GB 31613.1-2021National food safety standard--Determination of amprolium residues in cattle-derived products by liquid chromatographic-tandem mass spectrometry method and high performance liquid chromatographic method (English PDF)

1 Scope

This document specifies the sample preparation and liquid chromatography-tandem mass spectrometry methods for the detection of ampropion residues in cattle edible tissues: This document is applicable to the detection of amproline residues in beef muscle, liver, kidney and fat:

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 amproline in the sample was extracted with phosphate buffer (fat samples were extracted with acetonitrile), purified with HLB solid-phase extraction column, measured by liquid chromatography-tandem mass spectrometry, and quantified by the matrix calibration 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 Methanol (CH3OH): chromatographically pure:

5.1.2 Acetonitrile (CH3CN): chromatographically pure:

5.1.3 Formic acid (HCOOH): chromatographically pure:

5.1.4 Dipotassium hydrogen phosphate (K2HPO):

5.1.5 Potassium dihydrogen phosphate (KH2PO):

5.2 Standard products Amoprolium Hydrochloride (Cu4HiCIN4·HCl, CAS No:: 137-88-2), content >=98.0%:

5.3 Solution preparation

5.3.1 Phosphate buffer (pH 6:0): Take 2:0g of dipotassium hydrogen phosphate and 8:0g of potassium dihydrogen phosphate, add water to dissolve and dilute to 1000mL, and mix well:

5:3:20.1% formic acid solution: Take 1mL of formic acid, dilute it with water to 1000mL, and mix well:

5:3:30.1% formic acid solution-methanol: Take 900 mL of 0.1% formic acid solution and 100 mL of methanol, and mix well:

5.4 Preparation of standard solution

5.4.1 Standard stock solution (100 mg/L): Take an appropriate amount of amprolium hydrochloride standard (approximately equivalent to 10 mg of amprolium), weigh it accurately, dissolve it in methanol, dilute it to a 100mL volumetric flask, and shake well: That's it: Stored below -18°C, valid for 6 months:

5.4.2 Standard working solution: Accurately pipet an appropriate amount of standard stock solution, and dilute it step by step with 0.1% formic acid solution-methanol to a concentration of 5 µg/L, 20 µg/L, 50 pg/L,:200 µg/L, 500 µg/L and A series of standard working solutions of:2000 µg/L: Ready for use:

5.5 Materials

HLB solid phase extraction cartridge: 60 mg/3mL, or equivalent:

6 Instruments and equipment

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

6.2 Analytical balance: sensitive to 0:00001g and 0:01g:

6.3 Nitrogen blowing instrument:

6.4 Solid phase extraction device:

6.5 Homogenizer:

6.6 Vortex mixer:

6.7 Polypropylene plastic centrifuge tube: 50 mL:

6.8 Centrifuge: 8000 r/min or above:

7 Preparation and preservation of samples

7.1 Preparation of samples Take an appropriate amount of fresh or thawed air or test tissue, mince it, and homogenize it:

a) Take the homogenized test sample as the test material;

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 and conduct analysis and testing within 3 months:

8 Measurement steps

8.1 Extraction

8.1.1 Bovine muscle, liver and kidneys Take 2g of the sample (accurate to ±0:02g), add 10:0 mL of phosphate buffer, homogenize at 10000 r/min for 1 min, centrifuge at 8000 r/min for 5 min, collect the supernatant, and add 10:0 mL of phosphate buffer to the residue: , repeat the extraction once, combine the supernatants, and set aside:

8.1.2 Beef fat

Take 2g of the sample (accurate to ±0:02g), add 10:0 mL of acetonitrile, homogenize at 10000 r/min for 1 min, centrifuge at 8000 r/min for 5 min, and put the supernatant into a 50 mL plastic centrifuge tube: Add 10 mL of acetonitrile to the residue, repeat the extraction once, combine the two supernatants, blow to dryness with nitrogen in a 50°C water bath, dissolve the residue with 20:0 mL of phosphate buffer, and set aside:

8.2 Purification

The solid phase extraction column was activated with 3 mL each of methanol and water: Pass 5:0 mL of the backup solution through the column: When the liquid level reaches the surface of the column bed, rinse with 3 mL of water: Squeeze until it is almost dry: Add 2 mL of methanol and elute: The eluate is blown dry with nitrogen in a 40°C water bath: Use 0.1% formic acid solution-methanol (90:10, V/V) Dissolve the residue in 1:0 mL, pass it through a 0:22 µm filter membrane, and measure it with a liquid chromatography-tandem mass spectrometer:

8.3 Preparation of matrix matching standard curve Take the empty sample and process it in sequence according to 8:1 and 8:2 until it is blown dry with nitrogen, add 1:0mL of standard working solution of a series of concentrations to dissolve the residue, and make the concentrations of 5 µg/L, 20 µg/L, 50 µg/L,:200 µg/L, and 500 µg/L: and:2000 µg/L series matrix-matched standard solutions for liquid chromatography-tandem mass spectrometry measurement: Draw a standard curve with the peak area of the characteristic ion mass chromatogram as the ordinate and the concentration of the matrix matching standard solution as the abscissa: Find the regression equation and correlation coefficient:

8.4.1 Liquid Chromatography Reference Conditions

a) Chromatographic column: C1s chromatographic column (150mm×2:1mm, 3:5 µm) or equivalent;

b) Mobile phase: 0.1% formic acid solution-methanol (90:10, V/V);

c) Flow rate: 0:2mL/min;

d) Column temperature: 30°C;

e) Injection volume: 10 µL:

8.4.2 Mass spectrometry reference conditions

a) Ion source: electrospray (ESI) ion source;

b) Scanning method: positive ion scanning;

c) Detection method: multiple reaction monitoring (MRM);

d) Capillary voltage: 3000 V;

e) RF lens voltage: 0:5V;

f) Ion source temperature: 150°C;

g) Desolvation gas temperature: 350°C;

h) Cone air flow rate: 50 L/h;

i) Desolvation gas flow rate: 1000 L/h;

j) Secondary collision gas: argon;

k) Qualitative ion pairs, quantitative ion pairs, collision energy and cone voltage are shown in Table 1:

8.4.3 Determination method Take the sample solution and the corresponding matrix-matched standard solution for single-point or multi-point calibration, and quantify the chromatographic peak area according to the external standard method: The characteristic ion mass chromatographic peak areas of the matrix-matched standard solution and the target drug in the sample solution should be within the linear range of instrument detection: The retention time of ampropion in the sample solution is within ±2.5% of the retention time of amproline in the matrix-matched standard solution, and the relative ion abundance in the sample solution is consistent with the relative ion abundance in the matrix-matched standard solution: ratio, meeting the requirements of Table 2: Please see the attachment for the multiple reaction monitoring chromatogram of the standard solution:

Record A:

8.5 Blank test

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

9 Calculation and presentation of results

The residual amount of ampropion in the sample is calculated according to the standard curve or formula (1):