GB 31658.9-2021National food safety standard - Determination of estrogen residues in animal derived foods and animal urine by liquid chromatography-tandem mass spectrometry method (English PDF)

Foreword

This document complies with the provisions of GB/T 1:1-2020 "Standardization Work Guidelines Part 1: Structure and Drafting Rules of Standardization Documents"

Drafting:

1 Scope

This document specifies the estrogenic drugs (estriol, estrone, ethinyl estradiol, ethinyl estradiol, Sample preparation and liquid chromatography-tandem mass spectrometry method for detection of residual amounts of 17a-estradiol, 17beta-estradiol, diethylstilbestrol, hexylbestrol and diethylstilbestrol):

This document applies to estrogenic drugs (estriol, estrone, ethinylestradiol, Determination of residual amounts of 17a-estradiol, 17beta-estradiol, diethylstilbestrol, hexestrol and diethylstilbestrol):

2Normative reference documents

The contents of the following documents constitute essential provisions of this document through normative references in the text: Among them, for dated reference documents, only the version corresponding to the date applies to this document; for undated reference documents, the latest version (including all amendments) applies to this document:

GB/T 6682 Specifications and test methods for water used in analytical laboratories

3Terms and Definitions

There are no terms or definitions to be defined in this document:

4 Principles

The remaining drugs in the sample were extracted with acetonitrile after enzymatic hydrolysis, purified with a solid-phase extraction column, measured by liquid chromatography-tandem mass spectrometry, and quantified by the internal standard method:

5Reagents and materials

Unless otherwise specified, all reagents are of analytical grade and the water is first-grade water that complies with GB/T 6682:

5:1 Reagents

5:1:1 Acetonitrile (CH3CN): chromatographically pure:

5:1:2 Methanol (CH3OH):

5:1:3 Sodium hydroxide (NaOH):

5:1:4 n-Hexane (C6H1):

5:1:5 Dichloromethane (CH2Cl2):

5:1:6 Sodium chloride (NaCD):

5:1:7 Ammonium acetate (CH3COONH4):

5:1:8 Acetic acid (CH3COOH):

5:1:9 beta-glucuronidase/arylsulfatase (beta-glucuronidase/arylsulfatase): beta-glucuronidase 4:5 U/mL, arylsulfatase 14 U/mL

5:2 Preparation of solution

5:2:1 Dichloromethane-methanol solution: Take 70 mL of dichloromethane and 30 mL of methanol, and mix:

5:2:2 Methanol-water solution: Take 40 mL of methanol and 60 mL of water and mix:

5:2:3 Ammonium acetate solution (0:02 mol/L): Take 1:54g of ammonium acetate, add water to dissolve and dilute to about 800mL, adjust the pH to 5:2, dilute to 1000mL, and mix:

5:2:4 40% acetonitrile solution: Take:200 mL of acetonitrile and 300 mL of water, and mix well:

5:3 Standard products

Estriol, estrone, ethinyl estradiol, 17a-estradiol, 17beta-estradiol, diethylstilbestrol, hexestradiol, diethylstilbestrol Estriol-D3, estrone-D2, ethinylestradiol- D:, 17a-estradiol-D2, 17beta-estradiol-D2, diethylstilbestrol-Da, diethylstilbestrol-D and diethylstilbestrol-D6, the contents are all >=95%, see Appendix A:

5:4 Preparation of standard solution

5:4:1 Standard stock solution: Take 10 mg of each estrogen drug standard and isotope-labeled standard, weigh them accurately, add an appropriate amount of acetonitrile to dissolve and dilute to a 100 mL brown volumetric flask, and make a concentration of 100 µg/mL: Standard stock solution: Store at -18 degrees C, valid for 12 months:

5:4:2 Mixed standard stock solutions: Accurately pipette the standard stock solutions of estriol, estrone, ethinyl estradiol, 17a-estradiol, 17beta-estradiol, diethylstilbestrol, hexanebestrol and diethylstilbestrol respectively: 5mL, put in a 100mL brown volumetric flask, dilute to the mark with acetonitrile: Store at -18 degrees C, valid for 12 months:

