GB 31613.6-2022National food safety standards Determination of virginiamycin M1 residues in edible tissues of pigs and poultry by liquid chromatography-tandem mass spectrometry (English PDF)

5.1 Reagents

5.1.1 Acetonitrile (CH3CN). chromatographically pure.

5.1.2 Formic acid (HCOOH). chromatographically pure.

5.1.3 Acetonitrile (CH3CN).

5.1.4 n-Hexane (C6H14).

5.2 Solution preparation

5.2.1 Acetonitrile solution. Take 600mL of acetonitrile and 400mL of water, and mix them evenly.

5.2.2 0.1% formic acid solution. take 1mL of formic acid, dilute it with water to 1000mL, and mix well.

5.3 Standards

Virginiamycin M1 (Virginiamycin M1, C28H35N3O7, CAS number. 21411-53-0) content was >=90%.

5.4 Preparation of standard solution

5.4.1 Standard stock solution. Take 10 mg of virginiamycin M1 standard product, weigh it accurately, dissolve it with acetonitrile and dilute to volume.

200mL brown volumetric flask, prepared into a standard stock solution of virginiamycin M1 with a concentration of 50µg/mL. Store below 4°C, and the expiration date is

7d.

5.4.2 Standard intermediate solution. Accurately measure 0.1mL, 0.5mL, 1mL, 2mL and 3mL of standard stock solution, and put them into 50mL brown container

Diluted to the mark with acetonitrile to prepare vitamins with concentrations of 0.1 µg/mL, 0.5 µg/mL, 1 µg/mL, 2 µg/mL and 3 µg/mL.

Geniamycin M1 standard intermediate solution. Store below 4°C, with a validity period of 7 days.

5.5 Materials

5.5.1 Graduated test tube. 10mL.

5.5.2 Organic filter membrane. 0.22 µm.

6.2 Analytical balance. Sensitivity 0.00001g and 0.01g.

6.3 Homogenizer.

6.4 Ultrasound machine.

6.5 Centrifuge. >=4000r/min.

6.6 Vortex shaker.

6.7 Pipettes.

7.1 Preparation of samples

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

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

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 Storage of samples

Store below -40°C and avoid repeated freezing and thawing.

8.1 Extraction

Take 2 g of the sample (accurate to +/-0.05 g), add 4 mL of acetonitrile (add 2 mL of water to the muscle tissue first), vortex for 2 min, and sonicate for 30 min.

Centrifuge at 4000r/min for 10min, take the supernatant and transfer it to a 15mL centrifuge tube with a stopper. Add 2mL of acetonitrile to the residue and repeat the extraction once, and combine

The supernatant was set aside to obtain the extract.

8.2 Purification

Take the above extract, add water to about 9.5mL, vortex and mix for 30s, add n-hexane 3mL, vortex for 30s, centrifuge at 4000r/min for 10min,

Discard the upper layer of n-hexane, and repeat the degreasing once. Transfer the lower layer solution to a 10mL graduated test tube, add water to 10mL, vortex for 30s, and prepare

Use. Take the spare solution (liver. take 2.0mL of the spare solution, add 4.0mL of acetonitrile solution, mix well; kidney, sebum. take 2.0mL of the spare solution, add acetonitrile

solution (6.0 mL, mixed), filtered, and determined by liquid chromatography-tandem mass spectrometry.

8.3 Preparation of standard curve

8.3.1 Preparation of poultry muscle and liver matrix matching standard curve

Accurately measure virginiamycin M1 standards with concentrations of 0.1 µg/mL, 0.5 µg/mL, 1 µg/mL, 2 µg/mL and 3 µg/mL

Add.200 µL of each intermediate solution to the blank tissue extract after degreasing treatment with 7.1 and 7.2 n-hexane, add water to 10 mL, mix well,

Treated in the same way. Preparation of virginiamycin M1 concentration of 2ng/mL, 10ng/mL, 20ng/mL, 40ng/mL and 60ng/mL series

Match the standard solution with the column matrix, filter it, prepare it immediately before use, and use it for liquid chromatography coupled with mass spectrometer for determination. Take the measured characteristic ion peak area as the ordinate, and

The concentration of the corresponding standard solution is the abscissa, and the standard curve is drawn. Find the regression equation and correlation coefficient.

8.3.2 Preparation of standard curves of other tissues

Accurately measure virginiamycin M1 at concentrations of 0.1 µg/mL, 0.5 µg/mL, 1 µg/mL, 2 µg/mL and 3 µg/mL

Add acetonitrile solution to.200 µL of each standard intermediate solution to 10.0 mL, and prepare virginiamycin M1 with a concentration of 2 ng/mL, 10 ng/mL,

20ng/mL, 40ng/mL and 60ng/mL series of standard working solutions, prepared immediately before use, for determination by liquid chromatography coupled with mass spectrometer.

The area of the characteristic ion peak is the ordinate, and the corresponding concentration of the standard solution is the abscissa, and the standard curve is drawn. Find the regression equation and the correlation coefficient.

8.4 Determination

8.4.1 Chromatographic conditions

a) Chromatographic column. C18 (100mmx2.1mm, 1.7µm), or equivalent;

b) Column temperature. 30°C;

c) Injection volume. 5 µL;

d) Flow rate. 0.2mL/min;

e) Mobile phase. A is acetonitrile, B is 0.1% formic acid aqueous solution, and the gradient elution program is shown in Table 1.

8.4.2 Mass Spectrometry Conditions

a) Ion source. electrospray ion source;

b) Scanning mode. positive ion scanning;

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

d) Ion source temperature. 150°C;

e) Desolventization temperature..200°C;

f) Capillary voltage. 3.0kV;

g) Desolvation gas flow rate. 800L/h.

h) Cone back blowing air flow rate. 150L/h.

i) See Table 2 for qualifier ion pairs, quantifier ion pairs, cone voltage and collision energy.

8.5 Determination method

8.5.1 Qualitative determination

Under the same test conditions, the retention time of the chromatogram of virginiamycin M1 in the sample solution was the same as that in the corresponding standard working solution.

The deviation between them should not be greater than +/-2.5%; and the relative abundance of the detected ions should be consistent with the relative abundance of the standard solution with the same concentration.

The permissible deviation should meet the requirements in Table 3.

8.5.2 Quantitative determination

Take the sample solution and the corresponding matrix matching standard working solution/standard working solution for single-point or multi-point calibration, and quantify by peak area according to the external standard method.

Matrix matching standard working solution/response value of virginiamycin M1 in standard working solution and sample solution should be within the linear range of instrument detection

See Appendix A for the characteristic ion mass chromatograms of virginiamycin M1 matrix-matched standard solution under the above chromatography-mass spectrometry conditions.

8.6 Blank test

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

10.1 Sensitivity

The detection limits of virginiamycin M1 in muscle, liver, kidney and sebum were 2 µg/kg, 6 µg/kg, 8 µg/kg and

The quantitative limit of muscle was 10 µg/kg, and the quantitative limit of other tissues was 50 µg/kg.

10.2 Accuracy

In this method, virginiamycin M1 is effective in poultry and pig muscle tissue at 10 µg/kg~200 µg/kg, liver and kidney tissue at 50 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%.