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Basic data
| Standard ID | GB 31613.5-2022 (GB31613.5-2022) |
| Description (Translated English) | (National food safety standards-Determination of anticoccidial drug residues in chicken edible tissues-liquid chromatography-tandem mass spectrometry) |
| Sector / Industry | National Standard |
| Classification of Chinese Standard | X22 |
| Word Count Estimation | 11,174 |
| Date of Issue | 2022-09-20 |
| Date of Implementation | 2023-02-01 |
| Issuing agency(ies) | National Health Commission of the People's Republic of China, State Administration for Market Regulation |
GB 31613.5-2022: (National food safety standards-Determination of anticoccidial drug residues in chicken edible tissues-liquid chromatography-tandem mass spectrometry)
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National Health Commission of the People's Republic of China
National Food Safety Standards
Determination of Residues of Coccidiostats in Edible Tissues of Chickens
Liquid Chromatography-Tandem Mass Spectrometry
National Standards of People's Republic of China
release
State Administration for Market Regulation
Ministry of Agriculture and Rural Affairs of the People's Republic of China
foreword
This document is in accordance with the provisions of GB/T 1.1-2020 "Guidelines for Standardization Work Part 1.Structure and Drafting Rules for Standardization Documents"
drafting.
This document is published for the first time.
National Food Safety Standards
Determination of anticoccidial drug residues in chicken edible tissues by liquid chromatography-tandem mass spectrometry
1 Scope
This document regulates the levels of hemosanone, chlorhexenidine, salinomycin, monensin, methyl salinomycin, maduramicin ammonium and lasaloxil in the edible tissues of chickens
Sample preparation and liquid chromatography-tandem mass spectrometry method for the detection of seven kinds of coccidial drug residues.
This document is applicable to poultry muscle, liver and sebum (skin + fat) containing ketone, chlorhexenidine, salinomycin, monensin, methyl salinomycin, and madomi
Determination of residues of seven coccidiostats including startrimonium and lasaloxil.
2 Normative references
The content in the following documents constitutes the essential provisions of this document through normative references in the text. Among them, the dated reference documents,
Only the version corresponding to the date applies to this document; for undated references, the latest version (including all amendments) applies to this document
document.
GB/T 6682 Analytical laboratory water specifications and test methods
3 Terms and Definitions
This document does not have terms and definitions that need to be defined.
4 principles
The residual coccidiostats in the sample were hydrolyzed by trypsin, extracted with ethyl acetate, purified by solid phase extraction column, and detected by liquid chromatography-tandem mass spectrometry.
Quantification by matrix-matched external standard method.
5 Reagents and materials
Unless otherwise specified, all reagents are of analytical grade, and the water is first-class water in accordance with GB/T 6682.
5.1 Reagents
5.1.1 Methanol (CH3OH). chromatographically pure.
5.1.2 Acetonitrile (CH3CN). chromatographically pure.
5.1.3 Acetic acid (CH3COOH). chromatographically pure.
5.1.4 Formic acid (HCOOH). chromatographically pure.
5.1.5 Ethyl acetate (CH3CH2OOCCH3). chromatographically pure.
5.1.6 Trypsin. derived from bovine pancreas, ≥10000U/mg.
5.1.7 Sodium carbonate (Na2CO3).
5.2 Solution preparation
5.2.1 10% sodium carbonate solution. Take 20g of sodium carbonate, add appropriate amount of water to dissolve and dilute to.200mL, mix well.
5.2.2 1% acetic acid solution. Take 1 mL of acetic acid, dilute it with water to 100 mL, and mix well.
5.2.3 20% methanol solution. Take 20mL of methanol, dilute to 100mL with water, and mix well.
5.2.4 Eluent. take methanol 100mL, add ethyl acetate.200mL, mix well.
5.2.5 Complex solution. take methanol 50mL, add water 50mL, formic acid 0.1mL, mix well.
5.3 Standards
The content of fushenone, chlorhexidine, salinomycin, monensin, methyl salinomycin, maduromycin ammonium, and lasaloxil was ≥95%. See Appendix A for details.
5.4 Preparation of standard solution
5.4.1 Standard stock solution (1 mg/mL). take hemosanone, chlorhexenidine, salinomycin, monensin, methyl salinomycin, madomicin ammonium, lasalol
An appropriate amount of western standard substance (equivalent to 10 mg of each active ingredient) was accurately weighed, and an appropriate amount of methanol was added to dissolve and dilute to a volume of 10 mL.
The standard stock solution with a concentration of 1 mg/mL was prepared and stored in the dark below -18 ℃, and the validity period was 6 months.
5.4.2 Mixed standard working solution (10μg/mL). Accurately measure 0.1mL of each standard stock solution, put it in a 10mL volumetric flask, dilute with methanol
Diluted to the mark, and prepared into a mixed standard working solution with a concentration of 10 μg/mL. Stored in the dark below -18°C, the validity period is 6 months.
5.4.3 Mixed standard working solution (1μg/mL). Accurately measure 1mL of 10μg/mL mixed standard working solution in a 10mL volumetric flask,
Dilute to the mark with methanol, and prepare a mixed standard working solution with a concentration of 1 μg/mL. Store in the dark below -18°C, and the validity period is 3 months.
5.4.4 Mixed standard working solution (0.1μg/mL). Accurately measure 1mL of mixed standard working solution of 1μg/mL in a 10mL volumetric flask.
Dilute with methanol to the mark, and prepare a mixed standard working solution with a concentration of 0.1 μg/mL. Store in the dark below -18°C, and the validity period is 1 month.