5:4:3 Mix internal standard stock solution: accurately pipette estriol-D2, estrone-D2, ethinylestradiol-D4, 17a-estradiol-D2, 17beta-estradiol-D2, diethylstilbestrol-Da, Diethylstilbestrol-D, and Diethylstilbestrol-D: Put 5 mL of each standard stock solution into a 100 mL brown volumetric flask and dilute to the mark with acetonitrile: Store at -18 degrees C, valid for 12 months:

5:4:4 Mixed internal standard working solution: Accurately transfer 0:2 mL of mixed internal standard stock solution into a 10 mL brown volumetric flask, and dilute to the mark with acetonitrile: The concentration of each internal standard in this solution is 100 µg/L: Ready for use:

5:4:5 Mixed standard working solution: Accurately pipet an appropriate amount of mixed standard stock solution and mixed internal standard working solution, and dilute it with 40% acetonitrile solution to the concentration:

It is a series of standard working solutions of 1:0µg/L, 2:0µg/L, 5:0µg/L, 20µg/L, 50µg/L and:200µg/L: Each standard working solution contains an internal standard concentration of 5:0µg/L: Ready for use:

5:5 Materials

5:5:1 HLB solid phase extraction column::200 mg/6 mL, or equivalent:

5:5:2 Amino solid-phase extraction column: 500 mg/6mL, or equivalent, activated with 6mL of dichloromethane-methanol solution before use:

5:5:3 Syringe filter (universal filter membrane): made of nylon, with a pore size of 0:22 µm or equivalent performance:

6Instruments and Equipment

6:1 Liquid chromatography tandem mass spectrometer: equipped with automatic injector and electrospray ion source:

6:2 Analytical balance: sensitive to 0:00001g and 0:01g:

6:3 Nitrogen blower:

6:4 Solid phase extraction device:

6:5 Centrifuge tube: polypropylene plastic centrifuge tube, 50 mL:

6:6 Centrifuge: 10000 r/min or above:

6:7 pH meter:

6:8 Vortex mixer:

7 Preparation and preservation of samples

7:1 Preparation of samples

Take an appropriate amount of fresh or thawed air or test tissue, mince it, and homogenize it:

a) Take the homogenized test sample as the test material;

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 degrees C and conduct analysis and testing within 3 months:

8 Measurement steps

8:1 Extraction

8:1:1 Animal tissue, milk, goat milk and eggs

Take 5g of sample (accurate to +/-0:05g), put it into a 50mL centrifuge tube, add exactly 50µL of mixed internal standard working solution, mix for 15 seconds, add 10mL of ammonium acetate solution, and 50µL of beta-glucuronidase/arylsulfatase , vortex for 1 min, cover, and shake for enzymatic hydrolysis at (37+/-1)°C for 12 h: Take it out, cool to room temperature, add 4g of sodium chloride, shake to dissolve, add 20mL of acetonitrile, vortex for 1 minute, centrifuge at 10000 r/min for 5 minutes, take the acetonitrile layer, add 15mL of acetonitrile, and repeat the extraction: Combine the two extracts, add 10 mL of n-hexane, vortex for 1 min, centrifuge at 10000 r/min for 2 min, take the acetonitrile layer, blow to dryness with nitrogen at 40°C, dissolve with 1 mL of methanol, add 9 mL of water, and set aside:

8:1:2 Animal urine

Take 5:00mL of the sample into a 50mL centrifuge tube, adjust the pH to 5:2 with acetic acid, accurately add 50µL of mixed internal standard working solution, mix for 15s, add 10mL of ammonium acetate solution, 50pL of beta-glucuronidase/arylsulfatase, and follow-up The extraction steps are the same as 8:1:1:

8:2 Purification

Take the HLB solid phase extraction column and activate it with 6 mL of dichloromethane-methanol, 6 mL of methanol and 6 mL of water: Take the reserve solution and pass it through the column: Control the flow rate to not exceed 2 mL/min: Elute with 3 mL of methanol-water and 3 mL of water: column, drain it, and then connect the activated amino solid-phase extraction column in series under the HLB solid-phase extraction column, elute with 8 mL of dichloromethane-methanol, collect the eluate, remove the HLB column, and then use dichloromethane-methanol 8mL: Wash the amino column with 2 mL of methanol, combine the eluants, blow dry with nitrogen in a 40°C water bath, dissolve the residue with 1:0 mL of 40% acetonitrile solution, vortex to mix, and filter with a 0:22 µm filter for liquid chromatography-tandem mass spectrometry measurement:

8:3 Preparation of standard curve

Take a series of mixed standard working solutions of 1:0 µg/L, 2:0 µg/L, 5:0 µg/L, 20 µg/L, 50 µg/L and:200 µg/L for liquid chromatography tandem mass spectrometry measurement: Taking the ratio of the peak area of hormone drugs and the corresponding internal standard substance as the ordinate and the concentration of the standard solution as the abscissa, draw a standard curve and find the regression equation and correlation coefficient:

8:4 Determination

8:4:1 Liquid Chromatography Reference Conditions

a) Chromatographic column: C column (100 mmx2:1mm, 1:7µm), or equivalent;

b) Mobile phase: A is water, B is acetonitrile, gradient elution conditions are shown in Table 1;

c) Flow rate: 0:4 mL/min;

d) Column temperature: 40 degrees C;

e) Injection volume: 10µL:

8:4:2 Mass spectrometry reference conditions

a) Ion source: electrospray ion source;

b) Scanning method: negative ion scanning;

c) Detection method: multiple reaction monitoring (MRM);

d) Capillary voltage: 2:5kV;

e) RF lens voltage: 0:5V;

f) Ion source temperature: 150 degrees C;

g) Desolvation gas temperature: 600 degrees C;

h) Cone air flow rate: 50 L/h;

i) Desolvation gas flow rate: 1100 L/h;

j) Multiplier voltage: 650 V;

k) Secondary collision gas: argon; D reference values for retention time, qualitative ion pair, quantitative ion pair, cone voltage and collision energy are shown in Table 2:

8:4:3 Determination method

Inject the mixed standard working solution and the sample solution alternately for single-point or multi-point calibration: The chromatographic peak area is used for quantification according to the internal standard method: For each estrogen, the corresponding isotope internal standard is selected for quantification: The response value of the substance to be measured in the sample solution should be within the linear range measured by the instrument: The deviation between the retention time of the substance to be tested and the retention time of the corresponding internal standard substance in the sample and the ratio of the retention time of the substance to be tested and the retention time of the corresponding internal standard substance in the standard solution is within +/-2:5%, and each group in the sample If the relative ion abundance of the sample is consistent with the relative ion abundance of a standard solution with a similar concentration, and the deviation does not exceed the range specified in Table 3, it can be determined that the corresponding analyte is present in the sample: See Appendix B for the multiple reaction monitoring chromatogram of the standard working solution:

8:5 Blank test

Take the empty sample and perform parallel operations using the same measurement steps except that no standard solution is added:

9Result calculation and presentation

The residual amount of the drug to be tested in the sample is calculated according to the standard curve or formula (1) to formula (3):

10 Sensitivity, accuracy and precision of detection methods

10:1 Sensitivity

The detection limit of this method in pig, cattle, sheep, chicken tissues (muscle, liver, kidney and fat), eggs, milk and goat milk is 0:50µg/kg:

The limit of quantification is 1:0 µg/kg; the detection limit in pig urine and cow urine is 0:50 µg/L, and the limit of quantitation is 1:0 µg/L:

10:2 Accuracy

The recovery rate of this method at the added concentration level of 1 µg/kg (L) to 5 µg/kg (L) is 80% to 130%:

10:3 Precision

The intra-batch relative standard deviation of this method is <=15%, and the inter-batch relative standard deviation is <=20%: