GB 31613.2-2021National food safety standard--Determination of tylvalosin and 3-acetyltylosin residues in swine and chicken tissues by liquid chromatography-tandem mass spectrometry method (English PDF)

1 Scope

This document specifies the sample preparation and liquid chromatography-tandem mass spectrometry methods for the detection of tivalisin and 3-acetyltylosin residues in edible tissues of pigs and chickens:

This document is applicable to the determination of tivalisin and 3-acetyltylosin residues in pig, chicken sebum, muscle, liver, kidney, and eggs:

2 Normative 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 used in analytical laboratories

3 Terms and definitions

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

4 Principles

The remaining tyversin and 3-acetyl tylosin in the sample were extracted with 50% acetonitrile, purified with n-hexane and acidic alumina, measured by liquid chromatography-tandem mass spectrometry, and quantified using the matrix-matched standard solution 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 Formic acid (HCOOH): chromatographically pure:

5:1:3 n-Hexane (C8Hu):

5.1.4 Anhydrous magnesium sulfate (MgSO4):

5.1.5 Sodium chloride (NaCl):

5.2 Solution preparation 5:2:1 0:02 mol/L tartaric acid aqueous solution: Take 0:3g of tartaric acid and add 100mL of water to dissolve:

5:2:20.1% formic acid aqueous solution: Take 0:5 mL of formic acid and dissolve it in 500 mL of water:

5:2:3 50% acetonitrile tartaric acid solution: Take 50mL of acetonitrile, dilute it to 100mL with 0:02 mol/L tartaric acid aqueous solution, and mix well:

5.3 Standard products

5.3.1 Tylvalosin (CssHaNO, CAS number: 63409-12-1), content >=93.0%:

5:3:23-acetyltylosin (3-acetyltylosin, Cis H NOis, CAS number: 63409-10-9), content >=95.0%:

5.4 Preparation of standard solution

5.4.1 Tylvalisin standard stock solution (1mg/mL): Take 10 mg of Tylvalisin reference substance, weigh it accurately, dissolve it in acetonitrile and dilute it to a 10mL brown volumetric flask, shake well, and it is ready: Stored below -18°C, valid for 6 months:

5:4:23-Acetyl Tylosin Standard Stock Solution (1 mg/mL): Take 10 mg of 3-Acetyl Tylosin reference substance, weigh it accurately, and place it in 10 mL of brown solution:

Color volumetric flask, dissolve with acetonitrile and dilute to volume, shake well: Stored below -18 °C, valid for 6 months:

5.4.3 Mixed standard working solution (10 µg/mL): Accurately draw 1 mL of each of the standard stock solutions of tyvalisin and 3-acetyl tylosin into 100 mL:

Brown volumetric flask, dilute to volume with acetonitrile, shake well, and it is ready: Stored below -18°C, valid for 3 months:

5.5 Materials

5.5.1 Acid alumina: 100 mesh to:200 mesh:

5.5.2 Microporous filter membrane: nylon, 0:22µm:

6 Instruments and equipment

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

6.2 Analytical balance: sensitivity 0:01g and sensitivity 0:00001 g:

6.3 Centrifuge tubes: 50 mL, 10 mL:

6.4 Vortex mixer:

6:5 horizontal oscillator,

6.6 High-speed centrifuge: the maximum speed is not less than 8000 r/min:

7 Preparation and preservation of samples

7.1 Preparation of samples Take an appropriate amount of fresh or refrigerated blank or test sample, homogenize and set aside:

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 and add standard working solution of appropriate concentration as a blank self-added sample:

7.2 Storage of samples Store below -20°C:

8 Measurement steps

8.1 Extraction

Take 2g of the sample (accurate to ±0:02g) in a 50mL centrifuge tube, add 5:0 mL of water and 5:0 mL of acetonitrile, vortex and mix, add 10mL of n-hexane, shake for 5 minutes, then add 2g each of anhydrous magnesium sulfate and sodium chloride: Shake for 5 minutes, centrifuge at 8000 r/min for 5 minutes, and transfer the middle acetonitrile layer to another centrifuge tube for later use:

8.2 Purification

Accurately pipette 0:5mL of the reserve solution and 0:5mL of the 0:02 mol/L tartaric acid solution, totaling 1:0mL: Add 0:2g of acidic alumina, vortex and mix for 30 s, centrifuge at 10000 r/min for 5 min, take the upper layer, pass through a 0:22 µm microporous filter, and place it in a brown injection bottle for liquid chromatography-tandem mass spectrometry measurement:

8.3 Preparation of matrix standard curve Take 2g of the empty sample (accurate to ±0:02g) in a 50mL centrifuge tube, and follow the steps in 8:1 and 8:2 to obtain the matrix solution of the empty sample: Precisely measure an appropriate amount of standard working solution, and use blank sample matrix solution to prepare a concentration of 0:5 ng/mL, 1:0 ng/mL, A series of standard working solutions of 2:0ng/mL, 5:0ng/mL, 10 ng/mL, and 20 ng/mL were measured on the machine: Draw a standard curve with the peak area of the characteristic ion mass chromatogram as the ordinate and the concentration of the standard solution as the abscissa: If you need to use single-point calibration, you must prepare the injection in this standard curve:

Calibrate with a matrix-matched reference solution within the concentration range (0:5ng/mL~100ng/mL) that is closest to the injection concentration of the sample solution: The linear correlation coefficient of the standard curve should not be less than 0:99:

8.4 Determination

8.4.1 Chromatographic conditions reference conditions

a) Chromatographic column: C (150mm×2:1mm, 3:0µm), or equivalent;

b) Column temperature: 30°C;

c) Injection volume: 10 µL;

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

e) Mobile phase: A is acetonitrile, B is 0.1% formic acid solution, and the gradient elution procedure is shown in Table 1:

8.4.2 Mass spectrometry conditions reference conditions

a) Ion source: electrospray ion source;

b) Scanning method: positive ion scanning;

c) Detection method: multiple reaction monitoring;

d) Ion source temperature: 350°C;

e) Atomization temperature: 350°C;

f) Ionization voltage: 3700 V;

g) Cone air flow rate: 300 L/h;

h) Auxiliary air flow rate: 500 L/h;

i) Purge gas flow rate: 100 L/h;

j) Collision gas: high purity argon,:200 Pa;

k) Qualitative and quantitative ion pairs and the corresponding lens voltage and collision energy reference values are shown in Table 2:

8.5 Determination method

8.5.1 Qualitative determination Under the same test conditions, the retention time of tivalisin and 3-acetyltylosin in the sample solution is the same as the retention time of the corresponding target substances in the standard working solution:

The retention time deviation should be within ±2.5%, and the relative ion abundance detected should be consistent with the relative ion abundance of the calibration standard solution of equivalent concentration: The allowable deviation should comply with the requirements of Table 3:

8.5.2 Quantitative determination Take the sample solution and the corresponding standard working solution, conduct single-point or multi-point calibration, and quantify by chromatographic peak area according to the external standard method: The response values of the target substances in the standard working solution and the sample solution should be within the linear range of the instrument detection: Inside: Under the above chromatography-mass spectrometry conditions, the characteristic ion mass chromatograms of the standard solutions of tivalisin and 3-acetyltylosin are shown in Appendix A:

8.6 Blank test

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

9 Result calculation and presentation

The residual amount of the drug to be tested in the sample is calculated according to formula (1):

10 Sensitivity, accuracy and precision of detection methods

10.1 Sensitivity

The detection limit of this method is 2:5µg/kg, and the quantitation limit is 5µg/kg:

10.2 Accuracy

The recovery rate of this method at the added concentration level of 5 µg/kg to:200 µg/kg is 60% to 110%:

10.3 Precision

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