GB 31658.7-2021National food safety standard - Determination of 17B-estradiol, estriol, ethinylestradiol and estrone residues in animal products by gas chromatography-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:

This document is published for the first time:

National food safety standards

17betaG estradiol, estriol, ethinyl estradiol and

Determination of estrone residue gas chromatography-mass spectrometry

1 Scope

This document stipulates the steroid hormone drugs (17beta-estradiol, estriol, ethinyl estradiol, ethinyl estradiol, etc:)

Sample preparation and gas chromatography-mass spectrometry method for detection of alcohol, estrone) residues:

This document is applicable to the determination of 17beta-estradiol, estriol, ethinyl estradiol, and estrone residues in beef, sheep, pig, and chicken muscles and pig liver, kidney, and fat:

Detection:

2 Normative reference documents

The contents of the following documents constitute essential provisions of this document through normative citations in the text: Among them, the cited documents with dates are:

Only the version corresponding to the date applies to this document; for undated referenced documents, the latest version (including all amendments) applies to this document:

document:

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

3 Terms and definitions

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

4 Principles

The remaining 17beta-estradiol, estriol, ethinyl estradiol and estrone in the sample were determined by gas chromatography-mass spectrometry after enzymatic hydrolysis, extraction, purification and derivatization:

Quantification by external standard method:

5 Reagents 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 Acetonitrile (CH3CN): chromatographically pure:

5:1:2 Methanol (CH3OH): chromatographically pure:

5:1:3 Ethyl acetate (CH3COOC2H5):

5:1:4 n-Hexane (C6H14):

5:1:5 Toluene (C6H5CH3):

5:1:6 Sodium hydroxide (NaOH):

5:1:7 Sodium acetate (NaC2H3O2:3H2O):

5:1:8 Ammonium acetate (NH4C2H3O2):

5:1:9 Glacial acetic acid (C2H4O2):

5:1:10 Dithioerythritol (C4H10O2S2, DTE):

5:1 Trimethyliodosilane (C3H9ISi, TMIS):

5:1:12 Glucuronidase/aryl sulfatase:

5:1:13 N-methyltrimethylsilyltrifluoroacetamide (MSTFA):

5:2 Standard products

5:17beta-Estradiol (17beta-Estradiol, C18H24O2, CAS number: 50-28-2): content >=98:0%:

5: 2: Estriol (Estriol, C18H24O3, CAS number: 50-27-1): content >= 98:0%:

5: Ethinylestradiol (C20H24O2, CAS No:: 57-63-6): Content >= 98:0%:

5: Estrone (C18H22O2, CAS number::200-164-5): content >= 98:0%:

5:3 Solution preparation

5:3:1 Sodium hydroxide solution (1:0mol/L): Take 40g of sodium hydroxide, dissolve it in water and dilute it to 1000mL:

5:3:2 Sodium acetate buffer (pH 5:2): Take 19:0g of sodium acetate, dissolve it in water and dilute it to 500mL, and adjust the pH to 5:2 with acetic acid:

5:3 Ammonium acetate solution (2:0mol/L): Take 46:25g of ammonium acetate, dissolve it in water and dilute it to 300mL:

5:3:4 Methanol aqueous solution: Take 400mL of methanol and add water to 1000mL:

5:3:5 Ethyl acetate n-hexane solution: Take 10mL of ethyl acetate, add n-hexane to dissolve and dilute to 100mL:

5:3:6 Derivatization reagent: Weigh 0:01g of dithioerythritol, dissolve it with 5mL of N-methyltrimethylsilyl trifluoroacetamide, and add it under the liquid surface:

Add 10 µL of trimethylsilane iodide, mix well, and place it overnight at 2°C to 8°C: Store it sealed and protected from light and moisture: The derivatization reagent should be colorless: If browning occurs,

Color changes such as red indicate that the reagent has failed:

5:4 Preparation of standard solution

5:4:1 Standard stock solution (100 µg/mL): Take about 10 mg each of 17beta-estradiol, estriol, ethinyl estradiol and estrone standards, weigh them accurately, and divide

Add an appropriate amount of methanol to dissolve and dilute to a 100mL volumetric flask to prepare a standard stock solution with a concentration of 100µg/mL: -18 degrees C or below

Save, valid for 6 months:

5:4:2 Mix standard working solution (1µg/mL): Precisely measure each standard stock solution of 17beta-estradiol, estriol, ethinyl estradiol and estrone:

Put 1 mL in a 100 mL volumetric flask, dilute to the mark with methanol, and prepare a mixed standard working solution with a concentration of 1 µg/mL: Keep at 2°C to 8°C:

Deposit, valid for 1 month:

5:5 Materials

5:5:1 C18 solid phase extraction column: 500mg/3mL, or equivalent:

5:5:2 Silica gel solid-phase extraction column: 500mg/3mL, or equivalent:

6 Instruments and equipment

6:1 Gas chromatograph-mass spectrometer: equipped with EI source:

6: Analytical balance: sensitivity 0:00001g and 0:001g:

6:3 Nitrogen blowing instrument:

6:4 Solid phase extraction device:

6: Pipette: range:200µL~1000µL:

6: Pipette: range 20µL~100µL:

6: Homogenizer:

6: Vortex mixer:

