GB 31658.16-2021National food safety standard - Determination of avermectins residues in animal derived food by high performance liquid chromatography and liquid chromatography-tandem mass spectrometric method (English PDF)

Foreword

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

This document is published for the first time:

1 Scope

This document specifies the sample preparation and HPLC determination methods for avermectin, ivermectin, doramectin and acetamidoavermectin in animal foods:

This document is applicable to the determination of avermectin, ivermectin, doramectin and acetamidoavermectin residues in muscles, livers, kidneys and adipose tissues of pigs, cattle and sheep:

2 Normative reference documents

The contents of the following documents constitute essential provisions of this document through normative citations in the text: Among them, the cited documents with dates are:

Only the version corresponding to the date applies to this document; for undated referenced documents, the latest version (including all amendments) applies to this document:

document:

GB/T 6682 Specifications and test methods for water used in analytical laboratories

3 Terms and definitions

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

4 Principles

The residual avermectin drugs in the sample were extracted with acetonitrile, purified with a C18 solid-phase extraction column, fluorescently derivatized, and determined by high-performance liquid chromatography:

Quantification by external standard method:

5 Reagents 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 (CH3CN): chromatographically pure:

5:1:2 Methanol (CH3OH): chromatographically pure:

5:1:3 n-hexane (C6H14): chromatographically pure:

5:1:4 Acetic acid (CH3COOH): chromatographically pure:

5:1:5 Trifluoroacetic anhydride (C4F6O3): chromatographically pure:

5:1:6 Triethylamine (C6H15N):

5:1:7 1-N-methylimidazole (C4H6N2):

5:2 Solution preparation

5:2:1 Derivatization reagent solution A: Take 5 mL of 1-N-methylimidazole and 5 mL of acetonitrile, and mix well:

5:2:2 Derivatization reagent B solution: Take 5 mL of trifluoroacetic anhydride and 10 mL of acetonitrile, and mix well:

5:2:3 Washing solution: Take 80 mL of acetonitrile, 120 mL of water, and 0:2 mL of triethylamine, and mix well:

5:3 Standard products

The contents of avermectin, ivermectin, doramectin and acetamido-avermectin are all >=95%, see Appendix A for details:

5:4 Preparation of standard solution

5:4:1 Standard stock solution (1 mg/mL): Take appropriate amounts of each of the avermectin, ivermectin, doramectin, and acetamido-avermectin standard products (equivalent to about 10 mg of each active ingredient), Weigh accurately, add acetonitrile to dissolve in a 10 mL volumetric flask and dilute to the mark to prepare a standard stock solution with a concentration of 1 mg/mL: Store in the dark below -18°C and is valid for 6 months:

5:4:2 Mixed standard working solution (10 µg/mL): Precisely measure 0:1 mL of each standard stock solution in a 10 mL volumetric flask, dilute to the mark with acetonitrile, and prepare a mixed standard working solution with a concentration of 10 µg/mL: : Store below -18 degrees C away from light, valid for 3 months:

5:4:3 Mixed standard working solution (1 µg/mL): Precisely measure 1 mL of the mixed standard working solution of 10 µg/mL, put it in a 10 mL volumetric flask, dilute it to the mark with acetonitrile, and prepare a mixed standard with a concentration of 1 µg/mL: Working fluid: Store in a dark place below -18°C and is valid for 3 months:

5:5 Materials

5:5:1 Solid phase extraction column: triple bonded C18, 500mg/6mL, or equivalent:

5:5:2 Microporous nylon filter membrane: 0:2µm:

6 Instruments and equipment

6:1 High performance liquid chromatograph: equipped with fluorescence detector:

6:2 Analytical balance: sensitivity 0:00001g and 0:01g:

6:3 Centrifuge:

6:4 Tissue homogenizer:

6:5 Vortex mixer:

6:6 Solid phase extraction device:

6:7 Nitrogen blower:

7: Preparation and preservation of samples

7:1 Preparation of samples

Take an appropriate amount of fresh or thawed blank or test tissue, mince it, and homogenize it:

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

Take 2g of the sample (accurate to +/-0:02g), put it into a 50mL centrifuge tube, add 8mL of acetonitrile, vortex for 2min, and centrifuge at 4000r/min:

5min: Collect the supernatant, add 8mL of acetonitrile to the residue, and repeat the extraction once: Combine the two extracts and mix well: Take 4mL of the extract and add 6 ml of water:

mL and 10 µL of triethylamine, mix well, and set aside:

8:2 Purification

The solid-phase extraction column was activated with 4 mL of acetonitrile and 4 mL of washing solution in sequence, and the backup solution was passed through the column, and then rinsed with 5 mL of washing solution and 3 mL of water:

Drain, rinse with 4 mL of n-hexane, drain, and elute with 8 mL of acetonitrile: Collect the eluate and blow dry with nitrogen in a 40°C water bath:

8:3 Derivatization

Add 100 µL of derivatization reagent A, 150 µL of derivatization reagent B, 50 µL of acetic acid, and 50 µL of triethylamine, vortex for 30 s, and let stand in the dark at 20°C for 15 min: Add 650 µL of methanol, vortex for 10 s, and let stand in the dark at 20°C for 15 min: , filtered and then measured by high performance liquid chromatography:

8:4 Preparation of standard curve

Precisely measure an appropriate amount of the mixed standard working solution, dilute it with methanol, and prepare the concentration of avermectin drugs at 2:5 µg/L, 5 µg/L, 12:5 µg/L, 25 µg/L, 50 µg/L, and 125 µg/L: and a series of standard working solutions of 250 µg/L, dried with nitrogen in a water bath at 40°C, and treated according to 8:3 derivatization steps for measurement by high performance liquid chromatography: Take the measured peak area as the ordinate and the concentration of the standard solution as the abscissa, Draw a standard curve and find the regression equation and correlation coefficient:

8:5 Determination

The reference conditions for liquid chromatography are as follows:

a) Chromatographic column: C18 (250mmx4:6mm, 5µm), or equivalent;

b) Mobile phase: acetonitrile: water (90:10, volume ratio);

c) Flow rate: 1:0mL/min;

d) Detection wavelength: excitation wavelength: 365nm, emission wavelength: 475nm;

e) Column temperature: 35 degrees C;

f) Injection volume: 50µL:

8:6 Determination method

Take the sample solution and standard solution, perform single-point or multi-point calibration, and quantify the chromatographic peak area according to the external standard method:

The peak area of vermectin drugs should be within the linear range of instrument detection: Under the above chromatographic conditions, the high performance liquid chromatogram of the standard solution is shown in Appendix B:

8:7 Blank test

Take a blank sample and perform parallel operations using exactly the same measurement steps:

9 Calculation and presentation of results

The residual amount of abamectin drugs in the sample is calculated according to the standard curve or formula (1):

10 Method sensitivity, accuracy and precision

10:1 Sensitivity

The detection limit of this method is 1:5 µg/kg, and the quantitation limit is 5 µg/kg:

10:2 Accuracy

The recovery rate of this method at the added concentration level of 5µg/kg~100µg/kg is 60%~120%:

10:3 Precision

The intra-batch relative standard deviation of this method is <=20%, and the inter-batch relative standard deviation is <=20%:

Method two liquid chromatography-tandem mass spectrometry

11 Scope

This document specifies the sample preparation and liquid chromatography-

Tandem mass spectrometry method:

This document is applicable to the determination of avermectin, ivermectin, doramectin and acetamidoavermectin residues in muscles, livers, kidneys and adipose tissues of pigs, cattle and sheep:

12 Normative reference documents

The contents of the following documents constitute essential provisions of this document through normative citations in the text: Among them, the cited documents with dates are:

Only the version corresponding to the date applies to this document; for undated referenced documents, the latest version (including all amendments) applies to this document:

document:

GB/T 6682 Specifications and test methods for water used in analytical laboratories

13 Terms and definitions

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

14 Principles

The residual avermectin drugs in the sample were extracted with acetonitrile, purified with a C18 solid phase extraction column, and determined by liquid chromatography-tandem mass spectrometry and external standard method:

Quantitative:

15 Reagents and materials

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

15:1 Reagents

15:1:1 Acetonitrile (CH3CN): chromatographically pure:

15:1:2 n-hexane (C6H14): chromatographically pure:

15:1:3 Formic acid (HCOOH): chromatographically pure:

15:1:4 Triethylamine (C6H15N):

15:2 Solution preparation

Washing solution: Take 80 mL of acetonitrile, 120 mL of water, and 0:2 mL of triethylamine, and mix well:

15:3 Standard products

The contents of avermectin, ivermectin, doramectin and acetamido-avermectin are all >=95%: See Appendix A for details:

15:4 Preparation of standard solution

15:4:1 Standard stock solution (1 mg/mL): Take appropriate amounts of each of the avermectin, ivermectin, doramectin, and acetamidoavermectin standard products (similar to

Equivalent to about 10 mg of each active ingredient), weigh it accurately, dissolve it in a 10 mL volumetric flask with acetonitrile and dilute it to the mark, and prepare it to a concentration of

1mg/mL standard stock solution: Store below -18 degrees C, valid for 6 months:

15:4:2 Mixed standard working solution (10 µg/mL): Precisely measure 0:1 mL of each standard stock solution in a 10 mL volumetric flask, dilute to the mark with acetonitrile, and prepare a mixed standard working solution with a concentration of 10 µg/mL: : Store below -18 degrees C away from light, valid for 3 months:

15:4:3 Mixed standard working solution (1 µg/mL): Precisely measure 1 mL of the mixed standard working solution of 10 µg/mL, put it in a 10 mL volumetric flask, dilute it to the mark with acetonitrile, and prepare a mixed standard with a concentration of 1 µg/mL: Working fluid: Store in a dark place below -18°C: Validity period is 3 months:

15:4:4 Mixed standard working solution (0:1 µg/mL): Precisely measure 0:1 mL of the mixed standard working solution of 10 µg/mL in a 10 mL volumetric flask, dilute to the mark with acetonitrile, and prepare a concentration of 0: Mixed standard working solution of 1 µg/mL: Store in a dark place below -18°C and is valid for 3 months:

15:5 Materials

15:5:1 Solid phase extraction column: triple bonded C18, 500mg/6mL, or equivalent:

15:5:2 Microporous nylon filter membrane: 0:2µm:

16 Instruments and equipment

16:1 Liquid chromatography-tandem mass spectrometer: equipped with electrospray ion source:

16:2 Analytical balance: sensitive to 0:00001g and 0:01g:

16:3 Centrifuge:

16:4 Tissue homogenizer:

16:5 Vortex mixer:

16:6 Solid phase extraction device:

16:7 Nitrogen blower:

17 Preparation and preservation of samples

17:1 Preparation of samples

Take an appropriate amount of fresh or thawed blank or test tissue, mince it, and homogenize it:

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:

17:2 Storage of samples

Store below -18 degrees C:

18 Measurement steps

18:1 Extraction

Weigh 2g of the sample (accurate to +/-0:02g), put it into a 50mL centrifuge tube, add 8mL of acetonitrile, vortex for 2min, and centrifuge at 4000r/min:

5min: Collect the supernatant, add 8mL of acetonitrile to the residue, and repeat the extraction once: Combine the two extracts and mix well: Take 4mL of the extract and add water:

6mL and 10µL of triethylamine, mix well and set aside:

18:2 Purification

The solid-phase extraction column was activated with 4 mL of acetonitrile and 4 mL of washing solution in sequence, and the standby solution was passed through the column, rinsed with 5 mL of washing solution and 3 mL of water, and extracted:

Dry, rinse with 4 mL of n-hexane, drain, and elute with 8 mL of acetonitrile: Collect the eluate and blow dry with nitrogen in a 40°C water bath: Add 0:5 mL of 50% acetonitrile aqueous solution, vortex for 1 min to dissolve the residue, and filter with microporous membrane Filter and prepare for liquid chromatography-tandem mass spectrometry measurement:

18:3 Preparation of matrix-matched standard curve

Precisely measure 5 µL and 10 µL of 10 µg/mL mixed standard working solution, 5 µL and 25 µL of 1 µg/mL mixed standard working solution,

Mix 5 µL and 25 µL of 0:1 µg/mL standard working solution, add 6 portions of the extracted and purified blank sample residue respectively, blow dry with nitrogen in a 40°C water bath, add 0:5 mL of 50% acetonitrile aqueous solution, vortex to dissolve the residue, and prepare A series of matrix-matched standard solutions with concentrations of 1 µg/L, 5 µg/L, 10 µg/L, 50 µg/L, 100 µg/L, and:200 µg/L were prepared, and filtered by microporous membranes for liquid chromatography-tandem mass spectrometry measurement: The measured characteristic ion peak area is the ordinate and the corresponding standard solution concentration is the abscissa: Draw a standard curve and find the regression equation and correlation coefficient:

18:4 Determination

18:4:1 Chromatographic reference conditions

a) Chromatographic column: C18 (50mmx2:1mm, 1:7µm), or equivalent;

b) Column temperature: 35 degrees C;

GB 31658:16-2021

c) Injection volume: 10µL;

d) Flow rate: 0:3mL/min;

e) Mobile phase: A is 0:1% formic acid aqueous solution, B is 0:1% formic acid acetonitrile solution, and the gradient elution conditions are shown in Table 1:

18:4:3 Qualitative determination

Under the same test conditions, the retention time of abamectins in the sample solution is the same as that in the matrix-matched standard working solution:

The retention time of the substance should have a deviation within +/-2:5%, and the relative ion abundance detected should be consistent with the relative ion abundance of the matrix-matched standard solution of equivalent concentration: The allowable deviation should meet the requirements of Table 3:

18:4:4 Quantitative determination

Set the instrument conditions according to 18:4:1 and 18:4:2, perform single-point or multi-point calibration, and calculate the residual amount of the drug in the sample according to the external standard method: The matrix-matched standard solution and the target drug in the sample solution are The characteristic ion mass chromatographic peak areas should all be within the linear range of instrument detection: See Appendix B for the characteristic ion mass chromatogram of the standard solution:

18:5 Blank test

Take a blank sample and perform parallel operations using exactly the same measurement steps:

19 Calculation and presentation of results

The residual amount of abamectin drugs in the sample is calculated according to the standard curve or formula (2):

20 Method sensitivity, accuracy and precision

20:1 Sensitivity

The detection limit of this method is 0:2 µg/kg, and the quantification limit is 1 µg/kg:

20:2 Accuracy

The recovery rate of this method at the added concentration level of 1µg/kg~100µg/kg is 60%~120%:

20:3 Precision

The intra-batch relative standard deviation of this method is <=20%, and the inter-batch relative standard deviation is <=20%: