SN/T 5121-2019 English PDF

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SN/T 5121-2019: Determination of Ivermectin residue in live animal and feed for import and export - LC-MS/MS method
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Standard ID	USD	BUY PDF	Lead-Days	Standard Title (Description)	Status
SN/T 5121-2019	239	Add to Cart	3 days	Determination of Ivermectin residue in live animal and feed for import and export - LC-MS/MS method	Valid

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Basic data

Standard ID: SN/T 5121-2019 (SN/T5121-2019)
Description (Translated English): Determination of Ivermectin residue in live animal and feed for import and export - LC-MS/MS method
Sector / Industry: Commodity Inspection Standard (Recommended)
Classification of Chinese Standard: B40
Word Count Estimation: 11,181
Date of Issue: 2019-09-03
Date of Implementation: 2020-03-01
Regulation (derived from): Natural Resources Department Announcement No. 7 of 2019
Issuing agency(ies): General Administration of Customs

SN/T 5121-2019: Determination of Ivermectin residue in live animal and feed for import and export - LC-MS/MS method

---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.
(Determination of ivermectin residues in foodstuffs and feeds for import and export, liquid chromatography-mass spectrometry/mass spectrometry) ICS 71.040.40B40 People's Republic of China Entry-Exit Inspection and Quarantine Industry Standard SN/T 5121-2019 Determination of Ivermectin Residues in Food Animals and Feeds for Import and Export Liquid chromatography-mass spectrometry/mass spectrometry Published.2019-2009-03 2020-03-01 implementation Published by the General Administration of Customs of the People's Republic of China ????? ????, ????, ??

Foreword

This standard was drafted according to GB/T 1.1-2009. Please note that some content of this document may involve patents, and the issuing authority of this document is not responsible for identifying these patents. This standard is proposed and managed by the General Administration of Customs of the People's Republic of China. This standard was drafted. Chongqing Customs, People's Republic of China. The main drafters of this standard. Li Guangman, Xia Mingxing, Chen Zongxiang, Tang Yan, Wang Anliang, Zheng Xiaoling, Xiong Ying, Zhou Shuyu, Li Yingguo. SN/T 5121-2019 ????? ????, ????, ?? Determination of Ivermectin Residues in Food Animals and Feeds for Import and Export Liquid chromatography-mass spectrometry/mass spectrometry

1 Scope

This standard specifies liquid chromatography-mass spectrometry/mass spectrometry for determination of ivermectin residues in imported and exported food animals and feed. This standard applies to the determination of ivermectin in the blood and animal feed of imported and exported food animals (such as pigs, cattle, sheep, rabbits, chickens, ducks) and animal feed. Confirmation.

2 Normative references

The following documents are essential for the application of this document. For dated references, only the dated version applies to this document. file. For undated references, the latest version (including all amendments) applies to this document. GB/T 6682 Analytical laboratory water specifications and test methods

3 Principle

After the sample was extracted by acetonitrile vortex, it was detected and confirmed by liquid chromatography-mass spectrometry/mass spectrometry, and quantified by internal standard peak area method.

4 reagent materials

Unless otherwise specified, the reagents used are of analytical grade, and the water is first-grade water specified in GB/T 6682. 4.1 Acetonitrile. HPLC grade. 4.2 Formic acid. 4.3 Ammonium acetate. 4.4 Sodium chloride. 4.5 5mol/L ammonium acetate + 0.1% formic acid aqueous solution. Weigh 0.3385g of ammonium acetate in a beaker, dissolve in water, and transfer to 1000mL In a volumetric flask, wash the beaker with water at least 3 times until it is washed, and transfer all the washing liquid to a 1000 volumetric flask, then add 1 mL of formic acid In a 1000mL volumetric flask, dilute with water. 4.6 Reference material. Ivermectin (CAS number. 7028-86-7), purity is greater than 96%; Ivermectin isotope internal standard (Ivermetin-d2), purity is greater than 98%. 4.7 Standard stock solution. Accurately weigh 0.1g of ivermectin standard (accurate to 0.0001g), fully dissolve with acetonitrile (4.1), and dilute to volume. In a 100mL volumetric flask, a standard stock solution with a concentration of 1mg/mL was prepared; in addition, the ivermectin internal standard standard was 10mg. Dissolve fully in acetonitrile (4.1), dilute to volume in a 10mL volumetric flask, and prepare an ivermectin internal standard with a concentration of 1mg/mL. For the standard stock solution, pour the volume of the standard stock solution into the brown reagent bottle, and store it in the dark below -18 ° C. 4.8 Standard intermediate stock solution. Accurately transfer 100 μL of ivermectin standard stock solution (4.7) and 100 μL of ivermectin internal standard stock solution (4.7) in two 100 mL volumetric flasks with acetonitrile Dilute to a constant volume and prepare 1 μ/mL ivermectin standard intermediate stock solution and 1 μg/mL ivermectin internal standard standard intermediate stock solution. The solution is placed in a brown reagent bottle, and stored in a dark place below -18 ° C... 4.9 Standard working solution with internal standard. Dilute ivermectin standard intermediate stock solution (4.8) with acetonitrile (4.1) to appropriate concentration as needed Mixed standard working curve solution, at the same time each standard solution is added with an equal amount of ivermectin internal standard standard intermediate stock solution standard intermediate SN/T 5121-2019 ????? ????, ????, ?? Stock solution (4.8). The concentration of the internal standard of ivermectin in each standard solution was 20 μg/L. The reference linear concentration range of ivermectin was 5 μg/L to 50 μg/L. 4.10 Matrix standard working solution. Select 5 samples of ivermectin-free sample and process according to this standard 7.1 to "take the supernatant 5mL nitrogen "Air blow dry" (without the addition of ivermectin internal standard), add 1 mL of standard working solution (4.9) with internal standard at different concentrations, dissolve The slag, after passing through a 0.22 μm filter membrane (4.11), is configured into a matrix standard working solution, which is now ready for use. 4.11 Microporous membrane. organic, 0.22 μm.

5 instruments and equipment

5.1 Liquid chromatography-mass spectrometry/mass spectrometer. Power distribution spray ion source (ESI). 5． Balance. Sensitivity is 0.0001g and 0.01mg. 5.3 Refrigerated centrifuge. The rotation speed is not less than 8000r/min. 5.4 Vortex mixer. 5.5 Ultrasonic cleaner. 5.6 Nitrogen blowing instrument.

6 Sample preparation and storage

6.1 Sample preparation 6.1.1 Feed. take an appropriate amount of a representative sample from the sample taken, grind and mix it, and put it into a clean plastic bag. The number of samples 15g, sealed and marked. 6.1.2 Blood. Animal blood was collected with heparin sodium anticoagulant blood collection tubes, the sample size was 15mL, sealed and labeled. 6.2 Sample preservation Feed was stored at room temperature. Blood samples to be tested were stored at 4 ° C. Blood samples were stored frozen at -18 ° C.

