GB 31656.16-2022National food safety standards - Determination of chloramphenicol, thiamphenicol, florfenicol and florfenicol amine residues in aquatic products - gas chromatography (English PDF)

5.1 Reagents

5.1.1 Acetonitrile (CH3CN). chromatographically pure.

5.1.2 Methanol (CH3OH). chromatographically pure.

5.1.3 n-Hexane (C6H14). chromatographically pure.

5.1.4 Acetic acid (CH3COOH). chromatographically pure.

5.1.5 Ethyl acetate (C4H8O2). chromatographically pure.

5.1.6 Toluene (C7H8). chromatographically pure.

5.1.7 Concentrated ammonia water (NH3.H2O).

5.1.8 N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA). C8H18F3NOSi2.

5.1.9 Trimethylchlorosilane (TMCS). C3H9ClSi.

5.2 Solution preparation

5.2.1 Ethyl acetate-ammonia water. Take 2mL of concentrated ammonia water and dilute it to 100mL with ethyl acetate.

5.2.2 Methanol-ammonia water. take 2mL of concentrated ammonia water and dilute to 100mL with methanol.

5.2.3 5% acetic acid solution. Take 5mL of acetic acid and dilute it with water to 100mL.

5.2.4 Derivatization reagent. take N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and trimethylchlorosilane (TMCS),

Ratio of 99.1 mixed. Now ready to use.

5.3 Standards

Chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine, the content of which was >=98%. See Appendix A for details.

5.4 Preparation of standard solution

5.4.1 Standard stock solution. take 15 mg each of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine standard products, weigh them accurately, add acetonitrile

Appropriate amount to dissolve and dilute to a 100mL brown volumetric flask, and prepare a standard stock solution with a concentration of 150µg/mL. Below -18°C

Stored and valid for 3 months.

5.4.2 Mixed standard working solution. Accurately measure 1 mL of chloramphenicol standard stock solution, thiamphenicol, florfenicol and florfenicol amine respectively.

10mL in a 50mL volumetric flask, dilute to the mark with acetonitrile to make chloramphenicol concentration 3µg/mL, thiamphenicol, florfenicol and florfenicol

The mixed standard working solution with chloramine concentration of 30 µg/mL was stored below -18 degrees C, and the validity period was 1 month.

5.5 Materials

5.5.1 MCX solid-phase extraction column. 60mg/3mL, or equivalent.

5.5.2 Pipette. 20µL~200µL,.200µL~1000µL.

5.5.3 Chicken heart bottle. 100mL.

5.5.4 Polyethylene centrifuge tubes. 15mL, 50mL, with stoppers.

5.5.5 Glass centrifuge tube. 10mL, with stopper.

5.5.6 Brown reaction bottle. specification 2mL.

6.1 Gas chromatograph equipped with 63Ni electron capture detector.

6.2 Homogenizer.

6.3 Rotary evaporator.

6.4 Centrifuge.

6.5 Vortex mixer.

6.6 Analytical balance. Sensitivity 0.00001g and 0.01g.

6.7 Nitrogen blowing instrument.

6.8 Ultrasonic cleaner.

6.9 Solid phase extraction device.

7.1 Preparation of samples

Prepare samples according to the requirements of Appendix B of GB/T 30891-2014.

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

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.

8.1 Extraction

Take 5g of the sample (accurate to +/-0.05g), put it in a 50mL stoppered glass centrifuge tube, add 20mL of ethyl acetate-ammonia water, vortex immediately

1 min, ultrasonication for 15 min, centrifugation at 4000r/min for 5 min, the supernatant was taken into a 100 mL chicken heart bottle, the extraction was repeated once, and the supernatant was combined.

Add 3 mL of 5% acetic acid solution, oscillate to mix, and evaporate at 40 °C to about 1.5 mL, set aside.

8.2 Purification

Take the spare solution in a 10mL centrifuge tube, wash the chicken heart bottle with 1.5mL of 5% acetic acid, and put the washing solution in the centrifuge tube. Add 5mL of n-hexane, vortex

Mix for 1 min, centrifuge at 4000r/min for 5 min, take the water layer, repeat degreasing once with 5 mL of n-hexane, take the water layer, and set aside.

