GB 31658.15-2021National food safety standard - Determination of xylazine and metabolite(2, 6-dimethylaniline)in animal derived food by 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 liquid chromatography-tandem mass spectrometry methods for the detection of xylazine and metabolite 2,6-dimethylaniline residues in animal foods:

This document is applicable to the detection of xylazine and 2,6-dimethylaniline residues in muscle, fat, liver and kidney 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

Extract xylazine and 2,6-dimethylaniline remaining in the sample with acetonitrile or ammonia-acetonitrile, extract with n-hexane saturated with acetonitrile, and purify:

Determination by liquid chromatography-tandem mass spectrometry in positive ion mode and 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 Methanol (CH3OH): chromatographically pure:

5:1:2 Acetonitrile (CH3CN): chromatographically pure:

5:1:3 n-Hexane (C6H14):

5:1:4 Ammonia (NH3:H2O):

5:1:5 Formic acid (HCOOH):

5:1:6 Anhydrous sodium sulfate (Na2SO4):

5:2 Solution preparation

5:2:1 5% ammonia water and acetonitrile: Take 50 mL of ammonia water and dilute it with acetonitrile to 1000 mL:

5:2:2 20% acetonitrile aqueous solution: Take 20 mL of acetonitrile and dilute to 100 mL with water:

5:2:3 0:1% formic acid solution: Take 1mL of formic acid and dilute it with water to 1000mL:

5:2:4 Acetonitrile-saturated n-hexane: Take 30 mL of n-hexane, add 5 mL of acetonitrile, vortex to mix, shake for 10 min, separate with a separatory funnel, and take the upper liquid:

5:3 Standard products

5:3:2 2,6-dimethylaniline [2,6-dimethylaniline, (CH3)2C6H3NH2, CAS number: 87-62-7], content >=99:0%, see Appendix A:

5:4 Preparation of standard solution

5:4:1 Standard stock solution: Take 10 mg each of xylazine hydrochloride and 2,6-dimethylaniline standard products, weigh them accurately, add an appropriate amount of acetonitrile respectively to dissolve and dilute to a 10 mL volumetric flask, shake well: Prepare standard stock solutions of xylazine and 2,6-dimethylaniline with a concentration of 1 mg/mL: Store below -18°C and have a validity period of 3 months:

5:4:2 Standard working solution: Precisely measure appropriate amounts of xylazine and 2,6-dimethylaniline standard stock solutions into 10 mL volumetric flasks, dilute to volume with acetonitrile, shake well, and prepare respectively: Standard working solution with perazine concentration of 0:4 µg/mL and 2,6-dimethylaniline concentration of 1 µg/mL: Store at 2 degrees C~8 degrees C and prepare for immediate use:

5:5 Materials

Filter membrane: 0:22µm, water phase:

6 Instruments and equipment

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

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

6:3 Homogenizer:

6:4 Vortex mixer:

6:5 Oscillator:

6:6 Refrigerated centrifuge:

6:7 Nitrogen blower:

6:8 Ultrasound instrument:

7: Preparation and preservation of samples

7:1 Preparation of samples

Take an appropriate amount of fresh or thawed blank or test tissue 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 standard working solution of appropriate concentration, and add the sample as a blank:

7:2 Storage of samples

Store below -20 degrees C:

8 Measurement steps

8:1 Extraction

Take 5g of the sample (accurate to +/-0:02g), add 2g of anhydrous sodium sulfate and 10mL of acetonitrile, vortex to mix, shake for 10min, centrifuge at 4°C, 10000r/min for 10min (for the fat sample, add 10mL of 5% ammonia aqueous acetonitrile, Centrifuge at 4°C, 8000r/min for 1 min), take the supernatant, repeat the extraction once, and combine the two supernatants: Blow nitrogen at 40°C until nearly dry, add 1:0 mL of acetonitrile to dissolve, vortex and mix, and set aside:

8:2 Purification

Take the reserve solution, add 0:5 mL of n-hexane saturated with acetonitrile, vortex to mix, centrifuge at 4°C, 10000 r/min for 5 min, and remove the layer solution

200 µL, in a 5 mL centrifuge tube, add 800 µL of water, vortex to mix, centrifuge at 4°C, 10000 r/min for 5 min, take the solution, filter, and use for liquid chromatography-tandem mass spectrometry measurement:

8:3 Preparation of standard curve

Precisely measure appropriate amounts of standard working solutions of xylazine and 2,6-dimethylaniline, dilute with 20% acetonitrile aqueous solution, and prepare xylazine concentrations of 0ng/mL, 0:2ng/mL, 1ng/mL, and 5ng: /mL, 10ng/mL, 50ng/mL, 100ng/mL, 2,6-dimethylaniline concentration series: 0ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL Mix standard solutions for liquid chromatography - string

For mass spectrometry measurement, take the characteristic ion mass chromatographic peak area as the ordinate and the corresponding standard solution concentration as the abscissa, draw a standard curve, and find the regression equation and correlation coefficient:

8:4 Determination

8:4:1 Chromatographic conditions

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

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

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

d) Injection volume: 5µL;

e) Column temperature: 30 degrees C;

f) The mobile phase gradient elution conditions are shown in Table 1:

8:5 Determination method

8:5:1 Qualitative determination

Through the retention time of the sample chromatogram and the retention time of the corresponding standard, the characteristic ions of each chromatographic peak and the corresponding concentration standard solution:

The characteristic ions of the chromatographic peaks are qualitatively compared with each other, and the relative deviation between the retention time of the sample and the standard is not greater than +/-2:5%; the relative abundance of the characteristic ions of the sample is consistent with the relative abundance of the mixed standard solution of comparable concentration, and the relative ion abundance is If the degree deviation does not exceed the requirements in Table 3, it can be judged that there is a corresponding measured object in the sample:

8:5:2 Quantitative determination

Take the sample solution and standard solution, and quantify the xylazine and 2,6-dimethyl in the standard solution and sample solution according to the chromatographic peak area according to the external standard method:

The response values of xylaniline should be within the linear range of instrument detection: Under the above chromatography-mass spectrometry conditions, xylazine and 2:6-dimethylaniline standard solutions

See Appendix B for the liquid characteristic ion mass chromatogram:

8:6 Blank test

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

9 Calculation and presentation of results

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 xylazine in this method is 0:06 µg/kg, and the limit of quantitation is 0:2 µg/kg; the detection limit of 2,6-dimethylaniline is 1:5 µg/kg, and the limit of quantification is 5 µg/kg:

10:2 Accuracy

The recovery rate of xylazine in this method is 60% to 120% at the added concentration level of 0:2µg/kg~100µg/kg; the recovery rate of 2,6-dimethylaniline at the added concentration level of 5µg/kg~100µg/kg The recovery rate is 60%~120%:

10:3 Precision

The intra-batch relative standard deviation of this method is <=15%, and the inter-batch relative standard deviation is <=20%: