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GB 31656.15-2022: (National Food Safety Standards Determination of Mebendazole and Its Metabolite Residues in Aquatic Products-Liquid Chromatography-Tandem Mass Spectrometry)
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(National Food Safety Standards Determination of Mebendazole and Its Metabolite Residues in Aquatic Products-Liquid Chromatography-Tandem Mass Spectrometry)
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GB 31656.15-2022
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Basic data | Standard ID | GB 31656.15-2022 (GB31656.15-2022) | | Description (Translated English) | (National Food Safety Standards Determination of Mebendazole and Its Metabolite Residues in Aquatic Products-Liquid Chromatography-Tandem Mass Spectrometry) | | Sector / Industry | National Standard | | Classification of Chinese Standard | X04 | | Word Count Estimation | 10,185 | | Date of Issue | 2022-09-20 | | Date of Implementation | 2023-02-01 | | Issuing agency(ies) | National Health Commission of the People's Republic of China, State Administration for Market Regulation | GB 31656.15-2022: (National Food Safety Standards Determination of Mebendazole and Its Metabolite Residues in Aquatic Products-Liquid Chromatography-Tandem Mass Spectrometry)
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National Health Commission of the People's Republic of China
National Food Safety Standards
Mebendazole and its metabolite residues in aquatic products
Determination of liquid chromatography-tandem mass spectrometry
National Standards of People's Republic of China
release
State Administration for Market Regulation
Ministry of Agriculture and Rural Affairs of the People's Republic of China
foreword
This document is in accordance with the provisions of GB/T 1.1-2020 "Guidelines for Standardization Work Part 1.Structure and Drafting Rules for Standardization Documents"
drafting.
This document is published for the first time.
National Food Safety Standards
Determination of residues of mebendazole and its metabolites in aquatic products
Liquid chromatography-tandem mass spectrometry
1 Scope
This document specifies the sample preparation and procedures for the detection of mebendazole and its main metabolites, hydroxy-mebendazole and amino-mebendazole residues in aquatic products.
Liquid chromatography-tandem mass spectrometry method.
This document is applicable to the residues of mebendazole and its main metabolites hydroxy-mebendazole and amino-mebendazole in edible tissues of fish, shrimp and turtle
detection.
2 Normative references
The content in the following documents constitutes the essential provisions of this document through normative references in the text. Among them, the dated reference documents,
Only the version corresponding to the date applies to this document; for undated references, the latest version (including all amendments) applies to this document
document.
GB/T 6682 Water rules and test methods for analytical laboratories
GB/T 30891-2014 Sampling Specifications for Aquatic Products
3 Terms and Definitions
This document does not have terms and definitions that need to be defined.
4 principles
The residual mebendazole, hydroxymesbendazole and aminomesbendazole in the sample were extracted with ethyl acetate, degreased with n-hexane, and analyzed by liquid chromatography-
Detection by tandem mass spectrometry and quantification by internal standard method.
5 Reagents and materials
Unless otherwise specified, all reagents are of analytical grade, and the water is first-class water in accordance with GB/T 6682.
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 Instruments and equipment
6.1 Liquid chromatography-tandem mass spectrometry (LC-MS/MS). with electrospray ionization source (ESI).
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 Preparation and storage of samples
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 Measurement steps
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 (100mm×2.1mm, 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.
9 Calculation and presentation of results
The residual amount of the analyte in the sample was calculated according to the standard curve or formula (1).
10 Detection method sensitivity, accuracy and precision
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%.
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