GB 31659.6-2022National food safety standards-Determination of cloprostinol residues in milk by liquid chromatography-tandem mass spectrometry (English PDF)

5.1 Reagents

5.1.1 Acetonitrile (CH3CN). chromatographically pure.

5.1.2 Ammonia (NH3.H2O).

5.1.3 Formic acid (CH2O2).

5.1.4 Anhydrous sodium sulfate (Na2SO4).

5.1.5 Ammonium acetate (CH3COONH4). chromatographically pure.

5.2 Standards

5.2.1 Cloprostenol (C22H29ClO6, CAS number. 40665-92-7), content >= 98%.

5.3 Solution preparation

5.3.11 0.1% formic acid solution. take 1mL of formic acid, dilute it with water to 1000mL, and mix well.

5.3.2 5% ammonia solution. take 50mL of ammonia water, dilute to 1000mL with water, and mix well.

5.3.3 2% formic acid in acetonitrile solution. take 20mL of formic acid, dilute to 1000mL with acetonitrile, and mix well.

5.3.4 0.1mol/L ammonium acetate solution. Weigh 7.70g of ammonium acetate, dissolve in water and dilute to 1000mL, mix well.

5.3.5 5mmol/L ammonium acetate solution. take 50mL of 0.1mol/L ammonium acetate solution, dilute with water to 1000mL, and mix well.

5.3.6 Acetonitrile ammonium acetate solution. Take 30mL of acetonitrile, add 5mmol/L ammonium acetate solution to 100mL, mix well.

5.4 Preparation of standard solution

5.4.1 Standard stock solution. take about 10 mg of cloprostenol sodium reference substance, weigh it accurately, add an appropriate amount of acetonitrile to dissolve and dilute to 10 mL

A volumetric flask was prepared to make a standard stock solution of cloprostinol with a concentration of 1 mg/mL. It was stored below -18 degrees C and the validity period was 3 months.

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

5.5 Materials

5.5.1 Mixed anion exchange solid phase extraction column. 60mg/3mL or its equivalent.

5.5.2 Membrane. 0.22 µm, organic phase.

5.5.3 Centrifuge tube. 10mL, 50mL.

6.2 Analytical balance. Sensitivity 0.01g and 0.00001g.

6.3 Vortex mixer.

6.4 Ultrasound machine.

6.5 Refrigerated centrifuge.

6.6 Nitrogen blowing instrument.

6.7 Solid phase extraction device.

7.1 Preparation of samples

Take an appropriate amount of fresh or thawed blank or test sample, 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 -18°C.

8.1 Extraction

Take 2 g of the test material (accurate to +/-0.05 g) in a 50 mL centrifuge tube, add 5 mL of acetonitrile, 2 g of anhydrous sodium sulfate, vortex and mix, and ultrasonically extract

10 min, centrifuged at 10000 r/min at 4 °C for 10 min, the supernatant was taken into a 10 mL centrifuge tube, the extraction was repeated once, and the supernatant was combined twice.

Blow dry at 40°C with nitrogen, dissolve the residue in 1 mL of acetonitrile, add 3 mL of 0.1% formic acid aqueous solution, vortex and mix well, and set aside.

8.2 Purification

Take a mixed anion-exchange solid-phase extraction column, activate it with 3 mL of acetonitrile, and equilibrate with 3 mL of water. Take the spare liquid to pass through the column, and use 5% ammonia solution to

Rinse with 3mL and drain. Elute with 3mL of 2% formic acid acetonitrile solution, collect the eluent, blow dry with nitrogen at 40°C, and wash the residue with 1mL of acetonitrile acetic acid

Ammonium solution was dissolved, filtered with a microporous membrane, and determined by liquid chromatography-tandem mass spectrometry.

8.3 Preparation of matrix-matched standard curve

Accurately measure 1mL each of the standard working solution of cloprostinol, dissolve the residues of the blank sample after extraction, purification and drying, respectively, and filter through the filter membrane.

filtered, and prepared into a series of bases with concentrations of 5 ng/mL, 10 ng/mL, 20 ng/mL, 50 ng/mL, 100 ng/mL and 500 ng/mL.

Mass matching standard solution for liquid chromatography-tandem mass spectrometry determination, with the characteristic ion mass chromatographic peak area as the ordinate, the corresponding standard solution concentration

As the abscissa, draw a standard curve. Find the regression equation and correlation coefficient.

8.4 Determination

8.4.1 Chromatographic conditions reference conditions

a) Chromatographic column. C18 column with a length of 150 mm, an inner diameter of 2.1 mm, and a particle size of 5 µm, or those with equivalent performance;

b) Mobile phase. A is 5mmol/L ammonium acetate solution; B is acetonitrile;

c) Flow rate. 0.3mL/min;

d) Injection volume. 5 µL;

e) Column temperature. 30°C;

f) Mobile phase gradient and elution program are shown in Table 1.

8.4.2 Reference conditions for mass spectrometry

a) Ion source. electrospray ion source, negative ion mode;

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

c) Sheath gas pressure (GS1). 344.75kPa (50psi);

d) Auxiliary air pressure (GS2). 344.75kPa (50psi);

e) Collision gas pressure (colision gas). 34.475kPa (5psi);

f) curtain gas pressure (curtaingas). 206.85kPa (30psi);

g) Negative ion mode electrospray voltage (IS). -4500V;

h) Atomization temperature. 500°C;

i) The sheath gas, auxiliary gas, and collision gas are all high-purity nitrogen. Parameters such as spray voltage and collision energy should be optimized to optimal sensitivity;

j) The monitored ion parameters are shown in Table 2.

8.4.3 Assay

Take the sample solution and matrix-matched standard solution, set the instrument conditions according to 8.4.1 and 8.4.2, and perform single-point or multi-point calibration. External standard method

Calculation. The characteristic ion mass chromatographic peak area of the target drug in the matrix matching standard solution and the test solution should be within the linear range of the instrument detection.

Within the range. The ratio of the retention time of the substance to be tested in the sample solution to the retention time of the substance to be tested in the matrix matching standard working solution, the deviation is within +/-

2.5%, and the relative abundance of ions in the sample solution is compared with the relative abundance of ions in the matrix-matched standard solution, which meets the requirements of Table 3.

Then it can be judged that the corresponding substance to be tested exists in the sample. See Appendix A for the relevant spectrum.

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 1.5 µg/kg, and the limit of quantification was 5 µg/kg.

10.2 Accuracy

The recovery rate of this method was 80%-120% at the level of spiked concentration of 5 µg/kg-500 µg/kg.

10.3 Precision

The intra-assay relative standard deviation of this method was <=15%, and the inter-assay relative standard deviation was <=15%.