GB 31656.9-2021National food safety standard - Determination of pendimethalin residue in aquatic products by liquid chromatography-tandem mass spectrometry method (English PDF)

Foreword

This document complies with the provisions of GB/T 1:1-2020 "Standardization Work Guidelines Part 1: Structure and Drafting Rules of Standardization Documents"

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National food safety standards

Determination of pendimethalin residues in aquatic products by liquid chromatography-tandem mass spectrometry

1 Scope

This document specifies the sample preparation and liquid chromatography-tandem mass spectrometry methods for detecting pendimethalin residues in aquatic products: This document is applicable to the detection of pendimethalin residues in edible tissues of fish, shrimps, crabs, shells, sea cucumbers, and turtles:

2Normative reference documents

The contents of the following documents constitute essential provisions of this document through normative references in the text: Among them, for dated reference documents, only the version corresponding to the date applies to this document; for undated reference documents, the latest version (including all amendments) applies to this document:

GB/T 6682 Specifications and test methods for water use in analytical laboratories GB/T 30891-2014 Specifications for sampling of aquatic products

3Terms and Definitions

There are no terms or definitions to be defined in this document:

4 Principles

The remaining pendimethalin in the sample was extracted with acetonitrile, purified with HLB solid-phase extraction column, lipid impurities were removed with Ci8 adsorbent, measured by liquid chromatography-tandem mass spectrometry, and quantified by the internal standard method:

5Reagents and materials

Unless otherwise specified, all reagents are of analytical grade and the water is first-grade water that complies with GB/T 6682:

5:1 Reagents

5:1:1 Acetonitrile (C2H3N): chromatographically pure:

5:1:2 Methanol (CH4O): chromatographically pure:

5:1:3 Formic acid (CH2O2): chromatographically pure:

5:1:4 n-hexane (C6H): chromatographically pure:

5:1:5 Acetone (C3H8O): chromatographically pure:

5:1:6 Sodium chloride (NaCl),

5:2 Solution preparation

5:2:1 n-hexane-acetone solution: Take 500 mL of n-hexane and 500 mL of acetone, and mix well:

5:2:2 Formic acid aqueous solution (0:1%): Take 1mL of formic acid and dilute with water to 1000mL:

5:3 Standard products

5:3:1 Pendimethalin (Ci3Hi9N3O4, CAS No:: 40487-42-1), content >=98:0%:

5:3:2 Pendimethalin-Ds, C3H14N3O4D5, CAS number: 1219803-39-0), content >=98:0%:

5:4 Preparation of standard solution

5:4:1 Standard stock solution (1mg/mL): Take about 10mg of pendimethalin standard, weigh it accurately, dissolve it in acetonitrile and dilute it to a 10mL volumetric flask, shake well, and it is ready: Store away from light at 4 degrees C, valid for 6 months:

5:4:2 Internal standard stock solution (1 mg/mL): Take about 10 mg of the deuterated pendimethalin standard, weigh it accurately, dissolve it with B and dilute it to a 10 mL volumetric flask, shake well, and it is ready: Store away from light at 4 degrees C, valid for 6 months:

5:4:3 Standard intermediate solution (1 µg/mL): Accurately transfer 0:1 mL of the pendimethalin standard stock solution into a 100 mL volumetric flask, dilute to the mark with acetonitrile, and prepare a standard intermediate solution with a concentration of 1 µg/mL: Store at 4°C away from light, valid for 2 weeks:

5:4:4 Internal standard intermediate solution (1 µg/mL): Accurately transfer 0:1 mL of the deuterated pendimethalin standard stock solution into a 100 mL volumetric flask, dilute to the mark with acetonitrile, and prepare an internal standard with a concentration of 1 µg/mL: Intermediate liquid: Store at 4°C away from light, valid for 2 weeks:

5:5 Materials

5:5:1 HLB solid phase extraction column: 60 mg/3mL, or equivalent:

5:5:2 Cs adsorbent: 40µm～60µm:

5:5:3 Microporous filter membrane: 0:22µm, resistant to organic reagents:

6Instruments and Equipment

6:1 Liquid chromatography-tandem mass spectrometer: equipped with electrospray ion source (ESI source):

6:2 Analytical balance: sensitive to 0:01g and 0:00001g:

6:3 Nitrogen blowing instrument:

6:4 Homogenizer:

6:5 Vortex mixer:

6:6 Centrifuge: 10000 r/min or above:

6:7 Ultrasonic oscillator:

6:8 Rotary evaporator:

6:9 Solid phase extraction device:

7 Preparation and preservation of samples

7:1 Preparation of samples

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

a) Take a homogeneous test sample as a test material;

b) Take a homogeneous blank sample as a blank sample;

c) Take a homogeneous blank sample, add a standard solution of appropriate concentration, and add the sample as a blank:

7:2 Storage of samples

Store below -18 degrees C:

8 Measurement steps

8:1 Extraction

Weigh 5:0g of the sample (accurate to +/-0:02 g), add 50 µL of internal standard working solution, mix well, add 6 mL of water, 1 g of sodium chloride, and 20 mL of acetonitrile in sequence, vortex at 3000 r/min for 1 min, and ultrasonic extraction for 20 min, 4000 Centrifuge at r/min for 10 min: Take the supernatant, add 20 mL of acetonitrile to the residue and repeat the extraction once, and combine the supernatants: Concentrate to near dryness by rotary evaporation at 40°C, add 3 mL of acetonitrile to dissolve the residue, and set aside:

8:2 Purification

Activate the solid phase extraction column with 3 mL of acetonitrile, pass the reserve solution through the column, control the flow rate to not exceed 1 mL/min, elute with 3 mL of acetonitrile, and add 8 mL of n-hexane-acetone solution for elution: Collect the eluate, blow with nitrogen at 40°C until it is nearly dry, add 2 mL of acetonitrile accurately to reconstitute, add 0:5 g of C adsorbent, vortex at 3000 r/min for 1 min, centrifuge at 4000 r/min for 10 min, take the supernatant and 0:22µm filter membrane for liquid chromatography-tandem mass spectrometry measurement:

8:3 Preparation of standard curve

Accurately pipet appropriate amounts of standard working solution and internal standard working solution, dilute with acetonitrile to prepare pendimethalin concentration of 2:5 µg/L,

5:0µg/L, 10:0µg/L, 20:0 µg/L, 50:0µg/L, 100:0µg/L, deuterated pendimethalin concentration is 25:0µg/L series mixture 2

Standard working solution for determination by liquid chromatography-tandem mass spectrometer: Ready to use: Taking the peak area ratio of the test substance and the internal standard substance as the ordinate and the corresponding pendimethalin concentration as the abscissa, draw a standard working curve and find the regression equation and correlation coefficient:

8:4 Determination

8:4:1 Liquid Chromatography Reference Conditions

a) Chromatographic column: Cis column (100mmx2:1 mm, 1:7µm), or equivalent;

b) Column temperature: 40 degrees C;

c) Injection volume: 5pL;

d) Flow rate: 0:5mL/min;

e) Mobile phase: A is acetonitrile, B is formic acid aqueous solution (0:1%);

f) Gradient elution conditions are shown in Table 1:

8:4:2 Mass spectrometry reference conditions

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

b) Scanning method: positive ion scanning;

c) Detection method: multiple reaction monitoring (MRM);

d) Electrospray voltage: 3000 V;

e) Ion source temperature: 150 degrees C;

f) Desolvation gas temperature: 450 degrees C;

g) The flow rates of desolvation gas, collision gas, auxiliary gas and other gases should be adjusted before use so that the mass spectrometry sensitivity meets the detection requirements;

h) Precursor ions, product ions, cone voltage and collision energy are shown in Table 2:

8:4:3 Determination method

a) Qualitative methods

Qualification is carried out by comparing the retention time and characteristic ions of each component in the sample with the retention time and characteristic ions of each component in the corresponding standard working solution: Under the same test conditions, the relative deviation between the retention time of the positive sample and the retention time of the standard material should be within +/-2:5%, and the relative ion abundance detected should be consistent with the relative abundance of the calibration standard of equivalent concentration: The abundance ratio of quantitative and qualitative fragment ions should meet the requirements of Table 3:

b) Quantitative methods

The sample solution and the standard working solution are measured under the same chromatography and mass spectrometry conditions, single-point or multi-point calibration is performed, and the chromatographic peak area is used for quantification and internal standard method calculation: The response values of the target substances in the standard working solution and sample solution should be within the detection linear range of the instrument: See Appendix A for the multiple reaction monitoring spectrum of the standard solution:

8:5 Blank test

Take a blank sample and perform parallel operations using the same measurement steps except that no standard solution is added:

9Result calculation and presentation

The residual amount of the substance to be tested in the sample is calculated according to the standard curve or formula (1):

10 Sensitivity, accuracy and precision of detection methods

10:1 Sensitivity

The detection limit of pendimethalin is 1:0µg/kg, and the limit of quantitation is 2:0µg/kg:

10:2 Accuracy

The recovery rate of pendimethalin at the added concentration of 2 µg/kg to 20 µg/kg is 70% to 120%:

10:3 Precision

The intra-batch relative standard deviation of this method is <=15%, and the inter-batch relative standard deviation is <=15%: