GB 31658.4-2021National food safety standard - Determination of cephalosporins residues in animal derived food by liquid chromatography-tandem mass spectrometry method (English PDF)

Foreword

This document complies with the provisions of GB/T 1:1-2020 "Standardization Work Guidelines Part 1: Structure and Drafting Rules of Standardization Documents"

Drafting:

This document is published for the first time:

National food safety standards

Determination of cephalosporin residues in animal foods

Liquid chromatography-tandem mass spectrometry

1 Scope

This document specifies sample preparation and liquid chromatography G-string for detection of cephalosporin residues in pig, cattle muscle, liver, kidney, fat and milk:

Mass spectrometric determination method:

This document applies to cephalosporin drugs (cephalexin, cefradine, cefazolin,

Detection of residues of cefoperazone, cefacetonitrile, cefpirin, ceftronine, cefquinoxime, and cefotaxime):

2 Normative reference documents

The contents of the following documents constitute essential provisions of this document through normative citations in the text: Among them, the cited documents with dates are:

Only the version corresponding to the date applies to this document; for undated referenced documents, the latest version (including all amendments) applies to this document:

document:

GB/T 6682 Specifications and test methods for water used in analytical laboratories

3 Terms and definitions

There are no terms or definitions that need to be defined in this document:

4 Principles

The residual cephalosporins in the sample were extracted with acetonitrile-water and phosphate buffer, purified with a solid-phase extraction column, and measured by liquid chromatography-tandem mass spectrometry:

Determination, external standard method quantification:

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 Potassium dihydrogen phosphate (KH2PO4):

5:1:5 Sodium hydroxide (NaOH):

5:1:6 Sodium chloride (NaCl):

5:2 Solution preparation

5:2:1 Sodium hydroxide solution: Take 50g of sodium hydroxide, add water to dissolve and dilute to 500mL:

5:2:2 Acetonitrile-water solution (15:2, V/V):

5:2:3 30% acetonitrile solution: Take 30mL of acetonitrile and dilute to 100mL with water:

5:05mol/L phosphate buffer (pH=8:5): Take 6:8g of potassium dihydrogen phosphate, dissolve it in water and dilute it to 1000mL, add hydroxide

Adjust the pH of the sodium solution to 8:5+/-0:1:

5:2:5 0:1% formic acid solution: Take 1mL of formic acid, dissolve and dilute to 1000mL with water, and mix well:

5:3 Standard products

Cephalexin, cefradine, cefazolin, cefoperazone, cefacetonitrile, cefpirin, desacetylcefapirin, ceftronin, cefquine

Oxime and cefotaxime standard products, the contents are >=95:0%, see Appendix A:

5:4 Preparation of standard solution

5:4:1 Mixed standard stock solution: take cephalexin, cefradine, cefazolin, cefoperazone, cefacetonitrile, cefpirin, deacetylcephalosporin

Take 10 mg each of Pirin, ceftronine, cefquinoxime, and cefotaxime standard standards, weigh them accurately, dissolve them with 30% acetonitrile solution, and dilute to a constant volume:

25 mL volumetric flask, prepare a solution with a concentration of 400 µg/mL: Store in -18°C protected from light, valid for 1 month:

5:4:2 Mixed standard working solution: Precisely measure an appropriate amount of mixed standard stock solution, put it into a 10mL volumetric flask, and dilute it with water to the mark: Prepare

Mix standard working solutions with concentrations of 0:05 µg/mL, 0:50 µg/mL, 2:0 µg/mL and 20 µg/mL: Store in the dark at 4°C:

Validity period 7d:

5:5 Materials

5:5:1 HLB solid phase extraction column, 500mg/6mL, or equivalent:

5:5:2 Filter membrane: nylon material, pore size 0:22µm, or equivalent performance:

6 Instruments and equipment

6:1 Liquid chromatography-tandem mass spectrometer: equipped with electrospray ion source:

6:2 Analytical balance: sensitivity 0:00001g and 0:01g:

6:3 Rotary evaporator:

6:4 Solid phase extraction device:

6: Homogenizer:

6: Vortex mixer:

6:7 Polypropylene plastic centrifuge tubes: 10mL, 50mL:

6: Centrifuge: 5000r/min or above:

6:9 Chicken heart bottle: 50mL:

7: Preparation and preservation of samples

7:1 Preparation of samples

Take an appropriate amount of fresh or thawed blank 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 Preservation of samples

Store below -18 degrees C and conduct analysis and testing within 3 months:

8 Measurement steps

8:1 Extraction

Take 2g of the sample (accurate to +/-0:02g), put it in a 50mL centrifuge tube, add 10mL of acetonitrile-G aqueous solution or 10mL of acetonitrile (only applicable to cattle

Milk), homogenize at 10000r/min for 1min, centrifuge at 5000r/min for 2min, collect the supernatant and put it in a chicken heart bottle: Use 10mL of the above solution for the residue

Repeat the extraction once, combine the extracts, and rotary evaporate in a 40°C water bath to remove acetonitrile (if there is foam, add 4 mL of saturated sodium chloride aqueous solution), and immediately

That is, add 25 mL of phosphate buffer solution and set aside:

8:2 Purification

Take the solid phase extraction column and activate it with 5 mL of methanol and 10 mL of phosphate buffer: Pass the backup solution through the column: When the liquid level reaches the surface of the column bed, use phosphorus:

Elute with 3 mL of acetonitrile buffer solution, rinse with 2 mL of water, elute with 3 mL of acetonitrile in a 10 mL centrifuge tube, blow dry with nitrogen in a 40°C water bath, and add water:

Dissolve 1:0 mL and pass through a 0:22 µm filter membrane for liquid chromatography-tandem mass spectrometry measurement:

8:3 Preparation of matrix matching standard curve

Precisely measure an appropriate amount of the mixed standard working solution, dilute it with the extracted and purified blank sample solution to prepare concentrations of 5 µg/L, 10 µg/L,

A series of matrix calibration working solutions of 20 µg/L, 50 µg/L, 100 µg/L and:200 µg/L were passed through a 0:22 µm filter membrane for liquid chromatography-tandem connection:

Mass spectrometry: Use the quantitative ion pair peak area as the ordinate and the standard solution concentration as the abscissa to draw a standard curve: Find the regression equation and correlation

coefficient:

8: Measurement

8:4:1 Liquid Chromatography Reference Conditions

a) Chromatographic column: C18 chromatographic column (50mmx2:0mm, 1:7µm) or equivalent;

b) Mobile phase: A is 0:1% formic acid solution, B is methanol, and the gradient elution conditions are shown in Table 1;

c) Flow rate: 0:3mL/min;

d) Column temperature: 35 degrees C;

e) Injection volume: 10µL:

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::2000V;

e) RF lens voltage: 0:5V;

f) Ion source temperature: 150 degrees C;

g) Desolvation gas temperature: 500 degrees C;

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

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

j) Secondary collision gas: argon;

k) Qualitative ion pairs, quantitative ion pairs, collision energy and cone voltage are shown in Table 2:

8: 4: Determination method

Take the sample solution and the matrix-matching standard solution, conduct single-point or multi-point calibration, and quantify the chromatographic peak area according to the external standard method: The matrix-matching standard solution

The characteristic ion mass chromatographic peak areas of the target drug in the sample solution and the sample solution should be within the linear range of the instrument detection: Cephalosporins in the sample solution

The ratio of the retention time of cephalosporins to the retention time of cephalosporins in the matrix-matched standard working solution is within +/-2:5%, and the sample solution is

Compared with the relative ion abundance in the matrix matching standard solution, the relative ion abundance in the solution meets the requirements of Table 3: Matrix matching standard solution

See Appendix B for multiple reaction monitoring chromatograms:

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 the drug to be tested in the sample is calculated according to the standard curve or formula (1):

10 Sensitivity, accuracy and precision of detection methods

10:1 Sensitivity

The detection limit of this method in muscle, liver, kidney, fat and milk of pigs and cattle is 2:0 µg/kg, and the limit of quantification is 5:0 µg/kg:

10:2 Accuracy

The recovery rate of this method is 60% to 110% at the added concentration level of 5:0µg/kg~2000µg/kg:

10:3 Precision

The intra-batch relative standard deviation of this method is <=15%, and the inter-batch relative standard deviation is <=20%: