GB 31659.4-2022National Food Safety Standard Determination of Abamectin Drug Residues in Milk and Milk Powder by Liquid Chromatography-Tandem Mass Spectrometry (English PDF)

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 (CH2O2). chromatographically pure.

5.1.4 Triethylamine (C6H15N).

5.1.5 Isooctane (C8H18).

5.2 Solution preparation

5.2.11 0.1% formic acid in acetonitrile solution. Take 1 mL of formic acid and dilute it to 1000 mL with acetonitrile.

5.2.2 0.1% formic acid solution. Take 1mL of formic acid and dilute it with water to 1000mL.

5.2.3 Rinse solution. take 30 mL of acetonitrile, 70 mL of water and 20 µL of triethylamine, and mix them evenly.

5.2.4 50% acetonitrile solution. Take 50mL of acetonitrile and dilute with water to 100mL.

5.3 Standards

Avermectin (Avermectin, C48H72O14.C47H70O14, CAS number. 71751-41-2), content >= 98.0%; ivermectin (Iver-mectin, C48H74O14, CAS number. 70288-86-7), content >=98.0%; doramectin (C50H74O14, CAS number.

117704-25-3), content >=94.3%; acetamidoabamectin (Eprinomectin, C50H75NO14, CAS number. 123997-26-2), content >=98.0%.

5.4 Preparation of standard solution

5.4.1 Standard stock solution of abamectin drugs. Accurately weigh abamectin (calculated as avermectin B1a), ivermectin (calculated as 22,23-dihydro Abamectin B1a), doramectin and acetamidoabamectin (calculated as acetamidoabamectin B1a) each about 10 mg standard, in different In the same 10mL volumetric flask, dissolve it with acetonitrile and adjust the volume to the mark to prepare avermectin B1a, 22,23-dihydro-alpha Vermectin B1a, doramectin and acetamidoabamectin B1a standard stock solution. Stored below -18 °C, valid for 12 months.

5.4.2 Standard working solution of abamectin drugs. Accurately measure 1 mg/mL of abamectin B1a, 22,23-dihydroabamectin B1a, dora 100 µL of standard stock solution of mycocin and acetamidobamectin B1a was diluted to the mark with acetonitrile in different 10mL volumetric flasks, and prepared into Abamectin B1a, 22,23-dihydroabamectin B1a, doramectin and acetamidoabamectin B1a standard lab with a concentration of 10 µg/mL As solution. Store below -18°C, valid for 6 months.

5.5 Materials

5.5.1 C18 solid phase extraction column. 500mg/6mL, or equivalent.

5.5.2 Hydrophilic polytetrafluoroethylene microporous membrane. 0.22 µm.

7.1 Preparation of samples

Take an appropriate amount of fresh or thawed blank or test milk or goat milk and milk powder, and mix well.

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

Milk and goat's milk. refrigerated and protected from light or stored below -18°C. Milk powder. Store in a cool and dry place.

8.1 Extraction

Weigh 2g of milk or goat milk (accurate to ±0.05g) or milk powder 0.5g (accurate to ±0.01g) into a 50mL centrifuge tube, add acetonitrile After vortexing, shake horizontally at 200r/min for 5min, centrifuge at 8000r/min for 8min, transfer the supernatant to another 50mL centrifuge tube, add Water 10mL, triethylamine 25µL, mix well, set aside.

8.2 Purification and concentration

The C18 solid-phase extraction column was activated with 5 mL of acetonitrile and 5 mL of eluent solution in turn, and the spare liquid was passed through the column, rinsed with 5 mL of eluent solution, and pumped.

Then rinse with 5 mL of isooctane and drain; elute with 5 mL of acetonitrile, collect the eluate in a 10 mL test tube, and blow dry with nitrogen at 50 °C.

Add 1.0 mL of 50% acetonitrile solution to the residue to fully dissolve. After passing through a microporous membrane, it is determined by liquid chromatography-tandem mass spectrometer.

8.3 Preparation of matrix-matched standard curve

Precisely measure an appropriate amount of abamectin standard working solution, and dilute it with 50% acetonitrile aqueous solution to form The concentrations of doramectin B1a, doramectin and acetaminobamectin B1a were 0.5 ng/mL, 1 ng/mL, 2 ng/mL, 5 ng/mL, A series of standard working solutions of 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL and 400ng/mL (for milk or goat milk)

0.5ng/mL, 1ng/mL, 2ng/mL, 5ng/mL, 10ng/mL, 20ng/mL and 50ng/mL concentration points, milk powder use 2ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 50ng/mL, 100ng/mL and 400ng/mL concentration points), 1.0mL was taken from each Add the blank sample to the residue after extraction, purification and drying, fully dissolve, and use it as a matrix matching standard solution.

Determination. Draw the standard curve with the characteristic ion mass chromatographic peak area as the ordinate and the matrix matching standard solution concentration as the abscissa.

8.4.1 Reference conditions for liquid chromatography

a) Chromatographic column. C18 (50mm×2.1mm, 1.7µm), or equivalent.

b) Mobile phase. A is 0.1% formic acid solution, B is 0.1% formic acid acetonitrile solution. Mobile phase gradient. 0min~1min to maintain 70% B; 1min~3min, 70% B changes linearly to 100% B; 3min~4min maintains 100%B; 4min~6min maintains 70%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. 500°C;

f) Auxiliary gas 1.50psi;

g) Auxiliary gas 2.50psi;

h) Air curtain gas. 30psi;

i) Collision gas. Medium.

j) See Table 1 for the qualitative and quantitative ion pairs of the drug to be tested and the corresponding declustering voltage and collision energy reference values.

8.4.3 Assay

The retention time of the sample solution is within ±2.5% of the retention time of the matrix-matched standard solution. The relative abundance of ions in the sample solution is related to Compared with the relative abundance of ions in the matrix-matched standard solution, it meets the requirements of Table 2.

Take the matrix-matched standard solution and sample solution for single-point or multi-point calibration, and calculate the peak area according to the external standard method. Matrix-matched standard solution The peak areas of avermectins in the sample solution and the sample solution should be within the linear range of the instrument detection, and when the linear range exceeds the linear range, the appropriate multiple Analyze after dilution. See Appendix A for the characteristic ion mass chromatogram in the matrix-matched standard solution.

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 for milk or goat milk is 0.2 µg/kg, the limit of quantification is 0.5 µg/kg; the detection limit of milk powder is 1 µg/kg, the limit of quantification The limit is 4 µg/kg.

10.2 Accuracy

Abamectin B1a in milk in this method is 0.5 µg/kg~5 µg/kg, 22,23-dihydroabamectin B1a in milk is 0.5 µg/kg~ 20 µg/kg, doramectin at 0.5 µg/kg~30 µg/kg, acetamidobamectin B1a at 0.5 µg/kg~40 µg/kg In terms of levels, avermectins in goat milk were added at a concentration level of 0.5 µg/kg to 5 µg/kg, and abamectin B1a in milk powder was 4 µg/kg~40 µg/kg, 22,23-dihydroabamectin B1a at 4 µg/kg~160 µg/kg, doramectin at 4 µg/kg~240 µg/kg, The recoveries of acetamidoabamectin B1a were 60%-120% at the concentration levels of 4 µg/kg-320 µg/kg.

10.3 Precision

In this method, the relative standard deviation within the batch was <=15%, and the relative standard deviation between batches was <=20%.