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National food safety standard--Determination of tylvalosin and 3-acetyltylosin residues in swine and chicken tissues by liquid chromatography-tandem mass spectrometry method
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Basic data
| Standard ID | GB 31613.2-2021 (GB31613.2-2021) |
| Description (Translated English) | National food safety standard--Determination of tylvalosin and 3-acetyltylosin residues in swine and chicken tissues by liquid chromatography-tandem mass spectrometry method |
| Sector / Industry | National Standard |
| Classification of Chinese Standard | X22 |
| Classification of International Standard | 67.120 |
| Word Count Estimation | 12,129 |
| Date of Issue | 2021-09-16 |
| Date of Implementation | 2022-02-01 |
| Issuing agency(ies) | National Health Commission of the People's Republic of China, State Administration for Market Regulation |
| Summary | This standard specifies the method for sample preparation and liquid chromatography tandem mass spectrometry for the detection of tyvanectin and 3-acetyltylosin residues in edible tissues of pigs and chickens. This standard is applicable to the determination of the residues of Suvanmectin and 3-acetyltylosin in pigs, chicken skin fat, muscle, liver, kidney and eggs. |
GB 31613.2-2021: National food safety standard--Determination of tylvalosin and 3-acetyltylosin residues in swine and chicken tissues by liquid chromatography-tandem mass spectrometry method
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National food safety standards
Tylvalerin and 3-acetylvinacetate in edible tissues of pigs and chickens
Determination of lymectin residues
Liquid chromatography-tandem mass spectrometry
National Standards of People's Republic of China
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:
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 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:
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 (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℃, 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 ℃, 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℃, 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:
6Instruments 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℃:
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) 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℃;
e) Atomization temperature: 350℃;
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:
9Result 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%:
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