GB 31658.24-2022National food safety standards-Determination of seduromycin residues in animal foods by liquid chromatography-tandem mass spectrometry (English PDF)

5.1 Reagents

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 Dichloromethane (CH2Cl2).

5.2 Solution preparation

5.2.1 50% acetonitrile solution. take 50mL of acetonitrile, dissolve in water and dilute to 100mL, mix well.

5.2.2 80% dichloromethane methanol solution. Take 80mL of dichloromethane, add 20mL of methanol, and mix well.

5.2.3 Mobile phase A. take 100mL of water, add 0.1mL of formic acid, and mix well.

5.2.4 Mobile phase B. Take 100 mL of acetonitrile, add 0.1 mL of formic acid, and mix well.

5.3 Standards

Seduramicin (Semduramicin, molecular formula. C45H75O16, CAS number. 113378-31-7), content >=94.3%.

5.4 Preparation of standard solution

5.4.1 Standard stock solution. Accurately weigh the reference substance equivalent to 10 mg of seduromycin, dissolve it with acetonitrile and dilute it in a 10 mL volumetric flask,

Prepare seduramycin standard stock solution with a concentration of 1 mg/mL, store it below -18 degrees C, and have a validity period of 3 months.

5.4.2 10 µg/mL seduromycin standard working solution. Accurately measure 0.1 mL of the standard stock solution into a 10 mL volumetric flask, add 50% acetonitrile

The aqueous solution was diluted to the mark, and prepared into 10 µg/mL seduromycin standard working solution. Stored at 2°C~8°C, the validity period was 1 month.

5.4.3 1 µg/mL seduamycin standard working solution. accurately measure 1.0 mL of 10 µg/mL seduamycin standard working solution in a volume of 10 mL

Dilute to the mark with 50% acetonitrile aqueous solution to prepare 1 µg/mL seduromycin standard working solution. Store at 2°C~8°C, valid for 1

month.

5.5 Materials

5.5.1 Solid phase extraction column. graphitized carbon black amino solid phase extraction column. 500mg/6mL.

5.5.2 Membrane. organic phase, 0.22 µm.

6.2 Analytical balance. Sensitivity 0.01g and 0.00001g.

6.3 High speed centrifuge.

6.4 Vortex mixer.

6.5 Horizontal oscillator.

6.6 Homogenizer.

6.7 Solid phase extraction device.

6.8 Nitrogen blowing instrument.

7.1 Sample preparation

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

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 -20°C.

8.1 Extraction

Take 2 g of the sample (accurate to +/-0.05 g), add 12 mL of acetonitrile to a 50 mL centrifuge tube, vortex and mix for 1 min, shake for 10 min,

Centrifuge at 5000r/min for 5min (liver at 8000r/min), take the supernatant, add acetonitrile 12mL to repeat the extraction once, combine the two extracts,

spare.

8.2 Purification

The extraction column was activated with 5 mL of dichloromethane and 5 mL of acetonitrile in sequence, and the spare solution was taken and passed through the column at a controlled flow rate of 2 mL/min to 3 mL/min.

Drained for 10 min, eluted with 6 mL of 80% dichloromethane methanol solution, collected the eluate, and dried with nitrogen in a water bath at 40°C. Dissolved with 50% acetonitrile in water

The solution was dissolved and diluted to 2.0 mL, filtered, and determined by ultra-high performance liquid chromatography-tandem mass spectrometry.

8.3 Preparation of matrix-matched standard curve

Precisely measure 20 µL, 40 µL and 100 µL of 1 µg/mL sedumecin standard working solution, 10 µg/mL sedumecin standard working solution

20 µL, 100 µL and.200 µL were added to 6 parts of the blank sample residues that were treated according to the extraction and purification process, dissolved in 50% acetonitrile aqueous solution and

Diluted to 2mL, prepared at concentrations of 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL, 500ng/mL and 1000ng/mL

The matrix matching series standard solutions were centrifuged at 10,000 r/min at 4°C for 10 min, and the supernatant was taken, filtered through a filter membrane, and used for ultra-high performance liquid chromatography-tandem

Mass spectrometry. Take the measured characteristic ion chromatogram peak area as the ordinate, and the matrix matching standard solution concentration as the abscissa, draw the matrix matching standard curve

Line. Find the regression equation and correlation coefficient.

8.4 Determination

8.4.1 Chromatographic reference conditions

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

b) Mobile phase. A is 0.1% formic acid solution, B is 0.1% formic acid acetonitrile solution;

c) Gradient elution. the gradient elution program is shown in Table 1;

d) Flow rate. 0.3mL/min;

e) Column temperature. 30°C;

f) Injection volume. 10 µL.

8.4.2 Reference conditions for mass spectrometry

a) Ion source. electrospray ion source;

b) Scanning mode. positive ion scanning;

c) Detection method. multiple reaction monitoring;

d) Ionization voltage. 3.0kV;

e) Source temperature. 100°C;

f) Atomization temperature. 350°C;

g) Cone gas flow rate. 20L/h;

h) Atomized gas flow rate. 600L/h;

i) Refer to Table 2 for the reference values of test drug qualitative ion pair, quantitative ion pair, cone voltage and collision energy.

8.5 Determination method

Take the sample solution and matrix-matched standard solution for single-point or multi-point calibration, and calculate with the external standard method. Matrix-matched standard solution and sample solution

The characteristic ion mass chromatographic peak areas of seduromycin should be within the linear range of instrument detection. The relative ion abundance in the sample solution and the basic

Compared with the relative ion abundance in the mass-matched standard solution, it meets the requirements of Table 3.The characteristic ion mass chromatogram of the standard solution is shown in Appendix A.

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 limit of this method was 3 µg/kg, and the limit of quantification was 10 µg/kg.

10.2 Accuracy

The recovery rate of this method was 70%-120% at the spiked concentration level of 10 µg/kg-400 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%.