GB 31658.22-2022National food safety standard - Determination of beta-agonists residues in animal derived food by liquid chromatography-tandem mass spectrometric method (English PDF)

0.5 µg/kg.

Compared with GB/T 21313-2007, the main changes of this document are as follows.

a) Modify the text format to the national food safety standard text format;

b) Added testing organizations in the scope (see Chapter 1);

c) Added the category of drugs in the scope (see Chapter 1).

The release status of previous versions of this document and the documents it replaces are as follows.

---GB/T 22968-2008, GB/T 21313-2007.

National Food Safety Standards

Determination of beta-receptor agonist residues in food of animal origin

Liquid chromatography-tandem mass spectrometry

5.1 Reagents

The reagents used in the following are all analytical reagents unless otherwise specified, and the water is the first-class water in accordance with the provisions of GB/T 6682.

5.1.1 Acetonitrile (CH3CN). chromatographically pure.

5.1.2 Methanol (CH3OH). chromatographically pure.

5.1.3 Formic acid (HCOOH). chromatographically pure.

5.1.4 Perchloric acid (HClO4). 70%~72%.

5.1.5 Ammonia (NH3.H2O).

5.1.6 Ethyl acetate (CH3COOC2H5). chromatographically pure.

5.1.7 Tert-butyl methyl ether [CH3OC(CH3)3]. chromatographically pure.

5.1.8 beta-glucuronidase/arylsulfatase (beta-Glucuronidase/arylsulfatase). 30U/60U/mL.

5.2 Solution preparation

5.2.1 0.2mol/L ammonium acetate buffer solution. Take 15.4g of ammonium acetate, dissolve it in 1000mL water, adjust the pH to 5.2.

5.2.2 0.1mol/L perchloric acid solution. Take 8.7mL of perchloric acid and dilute with water to 1000mL.

5.2.3 10mol/L sodium hydroxide solution. Weigh 40g of sodium hydroxide, dissolve it with an appropriate amount of water and cool it down, then dilute to 100mL with water.

5.2.4 2% formic acid solution. Take 2mL of formic acid and dilute with water to 100mL.

5.2.5 5% ammoniated methanol solution. Take 5mL ammonia water and dilute it to 100mL with methanol.

5.2.6 0.1% formic acid in acetonitrile solution. Take 1 mL of formic acid and dilute it to 1000 mL with acetonitrile.

5.2.7 0.1% formic acid solution. Take 1mL of formic acid and dilute it with water to 1000mL.

5.2.8 Methanol-0.1% formic acid solution (10+90, V/V). Take 10mL of methanol and 90mL of 0.1% formic acid solution, mix well.

5.3 Standards

5.3.1 beta-receptor agonists. clenbuterol, ractopamine, salbutamol, cimaterol, zilpaterol, clorprenaline, terbutaline, sibuterol

Tributerol, Mabuterol, Bromobuterol, Bambuterol, Clemprorol, Tobuterol, Ritodrine, Clenbuterol, Mapenterol, Clumpantrol, and Oxymetholone

Diclonbuterol, content >= 98.0%, see Appendix A.

5.3.2 Isotope internal standard. clenbuterol-D9, ractopamine hydrochloride-D6, salbutamol-D3, cimaterol-D7, zilpaterol-D7, chlorprena

Lin-D7, terbutaline-D9, sibuterol-D9, mabuterol hydrochloride-D9, bambuterol hydrochloride-D9, clemprorol-D7, tobuterol hydrochloride-D9

and hydroxymethylclenbuterol-D6, the content of which was >=98.0%. See Appendix A.

5.4 Preparation of standard solution

5.4.1 Mixed standard stock solution. Accurately weigh about 10 mg of clenbuterol and other standard products, dissolve in methanol and dilute to a 10 mL volumetric flask,

Prepare a standard stock solution with a concentration of 1.0 mg/mL, store it below -18°C, and have a validity period of 12 months.

5.4.2 Mixed internal standard stock solution. Accurately weigh about 10 mg of isotope internal standard such as clenbuterol-D9, dissolve it in methanol and dilute to 10 mL capacity.

The internal standard stock solution with a concentration of 1.0 mg/mL was prepared in the vial and stored below -18°C, with a validity period of 12 months.

5.4.3 Mixed standard working solution. Accurately measure 100 µL of 1mg/mL mixed standard stock solution, put it in a 10mL volumetric flask, dilute it with methanol

To the mark, prepare a standard working solution with a concentration of 10 µg/mL, store it below -18°C, and have a validity period of 6 months.

5.4.4 Mixed internal standard working solution. Accurately measure 100 µL of 1 mg/mL mixed internal standard stock solution, put it in a 10 mL volumetric flask, and dilute it with methanol.

To the mark, the internal standard working solution with a concentration of 10 µg/mL was prepared. Stored below -18°C, the validity period was 6 months.