Take the MCX solid-phase extraction column, activate it with 3mL of methanol and 3mL of water successively, take the spare solution degreased by n-hexane, pass it through the column, and wash it with 5% ethyl alcohol.

acid solution 2mL, discard the eluent, elute with methanol-ammonia 6mL, collect the eluate, blow it with nitrogen at 65°C until it is nearly dry, and then wash it with methanol successively.

1 mL of ethyl acetate and 1 mL of ethyl acetate were used to wash the centrifuge tube wall, blown with nitrogen gas until nearly dry, and set aside.

8.3 Derivation

8.3.1 Derivatization of samples

Take the dried extract, add 100 µL of acetonitrile to redissolve, transfer to a 2 mL brown reaction bottle, wash with 100 µL and merge into the brown reaction bottle.

Add 100 µL of derivatization reagent to the reaction bottle, cap and vortex mix for 10 s, and react in an oven at 60 °C for 30 min. Cool to room temperature, 50 °C~

Blow dry with nitrogen at 55°C, add 1.0 mL of toluene accurately, vortex and mix well, and use for gas chromatograph determination.

8.3.2 Derivatization of standard working solution

Take an appropriate amount of mixed standard working solution of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine in a 2 mL brown reaction bottle, and place in nitrogen at 50 °C.

Blow dry. Follow the steps in 8.3.1 below.

8.4 Preparation of standard curve

Accurately measure the appropriate amount of standard working solutions of chloramphenicol, thiamphenicol, florfenicol, and florfenicol amine, and dilute the concentration of chloramphenicol with acetonitrile to

1.0 µg/L, 1.5 µg/L, 5.0 µg/L, 15.0 µg/L, 50.0 µg/L, 150.0 µg/L, thiamphenicol, florfenicol, florfenicol Concentrated amine

A series of standard solutions with concentrations of 10.0 µg/L, 15.0 µg/L, 50.0 µg/L, 150.0 µg/L, 500.0 µg/L and 1500.0 µg/L were obtained by derivatization.

After biochemistry, it is used for gas chromatography determination. With the measured peak area as the ordinate and the corresponding concentration of the standard solution as the abscissa, draw a standard curve. Return

Regression equations and correlation coefficients.

8.5 Determination

8.5.1 Chromatographic conditions

a) Chromatographic column. DB-5MS quartz capillary column (30mx0.25mm, film thickness 0.25µm), or equivalent;

b) Injection method. splitless injection;

c) Carrier gas. nitrogen, purity 99.999%;

d) Column flow rate. 1.0mL/min, makeup is 25mL/min;

e) The inlet temperature is 230°C, and the detector temperature is 310°C;

f) Column temperature program. initially keep at 80°C for 1 min, then raise the temperature to 280°C at 30°C/min, and keep for 5 min;

g) Injection volume. 1.0 µL.

8.5.2 Determination method

Take the sample solution and the corresponding standard solution for single-point or multi-point calibration, and calculate the peak area according to the external standard method. Standard solution and sample solution

The response values of chloramphenicol, thiamphenicol, florfenicol and florfenicol amine should be within the linear range of instrument detection. Under the above chromatographic conditions,

See Appendix B for the gas chromatogram of the standard solution.

8.5.3 Blank test

Take the blank sample, except that no drug is added, the same measurement steps are used for parallel operation.

10.1 Sensitivity

The detection limit of chloramphenicol in this method was 0.1 µg/kg, and the detection limits of thiamphenicol, florfenicol and florfenicol amine were all 1.0 µg/kg.

The limit of quantification of chloramphenicol was 0.3 µg/kg, and that of thiamphenicol, florfenicol and florfenicol amine were all 3.0 µg/kg.

10.2 Accuracy

The recovery rate of chloramphenicol in this method was 70%~120% at the level of 0.3µg/kg~30.0µg/kg; thiamphenicol, florfenicol and

The recoveries of florfenicol amine were 70%-120% at the spiked levels of 3.0 µg/kg-300 µg/kg.

10.3 Precision

In this method, the relative standard deviation within the batch was <=20%, and the relative standard deviation between batches was <=20%.