6: Centrifuge: 10000r/min:

6:10 oven:

6:11 pH meter:

6:12 Rotary evaporator:

6:13 Chicken heart bottle: 100mL:

7: Preparation and preservation of samples

7:1 Preparation of samples

Take an appropriate amount of fresh or thawed blank 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 Preservation of samples

Store below -18 degrees C and conduct analysis and testing within 3 months:

8 Measurement steps

8:1 Extraction

Take 5g of the sample (accurate to +/-0:02g), put it in a 50mL centrifuge tube, add 10mL of sodium acetate buffer, homogenize for 1 minute, and vortex

For 2 minutes, add 20 µL of glucuronidase/aryl sulfatase, enzymatically hydrolyze at 50°C for 2 hours, add 20 mL of ethyl acetate, and vortex for 3 minutes:

Centrifuge at 10000r/min for 5min, collect the supernatant in a 100mL chicken heart bottle, add 20mL of ethyl acetate to the residue, repeat the extraction once, and combine

The supernatant was rotary evaporated in a 40°C water bath until it was almost dry: Dissolve and wash the chicken heart bottle with 6 mL of sodium hydroxide solution three times: The washing liquid was transferred to a 50 mL centrifuge:

tube, add 20 mL of n-hexane, vortex for 1 min, centrifuge at 10000 r/min for 5 min, collect the lower supernatant, add 1 mL of ammonium acetate solution, and mix with

Adjust the pH to 5:0~5:2 with glacial acetic acid and set aside:

8:2 Purification

Take a C18 solid phase extraction column and activate it with 5 mL each of methanol and water: Take the reserve solution, pass it through the column, and elute with 5 mL each of water and methanol aqueous solution:

Wash, drain, add 5 mL of methanol, elute, collect the eluate, blow dry with nitrogen in a 50°C water bath: Add 5 mL of ethyl acetate n-hexane solution to dissolve, and prepare

Use: Take a silica gel solid-phase extraction column, add 5 mL of n-hexane to activate it, take the backup solution and pass it through the column, rinse, drain, add 5 mL of acetonitrile, elute, and collect the eluate:

Blow dry with nitrogen in a water bath at 50°C:

8:3 Derivatives

Add 100 µL each of toluene and derivatization reagent to the glass test tube blown dry by nitrogen, shake and mix, seal, and derivatize in an oven at 80°C:

60min, cool down, and prepare for GC-MS measurement:

8: 4 Preparation of standard working curve

Precisely measure an appropriate amount of the mixed standard working solution and dilute it with methanol to concentrations of 10 µg/L, 50 µg/L, 100 µg/L,:200 µg/L, and

For the 500 µg/L and 1000 µg/L series standard solutions, accurately measure 500 µL of each, blow dry with nitrogen in a 50°C water bath, perform derivatization according to step 7:3, and cool

After cooling, 300 µL of toluene was added for GC-MS measurement: Taking the concentrations of 17beta-estradiol, estriol, ethinyl estradiol and estrone as the abscissa, the corresponding

The quantitative ion peak area is the ordinate, draw a standard curve or find a linear regression equation:

8:5 Measurement

8:5:1 Chromatographic reference conditions

a) Chromatography column: MS quartz capillary chromatography column (30mx0:25mm, 0:25µm), or equivalent;

b) The carrier gas is high purity He, constant flow 1:0mL/min;

c) The injection port temperature is 220°C;

d) Splitless injection;

e) Injection volume: 1µL;

f) The initial temperature of the column is 100°C, maintained for 1 min; the temperature rise rate is 20°C/min to:200°C, maintained for 3 minutes; 20°C/min

Raise the temperature to 260°C at a heating rate of 20°C/min and hold for 5 minutes; then increase the temperature to 280°C at a heating rate of 20°C/min and hold for 3 minutes:

8:5:2 Mass Spectrometry Reference Conditions

a) Ion source (EI) temperature: 230 degrees C;

b) EM voltage::200V higher than the tuning voltage;

c) Electron energy: 70eV;

d) GC/MS transfer line temperature: 280 degrees C;

e) Quadrupole temperature: 150 degrees C;

f) Selected ion monitoring (SIM): The monitoring ions of 17beta-estradiol, estriol, ethinyl estradiol and estrone derivatives are shown in Table 1:

8:5:3 Measurement method

Take the sample solution and standard solution, conduct single-point or multi-point calibration, quantify by chromatographic peak area, and calculate by external standard method: Standard solution and sample solution

The characteristic ion mass chromatographic peak area of the drug to be tested should be within the linear range of instrument detection: The relative ion abundance in the sample solution is related to

Compared with the relative ion abundance in the standard solution, it meets the requirements of Table 2: The mass spectrum of the standard solution is shown in Appendix A:

8:6 Blank test

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

9 Calculation and presentation of results

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

10 Sensitivity, accuracy and precision of detection methods

10:1 Sensitivity

The detection limit of this method is 0:5 µg/kg, and the quantitation limit is 1:0 µg/kg:

10:2 Accuracy

The recovery rate of this method at the added concentration level of 1:0µg/kg~10:0µg/kg is 60%~110%:

10:3 Precision

The intra-batch variation coefficient CV of this method is <= 15%, and the inter-batch variation coefficient CV is <= 20%: