (DA, C15H21NO6, CAS No. 14277-97-5): Purity>=90%.

16.4 Preparation of standard solutions

16.4.1 Domoic acid standard stock solution (100 µg/mL): Accurately weigh 5.6

mg (accurate to 0.01 mg) of the domoic acid standard; USE acetonitrile solution (10%) to dissolve and dilute to 50 mL. The concentration of the domoic acid standard stock solution has been converted using the purity of the standard.

STORE at 4 °C in the dark, valid for 6 months.

16.4.2 Domoic acid standard intermediate solution (2.0 µg/mL): Accurately

pipette 1 mL of domoic acid standard stock solution (100 µg/mL) into a 50 mL volumetric flask; USE acetonitrile solution (10%) to dilute to the mark. STORE at 4 °C in the dark, valid for 3 months.

r/min for 10 min; PIPETTE the supernatant. ADD 5 mL of methanol solution (50%) to the residue to repeatedly extract twice. Combine the supernatant; USE methanol solution (50%) to dilute to 25 mL and mix well. After standing at -18 °C for 2 h, at 5 °C, centrifuge at 10000 r/min for 15 min. The supernatant is to be purified.

18.4 Sample purification

Accurately pipette 5 mL of the extract into the activated strong-anion solid- phase extraction column. CONTROL the effluent speed to about 1 drop per second. Then, successively use 5 mL of acetonitrile solution (10%) and 0.3 mL of formic acid solution (0.3%) to rinse; discard the effluent. USE 4 mL of formic acid solution (0.3%) to elute; collect the eluate; use formic acid solution (0.3%) to dilute to 4 mL (equivalent to 1 g of sample). After mixing well, it is filtered through a 0.22 µm aqueous-phase microporous filter membrane. The filtrate is subjected to liquid chromatography-tandem mass spectrometry.

18.5 Blank test

18.5.1 Reagent blank test: With the exception of no sample, USE the same

operation procedures as the sample to obtain the blank solution.

18.5.2 Blank matrix solution: SELECT a blank sample; USE the same operation

procedures as the sample to obtain the blank matrix solution.

18.6 Instrument reference conditions

18.6.1 Liquid chromatography reference conditions

a) Chromatographic column: C18 column. Column length is 100 mm. Inner diameter is 2.1 mm. Particle size is 5 µm. Or those with equivalent performance;

b) Mobile phase: Mobile phase A is methanol; mobile phase B is ammonium formate buffer solution (2 mmol/L); gradient elution. Gradient elution conditions are shown in Table 1;

c) Flow rate: 0.35 mL/min;

d) Column temperature: 30 °C;

e) Injection volume: 25 µL.

Table 1 -- Gradient elution conditions of mobile phase

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