GB 31656.15-2022National Food Safety Standards Determination of Mebendazole and Its Metabolite Residues in Aquatic Products-Liquid Chromatography-Tandem Mass Spectrometry (English PDF)

5.1 Reagents

5.1.1 Formic acid (HCOOH). chromatographically pure.

5.1.2 Ethyl acetate (CH3COOHC2H5). chromatographically pure.

5.1.3 Methanol (CH3OH). chromatographically pure.

5.1.4 n-Hexane (C6H14). chromatographically pure.

5.1.5 Dimethylsulfoxide [(CH3)2SO]. chromatographically pure.

5.2 Preparation of solution

5.2.1 50mmol/L disodium hydrogen phosphate solution. Take 8.95g of disodium hydrogen phosphate, dissolve it in water and dilute to 500mL.

5.2.2 0.1% formic acid solution. take 0.5mL formic acid, add water to dissolve and dilute to 500mL, mix well.

5.2.3 Methanol-0.1% formic acid solution (50.50, V/V). Take methanol and 0.1% formic acid solution and mix them in equal volumes.

5.3 Standards

Mebendazole (MBZ), content >=99.0%; hydroxy mebendazole (MBZ-OH), content >=99.0%; amino mebendazole (MBZ-OH)

NH2), content >=99.0%; deuterated mebendazole (MBZ-D3), content >=99.5%; deuterated hydroxy mebendazole (MBZ-OH-D3), content

>=99.0%. See Appendix A for details.

5.4 Preparation of standard solution

5.4.1 Standard stock solution (100 µg/mL). Take 10 mg of each standard substance of mebendazole, hydroxy-mesbendazole and amino-mesbendazole, and accurately weigh

Dissolve with 10mL dimethyl sulfoxide, add methanol to dilute to 100mL volumetric flask, shake well. Store below -18°C, valid for 3

month.

5.4.2 Mixed standard working solution. Accurately measure 1 mL each of standard stock solutions of mebendazole, hydroxy-mesbendazole and amino-methbendazole,

Alcohol-0.1% formic acid solution was diluted to prepare a mixed standard working solution with concentrations of 1000ng/mL, 100ng/mL and 10ng/mL.

Use it now.

5.4.3 Internal standard stock solution (100 µg/mL). Take 10 mg of deuterated mebendazole and deuterated hydroxymesbendazole standard substances, weigh them accurately,

Dissolve in 10mL dimethyl sulfoxide, dilute with methanol to 100mL volumetric flask, shake well. Store below -18°C, valid for 3

month.

5.4.4 Mixed internal standard working solution. Accurately measure 1 mL of internal standard stock solution of deuterated mebendazole and deuterated hydroxymesbendazole, and use methanol-

The 0.1% formic acid solution was diluted into a mixture containing deuterated mebendazole and deuterated hydroxymesbendazole at concentrations of 50 ng/mL and 10 ng/mL, respectively.

Internal standard working solution. Prepare now.

5.5 Materials

Microporous nylon filter membrane. 0.22 µm.

6.2 Analytical balance. Sensitivity 0.00001g and 0.01g.

6.3 Centrifuge. 8000r/min.

6.4 Vortex mixer.

6.5 Ultrasonic cleaner.

6.6 Nitrogen blowing instrument.

6.7 Polypropylene centrifuge tube with stopper. 50mL.

7.1 Preparation of samples

Prepare samples according to the requirements of Appendix B of GB/T 30891-2014.

a) Take the homogenized test sample as the test material;

b) Take the homogenized blank sample as the blank sample;

c) Take the homogenized blank sample, add the standard working solution of appropriate concentration, and add the sample as the blank.

7.2 Storage of samples

Store below -18°C.

8.1 Extraction

Take 2 g of the test material (accurate to +/-0.05 g), put it into a 50 mL centrifuge tube with stopper, accurately add 100 µL of mixed internal standard working solution, and vortex

2min, add 5mL of 50mmol/L disodium hydrogen phosphate solution, vortex for 1min, add ethyl acetate 6mL, vortex for 1min,

Centrifuge at 8000r/min for 6min, collect the supernatant (ethyl acetate layer), add 3 mL of ethyl acetate to the residue and repeat the extraction once, combine the supernatant,

spare.

