GB 31658.2-2021National Food Safety Standard Determination of Chloramphenicol 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 chloramphenicol 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 chloramphenicol residues in pig, chicken muscles, livers and edible tissues of fish and shrimp:

Determine the method:

This document is applicable to the determination of chloramphenicol residues in muscles, livers of pigs and chickens, and edible tissues of fish and shrimps:

2 Normative reference documents

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

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

For residual chloramphenicol in the sample, meta-chloramphenicol or deuterated chloramphenicol was used as the internal standard, followed by deproteinization with acetonitrile, 4% sodium chloride, and n-hexane:

Degreasing, ethyl acetate extraction, solid phase extraction column purification, nitrogen blow-drying, liquid chromatography-tandem mass spectrometry determination, and internal 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 Acetonitrile (CH3CN): chromatographically pure:

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

5:1:3 n-Hexane (C6H14):

5:1:4 Ethyl acetate (CH3COOC2H5):

5:1:5 Sodium chloride (NaCl):

5:2 Standard products

5: Chloramphenicol (CAP, Chloramphenicol, C11H12Cl2N2O5, CAS number: 56-75-7), content >= 98:0%:

5．

No:: 202480-68-0), content >= 98:0%:

5:3 Solution preparation

5:3:1 4% sodium chloride solution: Take 4g of sodium chloride, dissolve in water and dilute to 100mL, prepare before use:

5:3:2 Water-saturated ethyl acetate solution: Take 400mL of ethyl acetate, put it in a 500mL brown reagent bottle, add 50mL of water, cover, shake and let stand:

Place and take the upper solution when using:

5:4 Preparation of standard solution

5:4:1 Chloramphenicol standard stock solution: Take about 10 mg of chloramphenicol, weigh it accurately, dissolve it with appropriate amount of methanol and sonicate it, and dilute it to 100 mL:

Volumetric flask, make a standard stock solution with a concentration of 100 µg/mL: Store at -18°C, valid for 1 year:

5:4:2 Internal standard stock solution: Take 10 mg of the internal standard, weigh it accurately, dissolve it with an appropriate amount of methanol by ultrasonic and dilute it to a 100 mL volumetric flask to make a concentrated solution:

Internal standard stock solution with a concentration of 100 µg/mL: Store at -18°C and is valid for 1 year:

5:4:3 Chloramphenicol standard working solution: Precisely measure an appropriate amount of chloramphenicol standard stock solution and dilute it with mobile phase to a concentration of 10ng/mL:

50ng/mL standard working solution: Store at 4°C and is valid for 3 months:

5．

Save, valid for 3 months:

5:5 Materials

C18 solid phase extraction column: 500mg/3mL, or equivalent:

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 Vortex mixer:

6: Tissue homogenizer:

6: Low temperature centrifuge:

6:6 Sample concentrator:

6:7 Solid phase extraction device:

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

Take 5g of the sample (accurate to +/-0:02g), add 250µL of internal standard working solution, 5mL of acetonitrile, and 5mL of 4% sodium chloride solution, and vortex

Centrifuge for 2 minutes at 4000 r/min for 10 minutes, take the supernatant, repeat the extraction of the residue once, and combine the supernatants: Add 5 mL of n-hexane and vortex:

Centrifuge for 1 min at:2000 r/min for 10 min: Discard the supernatant and repeat the treatment with n-hexane once: Add 5 mL of water-saturated ethyl acetate solution and vortex:

Rotate for 1 min, centrifuge at:2000 r/min for 10 min, transfer the supernatant liquid to a 15 mL centrifuge tube, repeat the extraction once, combine the extracts, and use nitrogen:

Blow dry, add 3 mL of water-acetonitrile (95:5) to dissolve, and set aside:

8:2 Purification

Take the solid-phase extraction column and pre-wash it with 10 mL of methanol and 10 mL of water in sequence: Pass the backup solution through the column, add water to wash the column 2 times, 3 mL each time, and add mobile phase:

4 mL, elute at 1 mL/min, collect the eluate: Add 4 mL of water-saturated ethyl acetate solution, vortex for 1 min,:2000 r/min

Centrifuge for 5 minutes, take the supernatant liquid, repeat the process once, combine the supernatant solutions, and blow dry with nitrogen: Add 1:0 mL of mobile phase to dissolve, filter, and use for liquid phase chromatography:

Spectral G tandem mass spectrometry:

8:3 Preparation of standard curve

According to Table 1, accurately measure the appropriate amounts of chloramphenicol standard working solution and internal standard working solution, and dilute them with mobile phase to give chloramphenicol concentrations of

Standard solutions of 0:5ng/mL, 1:0ng/mL, 2:0ng/mL, 5:0ng/mL, 10ng/mL, and internal standard concentration of 5:0ng/mL are supplied to the liquid phase:

Chromatography-tandem mass spectrometry: Take the measured characteristic ion mass chromatographic peak area ratio of the external standard and the internal standard peak as the ordinate, and the corresponding standard solution concentration:

GB 31658:2-2021

Degree is the abscissa, draw a standard curve: Find the regression equation and correlation coefficient:

8: Measurement

8:4:1 Liquid chromatography conditions

a) Chromatographic column: C18 (150mmx3:12mm, 5µm);

b) Mobile phase: methanol-water (50:50);

c) Flow rate: 0:4mL/min;

d) Column temperature: 30 degrees C;

e) Injection volume: 20µL;

f) Running time: 10min:

8:4:2 Mass spectrometry conditions

a) Ion source: electrospray ion source;

b) Scanning method: negative ion scanning;

c) Detection method: multiple reaction monitoring;

d) Ionization voltage: 2:8kV;

e) Source temperature: 120 degrees C;

f) Collision gas: Argon gas 3:0x10-3mbar;

g) Desolvent gas speed: 500L/h;

h) Cone air velocity: 20L/h;

i) Data collection window: 10min;

j) Dwell time: 0:6s;

k) The qualitative ion pairs, quantitative ion pairs of the test drug and the corresponding cone voltage and collision energy are shown in Table 2:

8: 4: Determination method

Take the sample solution and the corresponding standard solution, perform single-point or multi-point calibration, and quantify the chromatographic peak area according to the internal standard method: The characteristic ion mass of chloramphenicol

The spectrum is shown in Figure A:1, and the mass chromatograms of each characteristic ion in the standard solution are shown in Figure A:2:

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 Result calculation

The residual amount of the substance 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 chloramphenicol in this method is 0:1 µg/kg, and the quantitation limit is 0:2 µg/kg:

10:2 Accuracy

The recovery rate of this method is 70% to 120% at the added concentration of 0:1µg/kg~10µg/kg:

10:3 Precision

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