5.5 Materials
5.5.1 Three-bond bonded C18 solid-phase extraction column. 500mg/6mL, or equivalent.
5.5.2 Nylon microporous membrane. 0.22 μm.
6 Instruments and equipment
6.1 Liquid chromatography-tandem mass spectrometer. with electrospray ion source.
6.2 Analytical balance. Sensitivity 0.01g and 0.00001g.
6.3 High speed refrigerated centrifuge. 10000r/min.
6.4 Tissue homogenizer.
6.5 Vortex mixer.
6.6 Solid phase extraction device.
6.7 Nitrogen blowing instrument.
6.8 water bath shaker.
6.9 pH meter.
7 Preparation and storage of samples
7.1 Preparation of samples
Take an appropriate amount of fresh or thawed blank or test tissue, mince and homogenize.
a) Take the homogenized test sample as the test sample;
b) Take the homogenized blank sample as the blank sample;
c) Take the homogenized blank sample, add the standard working solution of appropriate concentration, and add the sample as a blank.
7.2 Storage of samples
Store below -18°C.
8 Measurement steps
8.1 Extraction
Weigh 1 g of the sample (accurate to ±0.01 g), add 25 mg of trypsin, 5 mL of water, vortex for 1 min, and adjust the pH with 10% sodium carbonate solution.
to 7.5, 40°C water bath overnight for enzymatic hydrolysis. Take it out and let it cool to room temperature, add 1mL of 10% sodium carbonate solution, vortex for 1min, add 9mL of ethyl acetate, vortex
Rotate for 5 min, centrifuge at 4°C and 10000r/min for 10 min, transfer the supernatant, and repeat the extraction once for the residue.
Dilute the ester to 20.0mL and mix well. Take 2.0mL of the extract and dry it with nitrogen in a water bath at 35°C, add 1mL of acetonitrile and vortex for 1min, add water
10mL, mix well, set aside.
8.2 Purification
The solid-phase extraction column was activated with 5 mL of methanol and 5 mL of 1% acetic acid solution in turn, and all the spare liquid was passed through the column, and the flow rate was controlled to be 2-3 s every 2 s.
1 drop. Rinse with 3 mL of 1% acetic acid solution and 3 mL of 20% methanol solution, and elute with 10 mL of eluent.
Blow dry with nitrogen, vortex 1.0 mL of the complex solution for 1 min, centrifuge at -4 °C and 10000 r/min for 10 min, take the supernatant, filter the membrane, and supply the liquid phase.
Chromatography-tandem mass spectrometry.
8.3 Preparation of matrix-matched standard curve
After 6 blank samples were extracted and purified, an appropriate amount of standard working solution was added, dried in a water bath with nitrogen at 35°C, and 1.0 mL of complex solution was added to vortex.
Spin for 1 min to prepare matrix-matched standard solutions with concentrations of 0.5 μg/L, 1 μg/L, 5 μg/L, 10 μg/L, 25 μg/L and 50 μg/L.
Centrifuge at -4°C and 10,000r/min for 10min, take the supernatant, filter the membrane, and use it for liquid chromatography-tandem mass spectrometry. To measure the characteristic ion peak
The area is the ordinate, and the corresponding standard solution concentration is the abscissa, draw the standard curve, and find the regression equation and correlation coefficient.
8.4 Determination
8.4.1 Reference conditions for liquid chromatography
Chromatographic column. C18 (50mm×2.1mm, 1.7μm), or equivalent;
Mobile phase. A is 0.1% formic acid aqueous solution, B is 0.1% formic acid methanol solution;
Gradient elution. the gradient elution program is shown in Table 1;
Flow rate. 0.3mL/min;
Column temperature. 30°C;
Injection volume. 10 μL.
8.4.2 Reference conditions for mass spectrometry
a) Ion source. Electrospray (ESI) ion source;
b) Scanning mode. positive ion scanning;
c) Detection method. multiple reaction monitoring;
d) Ionization voltage. 4.0kV;
e) Ion source temperature. 100°C;
f) Atomization temperature. 350°C;
g) Cone gas flow rate. 30L/h;
h) Atomized gas flow rate. 600L/h;
i) Refer to Table 2 for the reference values of the qualitative ion pair, quantitative ion pair, cone voltage and collision energy of the drug to be tested.
8.4.3 Assay
8.4.3.1 Qualitative determination
Under the same test conditions, the retention time of coccidiostats in the test solution matched the retention time of coccidiostats in the standard working solution.
The deviation is within ± 2.5%, and the detected relative ion abundance should match the relative ion of the standard solution with a matrix of comparable concentration.
The abundance is consistent. The allowable deviation should meet the requirements of Table 3.
8.4.3.2 Quantitative determination
Take the sample solution and matrix-matched standard working solution for single-point or multi-point calibration, and quantify according to the external standard method. Matrix-matched standard working solution and sample
The response values of the target substances in the solution should be within the linear range of instrument detection. Under the above conditions of chromatography-mass spectrometry, the characteristic ion mass of the standard solution
For chromatograms see Appendix B.
8.5 Blank test
Take the blank sample, except that no drug is added, the same measurement steps are used for parallel operation.
9 Calculation and presentation of results
The residues of coccidiostats in the samples were calculated according to the standard curve or formula (1).
10 Method sensitivity, accuracy and precision
10.1 Sensitivity
The detection limit of this method was 5 μg/kg, and the quantification limit was 10 μg/kg.
10.2 Accuracy
The recovery rate of this method was 60%-120% at the spiked concentration level of 10 μg/kg-100 μg/kg.
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