5.5 Materials

5.5.1 Mixed cation exchange solid phase extraction column. 60mg/3mL, or equivalent.

5.5.2 Microporous membrane. 0.22 µm.

6.2 Analytical balance. Sensitivity 0.00001g.

6.3 Balance. Sensitivity 0.01g.

6.4 Constant temperature oscillating water bath shaker.

6.5 Vortex mixer.

6.6 High speed centrifuge.

6.7 Oscillators.

6.8 Nitrogen blowing instrument.

6.9 Solid phase extraction device.

7.1 Sample preparation

Take an appropriate amount of fresh or thawed blank or test sample, and homogenize.

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 Sample storage

Store below -18°C.

8.1 Enzymatic hydrolysis and extraction

Weigh 2 g of the sample (accurate to +/-0.05 g), put it in a 50 mL centrifuge tube, add 6 mL of 0.2 mol/L ammonium acetate buffer solution, beta-glucose

Aldolase/arylsulfatase 40 µL, vortexed, shaken in a water bath at 37°C protected from light for 16h, placed at room temperature, and set aside.

8.2 Extraction, purification and concentration

Take the spare solution, add 100 µL of 100 ng/mL internal standard working solution, vortex and mix well, centrifuge at 8000 r/min for 8 min, take the supernatant, add

0.1 mol/L perchloric acid solution 5mL, vortex and mix well, adjust the pH to 1.0+/-0.2 with perchloric acid, centrifuge at 8000r/min for 8min, and the supernatant

The pH of the solution was adjusted to 10+/-0.5 with 10mol/L NaOH solution. Add 15mL of ethyl acetate, oscillate at a medium speed for 5min, and centrifuge at 5000r/min.

After 5 min, take the upper organic phase. Add 10 mL of tert-butyl methyl ether to the lower aqueous phase, oscillate at a medium speed for 5 min, and centrifuge at 5000 r/min for 5 min.

The upper organic phases were combined, blown dry with nitrogen at 50 °C, dissolved in 5 mL of 2% formic acid solution, and set aside.

Take a mixed-type cation-exchange solid-phase extraction column, activate it with 3 mL each of methanol and 2% formic acid solution in turn, take the spare solution to pass through the column, and use 2% formic acid solution in turn to activate it.

Rinse with 3 mL of formic acid solution and 3 mL of methanol each, drain to dryness, and elute with 3 mL of 5% ammoniated methanol solution; the eluent was blown dry with nitrogen at 50 °C.

Add 0.5 mL of methanol-0.1% formic acid solution (10+90, V/V) to the residue, fully dissolve, pass through a 0.22 µm microporous membrane, and supply the liquid phase

Chromatography-tandem mass spectrometry.

8.3 Preparation of standard curve

Precisely measure an appropriate amount of mixed standard working solution and mixed internal standard working solution, and dilute with methanol-0.1% formic acid solution (10+90, V/V) to obtain the concentration

It is a series of standard working solutions of 1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL, 20ng/mL and 50ng/mL.

5ng/mL for liquid chromatography-tandem mass spectrometry. The peak area of the characteristic ion chromatogram peak of the drug to be tested and the characteristic ion chromatogram of the corresponding internal standard

The peak area ratio of the spectral peaks is the ordinate, and the corresponding standard solution concentration ratio is the abscissa, draw the standard working curve, and find the regression equation and correlation

coefficient.

8.4 Determination

8.4.1 Reference conditions for liquid chromatography

a) Chromatographic column. Pentafluorophenyl column (50mmx3.0mm, 2.6µm), or equivalent.

b) Mobile phase. A phase is 0.1% formic acid solution, B phase is 0.1% formic acid acetonitrile solution. The mobile phase gradient is 0min~0.5min.

Hold 5% B; 0.5min~5min, 5%B linearly change to 60%B, 5min~6.5min, keep 95%B, 6.5min~

8.5min to maintain 5% B.

c) Flow rate. 0.4mL/min.

d) Injection volume. 5 µL.

e) Column temperature. 30°C.

8.4.2 Reference conditions for tandem mass spectrometry

a) Ion source. electrospray ion source;

b) Scanning mode. positive ion scanning;

c) Detection method. multiple reaction ion monitoring (MRM);

d) Electrospray voltage. 5500V;

e) Ion source temperature. 550°C;

f) Auxiliary gas 1.50psi;

g) Auxiliary gas 2.60psi;

h) Air curtain gas. 30psi;

i) Collision gas. Medium.

8.4.3.2 Quantitative determination

Take the sample solution and standard working solution for single-point or multi-point calibration, and quantify according to the internal standard method. A series of standard working solutions and target substances in the sample solution

The response values should be within the linear range of instrument detection. Under the above conditions of liquid chromatography-tandem mass spectrometry, the mass of each characteristic ion in the standard solution

The chromatogram is shown in Figure B in Appendix B.

8.5 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 limit of detection of this method for muscle, liver and kidney of pigs, cattle and sheep is 0.2 µg/kg, and the limit of quantification is 0.5 µg/kg.

10.2 Accuracy

This method is effective for the return of beta-receptor agonists at the added concentration of 0.5 µg/kg~10 µg/kg in muscle, liver and kidney of pigs, cattle and sheep.

The yield is 60%~120%.

10.3 Precision

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