GB 31613.5-2022National food safety standards-Determination of anticoccidial drug residues in chicken edible tissues-liquid chromatography-tandem mass spectrometry (English PDF)

5.1 Reagents

5.1.1 Methanol (CH3OH). chromatographically pure.

5.1.2 Acetonitrile (CH3CN). chromatographically pure.

5.1.3 Acetic acid (CH3COOH). chromatographically pure.

5.1.4 Formic acid (HCOOH). chromatographically pure.

5.1.5 Ethyl acetate (CH3CH2OOCCH3). chromatographically pure.

5.1.6 Trypsin. derived from bovine pancreas, >=10000U/mg.

5.1.7 Sodium carbonate (Na2CO3).

5.2 Solution preparation

5.2.1 10% sodium carbonate solution. Take 20g of sodium carbonate, add appropriate amount of water to dissolve and dilute to.200mL, mix well.

5.2.2 1% acetic acid solution. Take 1 mL of acetic acid, dilute it with water to 100 mL, and mix well.

5.2.3 20% methanol solution. Take 20mL of methanol, dilute to 100mL with water, and mix well.

5.2.4 Eluent. take methanol 100mL, add ethyl acetate.200mL, mix well.

5.2.5 Complex solution. take methanol 50mL, add water 50mL, formic acid 0.1mL, mix well.

5.3 Standards

The content of fushenone, chlorhexidine, salinomycin, monensin, methyl salinomycin, maduromycin ammonium, and lasaloxil was >=95%. See Appendix A for details.

5.4 Preparation of standard solution

5.4.1 Standard stock solution (1 mg/mL). take hemosanone, chlorhexenidine, salinomycin, monensin, methyl salinomycin, madomicin ammonium, lasalol

An appropriate amount of western standard substance (equivalent to 10 mg of each active ingredient) was accurately weighed, and an appropriate amount of methanol was added to dissolve and dilute to a volume of 10 mL.

The standard stock solution with a concentration of 1 mg/mL was prepared and stored in the dark below -18 degrees C, and the validity period was 6 months.

5.4.2 Mixed standard working solution (10µg/mL). Accurately measure 0.1mL of each standard stock solution, put it in a 10mL volumetric flask, dilute with methanol

Diluted to the mark, and prepared into a mixed standard working solution with a concentration of 10 µg/mL. Stored in the dark below -18°C, the validity period is 6 months.

5.4.3 Mixed standard working solution (1µg/mL). Accurately measure 1mL of 10µg/mL mixed standard working solution in a 10mL volumetric flask,

Dilute to the mark with methanol, and prepare a mixed standard working solution with a concentration of 1 µg/mL. Store in the dark below -18°C, and the validity period is 3 months.

5.4.4 Mixed standard working solution (0.1µg/mL). Accurately measure 1mL of mixed standard working solution of 1µg/mL in a 10mL volumetric flask.

Dilute with methanol to the mark, and prepare a mixed standard working solution with a concentration of 0.1 µg/mL. Store in the dark below -18°C, and the validity period is 1 month.

5.5 Materials

5.5.1 Three-bond bonded C18 solid-phase extraction column. 500mg/6mL, or equivalent.

5.5.2 Nylon microporous membrane. 0.22 µm.

6.1 Liquid chromatography-tandem mass spectrometer. with electrospray ion source.

6.2 Analytical balance. Sensitivity 0.01g and 0.00001g.

6.3 High speed refrigerated centrifuge. 10000r/min.

6.4 Tissue homogenizer.

6.5 Vortex mixer.

6.6 Solid phase extraction device.

6.7 Nitrogen blowing instrument.

6.8 water bath shaker.

6.9 pH meter.

7.1 Preparation of samples

Take an appropriate amount of fresh or thawed blank or test tissue, mince 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 Storage of samples

Store below -18°C.

8.1 Extraction

Weigh 1 g of the sample (accurate to +/-0.01 g), add 25 mg of trypsin, 5 mL of water, vortex for 1 min, and adjust the pH with 10% sodium carbonate solution.

to 7.5, 40°C water bath overnight for enzymatic hydrolysis. Take it out and let it cool to room temperature, add 1mL of 10% sodium carbonate solution, vortex for 1min, add 9mL of ethyl acetate, vortex

Rotate for 5 min, centrifuge at 4°C and 10000r/min for 10 min, transfer the supernatant, and repeat the extraction once for the residue.

Dilute the ester to 20.0mL and mix well. Take 2.0mL of the extract and dry it with nitrogen in a water bath at 35°C, add 1mL of acetonitrile and vortex for 1min, add water

10mL, mix well, set aside.

8.2 Purification

The solid-phase extraction column was activated with 5 mL of methanol and 5 mL of 1% acetic acid solution in turn, and all the spare liquid was passed through the column, and the flow rate was controlled to be 2-3 s every 2 s.

1 drop. Rinse with 3 mL of 1% acetic acid solution and 3 mL of 20% methanol solution, and elute with 10 mL of eluent.

Blow dry with nitrogen, vortex 1.0 mL of the complex solution for 1 min, centrifuge at -4 °C and 10000 r/min for 10 min, take the supernatant, filter the membrane, and supply the liquid phase.

Chromatography-tandem mass spectrometry.

8.3 Preparation of matrix-matched standard curve

After 6 blank samples were extracted and purified, an appropriate amount of standard working solution was added, dried in a water bath with nitrogen at 35°C, and 1.0 mL of complex solution was added to vortex.

Spin for 1 min to prepare matrix-matched standard solutions with concentrations of 0.5 µg/L, 1 µg/L, 5 µg/L, 10 µg/L, 25 µg/L and 50 µg/L.

Centrifuge at -4°C and 10,000r/min for 10min, take the supernatant, filter the membrane, and use it for liquid chromatography-tandem mass spectrometry. To measure the characteristic ion peak

The area is the ordinate, and the corresponding standard solution concentration is the abscissa, draw the standard curve, and find the regression equation and correlation coefficient.

8.4 Determination

8.4.1 Reference conditions for liquid chromatography

Chromatographic column. C18 (50mmx2.1mm, 1.7µm), or equivalent;

Mobile phase. A is 0.1% formic acid aqueous solution, B is 0.1% formic acid methanol solution;

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

Flow rate. 0.3mL/min;

Column temperature. 30°C;

Injection volume. 10 µL.

8.4.2 Reference conditions for mass spectrometry

a) Ion source. Electrospray (ESI) ion source;

b) Scanning mode. positive ion scanning;

c) Detection method. multiple reaction monitoring;

d) Ionization voltage. 4.0kV;

e) Ion source temperature. 100°C;

f) Atomization temperature. 350°C;

g) Cone gas flow rate. 30L/h;

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

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

8.4.3 Assay

8.4.3.1 Qualitative determination

Under the same test conditions, the retention time of coccidiostats in the test solution matched the retention time of coccidiostats in the standard working solution.

The deviation is within +/- 2.5%, and the detected relative ion abundance should match the relative ion of the standard solution with a matrix of comparable concentration.

The abundance is consistent. The allowable deviation should meet the requirements of Table 3.

8.4.3.2 Quantitative determination

Take the sample solution and matrix-matched standard working solution for single-point or multi-point calibration, and quantify according to the external standard method. Matrix-matched standard working solution and sample

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

For chromatograms see 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 detection limit of this method was 5 µg/kg, and the quantification limit was 10 µg/kg.

10.2 Accuracy

The recovery rate of this method was 60%-120% at the spiked concentration level of 10 µg/kg-100 µg/kg.