GB 31658.5-2021National Food Safety Standard Determination of Florfenicol and Florfenicol Residues in Animal Foods Liquid Chromatography-Tandem Mass Spectrometry (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 florfenicol and florfenicolamide residues in animal foods

Liquid chromatography-tandem mass spectrometry

1 Scope

This document specifies sample preparation and liquid chromatography-tandem mass spectrometry for the detection of florfenicol and florfenicolamine residues in animal foods:

method:

This document is applicable to the determination of florfenicol and florfenicol amide residues in muscle, skin + fat, liver and kidney tissue of chicken, pig, cattle and sheep:

Determination:

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 remaining florfenicol and florfenicolamide in the sample were extracted with alkalized ethyl acetate, degreased with n-hexane, purified with a solid phase extraction column, and liquid chromatographed:

Determination by spectrum-G tandem mass spectrometry and quantification by internal 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 Acetonitrile (CH3CN): chromatographically pure:

5:1:2 Methanol (CH3OH): chromatographically pure:

5:1:3 Formic acid (HCOOH): chromatographically pure:

5:1:4 Ethyl acetate (CH3COOC2H5):

5:1:5 Ammonia (NH3:H2O):

5:1:6 Acetic acid (CH3COOH):

5:2 Solution preparation

5:2:1 5% acetic acid aqueous solution: Take 5mL of acetic acid, dissolve it in water and dilute it to 100mL:

5: 2: 10% ammonia-methanol solution: Take 5 mL of ammonia water, dissolve it in methanol and dilute it to 50 mL: Prepare it now:

5:2:3 30% acetonitrile aqueous solution: Take 30mL of acetonitrile, dissolve it in water and dilute it to 100mL:

5:2:4 Ethyl acetate-ammonia solution: Take 98mL of ethyl acetate, add 2mL of ammonia, and mix:

5:3 Standard products

5:3:1 Florfenicol (Florfenicol, C12H14Cl2FNO4S, CAS number: 73231-34-2): content >=99:0%:

5: Florfenicolamine (C10H14FNO3S, CAS number: 76639-93-5): Content >= 97:6%:

GB 31658:5-2021

5: Deuterated chloramphenicol (Chloramaphenicol D5, d5-CAP, CAS number: 202480-68-0): content is 100ng/mL:

5:4 Preparation of standard solution

5:4:1 Florfenicol and florfenicol amine standard stock solutions: Take 25 mg of florfenicol and florfenicol amine standard solutions, weigh them accurately, and add acetonitrile appropriately:

Dissolve and dilute to a 25mL volumetric flask to prepare standard stock solutions of florfenicol and florfenicolamine respectively: Store below -18°C:

Valid for 6 months:

5:4:2 10 µg/mL mixed standard working solution: Precisely measure 1 mL each of florfenicol and florfenicol amine standard stock solutions, and place in 100 mL

Dilute the volumetric flask with acetonitrile to the mark to make a mixed standard working solution with a concentration of 10 µg/mL: Store it at 2°C ~ 8°C and is valid for one month:

5:4:3 1µg/mL mixed standard working solution: Precisely measure 1mL of 10µg/mL mixed standard working solution, put it in a 10mL volumetric flask, and add acetonitrile to it:

Dilute to the mark to make a mixed standard working solution with a concentration of 1 µg/mL: Store at 2°C ~ 8°C and is valid for 1 month:

5:5 Materials

MCX solid phase extraction column 60mg/3mL, or equivalent:

6 Instruments and equipment

6:1 Ultra-high performance liquid chromatography-tandem mass spectrometer: equipped with electrospray ion source:

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

6: High speed centrifuge:

6: Vortex mixer:

6:5 horizontal oscillator:

6: Homogenizer:

6:7 Solid phase extraction device:

6: 8 Nitrogen blowing instrument:

6:9 Filter membrane: organic phase, 0:22µm:

6:10 chicken heart bottle: 100mL:

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 a homogeneous test sample as a test material;

b) Take a homogeneous blank sample as a blank sample;

c) Take a homogeneous blank sample, add standard working solution of appropriate concentration, and add the sample as a blank:

7:2 Preservation of samples

Store below -18 degrees C:

8 Measurement steps

8:1 Extraction

Weigh 2g of the sample (accurate to +/-0:02g), put it into a 50mL centrifuge tube, add 100µL of deuterated chloramphenicol internal standard solution, and vortex to mix:

Mix well and let stand for 15 minutes: Add 10 mL of ethyl acetate-ammonia solution, vortex for 1 minute, centrifuge at 3000 r/min for 5 minutes, and take the supernatant:

In a 100 mL chicken heart bottle, the residue was repeatedly extracted twice, and the three extracts were combined: Add 2 mL of 5% acetic acid to the chicken heart bottle, shake and mix, and

Concentrate to 1:5 mL in a 40°C water bath: Transfer to another 50 mL centrifuge tube, wash the chicken heart bottle with 2 mL of 5% acetic acid, and transfer the washing solution to

In the same centrifuge tube, add 5 mL of n-hexane for defatting, vortex for 1 min, and centrifuge at 3000 r/min for 5 min: Discard the upper layer and repeat the degreasing of the lower layer extract:

Once, spare:

8:2 Purification

The MCX solid-phase extraction column was activated with 2 mL of methanol and 2 mL of water in sequence: Pass the backup solution through the column, elute with 2 mL of 5% acetic acid, and rinse with 10% ammonia:

Elute with 5 mL of methanol: Collect the eluate and blow dry with nitrogen at 40°C: Dissolve the residue with 500 µL of 30% acetonitrile aqueous solution, filter with a membrane, and provide

Ultra-high performance liquid chromatography-tandem mass spectrometry:

8:3 Preparation of matrix matching standard curve

Precisely measure 5 µL, 25 µL and 50 µL of the 1 µg/mL mixed standard working solution, and 10 µL, 25 µL of the 10 µg/mL mixed standard working solution:

and 50 µL, and sequentially added to 6 portions of the extracted and purified blank sample concentrate, and at the same time, 100 ng/mL deuterated chloramphenicol was added:

Take 100 µL of the standard solution, add 30% acetonitrile aqueous solution to dissolve and dilute to 0:5 mL, and prepare the concentrations to 10 ng/mL, 50 ng/mL,

Matrix matching series standard solutions of 100ng/mL,:200ng/mL, 500ng/mL, and 1000ng/mL were filtered and used for liquid chromatography-tandem mass spectrometry:

Spectrum determination: Using the measured characteristic ion mass chromatographic peak area ratio as the ordinate and the corresponding standard solution concentration as the abscissa, draw the matrix matching standard:

Quasi-curve: Find regression equation and correlation coefficient:

8: Measurement

8:4:1 Chromatographic conditions

a) Chromatographic column: C18 (100mmx2:1mm, 1:7µm), or equivalent;

b) Mobile phase: A: aqueous solution, B: acetonitrile solution;

c) Gradient elution: see Table 1 for gradient elution conditions;

d) Flow rate: 0:25mL/min;

e) Column temperature: 30 degrees C;

f) Injection volume: 10µL:

8:4:2 Mass spectrometry conditions

a) Ion source: electrospray ion source;

b) Scanning method: positive ion scanning (florfenicol amide) and negative ion scanning (florfenicol and deuterated chloramphenicol);

c) Detection method: multiple reaction monitoring;

d) Ionization voltage: 2:8kV;

e) Source temperature: 80 degrees C;

f) Atomization temperature: 300 degrees C;

g) Cone air flow rate: 30L/h;

h) Atomizing gas flow rate: 600L/h;

i) The qualitative ion pairs, quantitative ion pairs and corresponding cone voltages and collision energies of the test drugs are shown in Table 2:

8: 4: Determination method

Take the sample solution and matrix-matching standard solution, perform single-point or multi-point calibration, and quantify the sample solution and matrix by chromatographic peak area according to the internal standard method:

The characteristic ion mass chromatographic peak areas of florfenicol and florfenicolamide in the matching standard solution should be within the linear range of instrument detection:

Compared with the relative ion abundance in the matrix-matched standard solution, the relative ion abundance in the sample solution meets the requirements of Table 3: The standard solution characteristics

See Appendix B for the characteristic ion mass chromatogram:

10 Sensitivity, accuracy and precision of detection methods

10:1 Sensitivity

The detection limit of this method is 3 µg/kg, and the quantification limit is 10 µg/kg:

10:2 Accuracy

The recovery rate of this method is 70% to 120% at the added concentration level of 10µg/kg~300µg/kg:

10:3 Precision

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