GB 31613.4-2022National Food Safety Standard Determination of Perilimycin Residues in Bovine Edible Tissues by Liquid Chromatography-Tandem Mass Spectrometry (English PDF)

5.1 Reagents

5.1.1 Acetonitrile (CH3CN). chromatographically pure.

5.1.2 Methanol (CH3OH). chromatographic alcohol.

5.1.3 Formic acid (HCOOH). chromatographically pure.

5.1.4 Anhydrous sodium sulfate (Na2SO4).

5.1.5 Sodium Chloride (NaCl).

5.2 Solution preparation

5.2.1 5% formic acid in acetonitrile solution. Take 5 mL of formic acid and dilute to 100 mL with acetonitrile.

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

5.2.3 0.1% formic acid solution. Take 1mL of formic acid and dilute it with water to 1000mL.

5.2.4 80% methanol solution. Take 80mL of methanol and dilute with water to 100mL.

5.3 Standards

Pirlimycin (Pirlimycin, C17H31ClN2O5S, CAS number. 79548-73-5), content >=99.0%.

5.4 Preparation of standard solution

5.4.1 Standard stock solution. Accurately weigh about 10 mg of standard pirinomycin, put it in a 10 mL volumetric flask, dissolve it with methanol and dilute to the mark.

Prepare the standard stock solution of pirinomycin with a concentration of 1 mg/mL, store it below -18 degrees C, and have a validity period of 12 months.

5.4.2 Standard intermediate solution. Accurately measure 0.1mL of standard stock solution of pirinomycin, put it in a 10mL volumetric flask, dilute with methanol to the mark, and prepare

A standard intermediate solution of pirimycin with a concentration of 10 µg/mL was prepared, stored at 2°C to 8°C, and valid for 6 months.

GB31613.4-2022

5.4.3 Standard working solution. Accurately measure 0.1mL of the standard intermediate solution of pirinomycin, put it in a 10mL volumetric flask, and dilute it with 80% methanol to the mark.

The standard working solution of pilimycin with a concentration of 100 ng/mL was prepared and stored at 2 degrees C to 8 degrees C, and the validity period was 6 months.

5.5 Materials

5.5.1 Solid-phase extraction column. matrix-enhanced fat removal column (300mg/3mL), or equivalent.

5.5.2 Homogeneous ceramics.

5.5.3 Hydrophilic polytetrafluoroethylene microporous membrane. 0.22 µm.

6.2 Analytical balance. Sensitivity 0.01g and 0.00001g.

6.3 Vortex mixer.

6.4 High speed centrifuge.

7.1 Preparation of samples

Take an appropriate amount of fresh or thawed blank or test sample, 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 2 g of the sample (accurate to +/-0.05 g) into a 50 mL centrifuge tube, add ceramic homogenizers, add 10 mL of 5% formic acid acetonitrile solution,

Vortex for 30 s, add 4 g of anhydrous sodium sulfate and 1 g of sodium chloride, vortex for 30 s, centrifuge at 8000 r/min for 8 min at 4 °C, and quickly pipette the upper layer B.

Add 2.4 mL of nitrile to another centrifuge tube, add 0.6 mL of water, mix well, and set aside.

8.2 Purification

Pass the spare liquid directly through the solid phase extraction column, flow out naturally, collect the filtrate, and squeeze it dry.

Spectrometer-tandem mass spectrometry.

8.3 Preparation of matrix-matched standard curve

Precisely measure an appropriate amount of standard working solution of pyrithycin, and dilute it with the extracted and purified blank sample solution to contain drug concentrations of

1 ng/mL, 2 ng/mL, 5 ng/mL, 10 ng/mL, 100 ng/mL and 400 ng/mL matrix-matched series of standard solutions, from which each

1.0mL, measured on the machine through a microporous membrane. The characteristic ion mass chromatographic peak area is the vertical axis, and the matrix matching standard solution concentration is the horizontal axis,

Draw a standard curve.

8.4 Determination

8.4.1 Reference conditions for liquid chromatography

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

b) Mobile phase. A is 0.1% formic acid solution, B is 0.1% formic acid acetonitrile solution. Mobile phase gradient. 0min~1min hold

10% B; 1min~3min, 10%B changes linearly to 90%B; 3min~4min maintains 90%B; 4min~5min maintains

10%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) Curtain air. 25psi.

i) Collision gas. Medium.

j) Refer to Table 1 for the qualitative and quantitative ion pairs of pirinomycin 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.

8.5 Blank test

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

10.1 Sensitivity

The detection limit of this method for bovine muscle, liver, kidney and fat is 2 µg/kg, and the limit of quantification is 10 µg/kg.

10.2 Accuracy

In this method, the concentration of pilinomycin in bovine muscle is 10 µg/kg~200 µg/kg, liver is 10 µg/kg~2000 µg/kg, kidney is 10 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%.