7 Analysis steps

7.1 Extraction 7.1.1 Blood Weigh 2.0g (accurate to 0.01g) sample and 0.5g sodium chloride (4.4) into a 50mL centrifuge tube, add 40μL of ivermectin internal standard intermediate stock solution (4.8, Equivalent to the internal standard mass of ivermectin is 4ng), 3mL of water and 10mL of acetonitrile (4.1), vortex Mix for 2 min, shake for 30 min, freeze and centrifuge for 5 min at 8000 r/min below 10 ° C, and take 5 mL of nitrogen from the supernatant. Dry, add 1.0 mL of acetonitrile (4.1), dissolve the residue with ultrasound, pass through a 0.22 μm filter membrane (4.11), and use for liquid chromatography-mass spectrometry/mass spectrometry for determination and confirmation. 7.1.2 Feed Weigh 2.0g (accurate to 0.01g) sample and 3.0g sodium chloride (4.4) into a 50mL centrifuge tube, add 40μL of ivermectin isotope internal standard intermediate stock solution (4.8, It is equivalent to the internal standard mass of ivermectin is 4ng), 7.5mL of water and 10mL of acetonitrile (4.1). Vortex for 2 min, shake to extract 30 min, 8000 r/min at 10 ° C or lower for 5 min, freeze and centrifuge the supernatant for 5 mL of nitrogen. Blow-dry, add 1.0 mL of acetonitrile (4.1), dissolve the residue with ultrasound, pass through a 0.22 μm filter membrane (4.11), and use for liquid chromatography-mass spectrometry/mass spectrometry for determination and confirmation. SN/T 5121-2019 ????? ????, ????, ?? 7.2 Measurement and confirmation 7.2.1 LC-MS/MS Reference Conditions 7.2.1.1 Reference conditions for liquid chromatography The reference conditions are as follows. a) Chromatographic column. C18 column, 100mm × 4.6mm (inner diameter), 2.6μm, or equivalent. b) Mobile phase. A. 5 mmol/L ammonium acetate + 0.1% formic acid aqueous solution (4.5); B. acetonitrile (4.1). See Table 1 for gradient elution procedure. c) Flow rate. 0.4mL/min. c) Column temperature. 30 ° C. d) Injection volume. 20 μL. Table 1 Mobile phase and gradient elution procedure Time (min) Flow rate (ml/min) 5mmol/L ammonium acetate + 0.1% formic acid aqueous solution (A)% Acetonitrile (B)% 0 0.4 50 50 1 0.4 5 95 12 0.4 5 95 12.1 0.4 50 50 15.0 0.4 50 50 7.2.1.2 Reference conditions for mass spectrometry The reference conditions are as follows. a) Ion source. Electrospray ion source (ESI); b) scanning method. positive ion; c) Monitoring method. multiple response monitoring (MRM); d) The atomizing gas (OS1), curtain gas (CUR), auxiliary gas (GS2), and collision gas (CAD) are all high-purity nitrogen or other suitable gases; Before use, the gas flow rate, ion source temperature, and electrospray voltage should be set to appropriate values, so that the sensitivity of the mass spectrometer can meet the detection requirements. See Appendix A for detailed conditions; e) The de-clustering voltage (DP), collision chamber inlet voltage (EP), collision voltage (CE), and collision chamber outlet voltage (CXP) should be optimized to the best Conditions, monitoring ions and quantifier ions, see Appendix A for detailed conditions. 7.2.2 Quantitative determination According to the content of the test substance in the sample, a matrix standard working solution with a suitable response value is prepared for chromatographic analysis. There should be 5 concentration levels. The response value of ivermectin in the matrix standard working solution and the test solution should be within the linear response range of the instrument. If so The content of the test substance in the product exceeds the linear range of the standard curve, and it should be diluted with acetonitrile (4.1) to an appropriate concentration for analysis. Under the above chromatographic conditions The reference retention time of ivermectin is 7.60 min, and the multiple reaction monitoring (MRM) chromatogram of ivermectin standard solution is shown in Figure B. 1. 7.2.3 Qualitative analysis Determine the sample and matrix standard working solution according to the conditions of liquid chromatography-mass spectrometry/mass spectrometry. The standard product is consistent, and the retention time deviation is within 5%; the relative abundance of the qualitative ion pair is a table of the percentage of the intensity relative to the strongest ion SN/T 5121-2019 ????? ????, ????, ?? It should be consistent with the relative abundance of a standard working solution with a fairly standard concentration. The allowable deviation of the relative abundance does not exceed the range specified in Table 2. The corresponding test object exists in the broken sample. Table 2 Maximum allowable deviation of relative ion abundance during qualitative confirmation Relative ion abundance /% ＞ 50 ＞ 20 ～ 50 ＞ 10 ～ 20 ≤10 Allowable relative deviation /% ± 20 ± 25 ± 30 ± 50 7.3 Blank test Except for not adding samples, all operations are carried out according to the above operation steps.