8.2 Purification

Take the spare solution, dry it with nitrogen at 40°C, dissolve the residue with 2.0 mL of methanol-0.1% formic acid solution, add 2 mL of n-hexane, and vortex

1 min, centrifuged at 8000r/min for 6 min, discarded the upper n-hexane layer, repeated degreasing once, took the clear liquid, passed through a 0.22 µm filter membrane, and used for liquid chromatography.

Spectrometer-tandem mass spectrometry.

8.3 Preparation of standard curve

Precisely measure the appropriate amount of standard working solution and internal standard working solution, and dilute with methanol-0.1% formic acid solution to form mebendazole, aminomethbendazole and

The concentrations of hydroxymebendazole were 0.5ng/mL, 1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL,

100ng/mL series standard working solution (internal standard deuterated mebendazole concentration is 2.5 ng/mL, deuterated hydroxy mebendazole is 0.5 ng/mL),

It was used for liquid chromatography-tandem mass spectrometry. The ratio of the concentration of each drug to the concentration of the internal standard was used as the abscissa (x), and the peak area of each drug was compared with the concentration of the same

The ratio of the peak area of the isotopic internal standard is the ordinate (y) to draw the standard curve. Find the regression equation and correlation coefficient.

8.4 Determination

8.4.1 Chromatographic reference conditions

a) Chromatographic column. C18 (100mmx2.1mm, particle size 3µm), or equivalent;

b) Column temperature. 30°C;

c) Injection volume. 10 µL;

d) Flow rate. 0.2mL/min;

e) Mobile phase. A is methanol; B is 0.1% formic acid solution, and the gradient elution program is shown in Table 1.

8.4.2 Reference conditions for mass spectrometry

a) Ion source. electrospray ion source (ESI);

b) Scanning mode. positive ion scanning;

c) Spray voltage. 3500V;

d) Sheath gas pressure. 30L/min;

e) Auxiliary air pressure. 15L/min;

f) Ion transfer tube temperature. 350°C;

g) In-source collision-induced dissociation voltage. 12V;

h) Detection method. Selected Reaction Monitoring (SRM), Selected Reaction Monitoring precursor ion, product ion, and collision energy are shown in Table 2;

i) Width at half peak of Q1.0.4 Da;

j) Half width of Q3.0.7Da;

k) Collision gas pressure. argon, 0.2Pa.

8.4.3 Assay

8.4.3.1 Qualitative determination

Under the same test conditions, the ratio of the retention time of mebendazole and its metabolites in the sample solution to the retention time in the standard working solution was more

The difference is within +/-2.5%, and the relative abundance of the detected ions should be consistent with the relative abundance of the calibration standard solution with the same concentration.

It should meet the requirements of Table 3.

8.4.3.2 Quantitative determination

Take the sample solution and the standard working solution for single-point or multi-point calibration. Mebendazole uses deuterated mebendazole as the internal standard, and hydroxymesbendazole and

Deuterium-methylbendazole is used as the internal standard, and the chromatographic peak area is used for quantification, and the internal standard method is used for calculation. Standard solution and sample solution

The characteristic ion mass chromatogram peak area of the target drug should be within the linear range of the instrument detection. The standard solution characteristic ion mass color

See Appendix B for spectrograms.

8.5 Blank test

Take the blank sample, except that no drug is added, the same measurement steps are used for parallel operation.

10.1 Sensitivity

The limit of detection of mebendazole, amino mebendazole and hydroxy mebendazole in this method was all 0.5 µg/kg, and the limit of quantification was 1 µg/kg.

10.2 Accuracy

When the addition concentration of this method is 1 µg/kg-40 µg/kg, the recovery rate is 70%-120%.

10.3 Precision

The intra-assay relative standard deviation of this method was <=15%, and the inter-assay relative standard deviation was <=15%.