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

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.

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 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 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 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

7 Analysis 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); --- the final constant volume of the sample solution, the unit is milliliters (mL);

The result retains three significant digits.

Determination of low limit and recovery

10 Precision

The absolute difference between the two independent results obtained under repeatability conditions does not exceed 10% of the arithmetic mean.

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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.

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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.

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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 ????? ????, ????, ??