8 Results calculation and expression

Use the instrument software to process the data or draw a standard curve according to the internal standard method. The value is the abscissa, and the standard curve is drawn with the concentration ratio of each matrix standard working solution to the internal standard solution as the ordinate. The peak area ratio of the standard is substituted into the curve equation to calculate the ratio of the concentration of the analyte in the sample to the internal standard. Since the internal standard concentration is known, The concentration of analyte C in the sample solution is substituted into formula (1), and the blank value must be deducted from the calculation result. In the formula. X --- content of test substance in sample, unit is microgram per kilogram (μg/kg); C --- solution concentration of test substance obtained from standard curve, unit is microgram per liter (μg/L) ; C0 --- the solution concentration of the test substance in the blank test obtained from the standard curve, the unit is micrograms per liter (μg/L); 犞 --- the final constant volume of the sample solution, the unit is milliliters (mL); The result retains three significant digits. 九 Determination of low limit and recovery 9.1 Low limit of determination Lower limit of determination by this method. the detection limit of blood and edible animal feed (such as pig, cow, sheep, rabbit, chicken, duck) and animal feed It is 10 μg/kg. 9.2 Recovery rate This method is used to test the recovery rate of blood, animal feed, and other substrates of food animals (such as pigs, cattle, sheep, rabbits, chickens, and ducks). See Appendix C for data. 10 precision The absolute difference between the two independent results obtained under repeatability conditions does not exceed 10% of the arithmetic mean. SN/T 5121-2019 ????? ????, ????, ??

Appendix A

(Informative appendix) Mass reference conditions 1) MS Reference Conditions a) Electrospray voltage (IS). 5500V; b) Impact gas pressure (CAD). Medium; c) Atomizing gas pressure (GS1). 50Psi; d) Curtain gas pressure (CUR). 20Psi; e) Auxiliary gas pressure (GS2). 50Psi; f) Ion source temperature (TEM). 600 ° C; g) For monitoring ion pairs, degroup voltage (DP), collision chamber inlet voltage (EP), collision voltage (CE), and collision chamber outlet voltage (CXP) Table A. 1. Table A. 1 Qualitative and quantitative transitions and CE, DP, EP, CXP reference values Compound parent ion (Q1) detection mode ion (Q3) CEV DP EP CXP Ivermectin 892.8 Positive ion 569.7  21.19 79.995 10 7 307.5 59.90 79.95 10 12 d2-Ivermella Prime internal standard 894.9 Positive ion 571.5 5 19.90 90 10 10 Note. The ions with gadolinium in the table are quantitative ions. For different mass spectrometers, the parameters of the instrument may differ. The mass spectrometer parameters should be optimized to the best before the determination. 1) Non-commercial statement. The reference mass spectrometry conditions listed in Appendix A were completed on an ABAPI4000/TRAP LC/MS instrument. The test instruments are listed here. The device model is for reference only and does not involve commercial purposes. Standard users are encouraged to try different manufacturers or models of instruments. SN/T 5121-2019 ????? ????, ????, ??

Appendix B

(Informative appendix) Multiple reaction monitoring (MRM) chromatograms of ivermectin standard solution and isotope internal standard solution Multireaction detection (MRM) chromatograms of ivermectin standard solution and isotope internal standard solution are shown in Figure B. 1. SN/T 5121-2019 ????? ????, ????, ?? SN/T 5121-2019 ????? ????, ????, ??

Appendix C

(Informative appendix) Experimental data on recovery of ivermectin in different matrices Matrix addition level μg/kg Recovery rate Pig blood 10 73.7 to 79.5 20 79.0 to 89.9 40 87.0 to 89.0 Cow blood 10 83.7 ～ 104 20 92.0 to 96.5 40 81.5 to 89.5 Sheep blood 10 74.0 to 83.5 20 80.5 to 88.3 40 86.5 to 94.2 Chicken blood 10 73.7 to 78.9 20 82.0 to 88.0 40 79.2 to 86.5 Duck blood 10 81.2 to 86.0 20 79.0 to 87.5 40 86.0 to 89.2 Matrix addition level μg/kg Recovery rate Rabbit blood 10 76.8 to 83.7 20 82.0 to 89.0 40 80.2 to 89.4 Pellet feed 10 72.8 to 85.6 20 81.5 to 93.5 40 85.8 to 90.2 Premix feed 10 71.5 to 82.7 20 81.0 to 92.0 40 84.8 to 90.5 Concentrated feed 10 78.6 to 85.9 20 74.0 to 79.5 40 87.2 to 91.0 SN/T 5121-2019 ????? ????, ????, ?? ......

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