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GB 31658.7-2021: National food safety standard - Determination of 17B-estradiol, estriol, ethinylestradiol and estrone residues in animal products by gas chromatography-mass spectrometry method
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National food safety standard - Determination of 17B-estradiol, estriol, ethinylestradiol and estrone residues in animal products by gas chromatography-mass spectrometry method
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GB 31658.7-2021
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Basic data | Standard ID | GB 31658.7-2021 (GB31658.7-2021) | | Description (Translated English) | National food safety standard - Determination of 17B-estradiol, estriol, ethinylestradiol and estrone residues in animal products by gas chromatography-mass spectrometry method | | Sector / Industry | National Standard | | Classification of Chinese Standard | X04 | | Word Count Estimation | 9,962 | | Issuing agency(ies) | National Health Commission of the People's Republic of China, State Administration for Market Regulation | GB 31658.7-2021: National food safety standard - Determination of 17B-estradiol, estriol, ethinylestradiol and estrone residues in animal products by gas chromatography-mass spectrometry method
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National food safety standards
17β-estradiol, estriol, ethinylestradiol in animal foods
Determination of alcohol and estrone residues by gas chromatography-mass spectrometry
National Standards of People's Republic of China
Released by the National Health Commission of the People's Republic of China
State Administration for Market Regulation
Ministry of Agriculture and Rural Affairs of the People's Republic of China
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
17βG estradiol, estriol, ethinyl estradiol and
Determination of estrone residue gas chromatography-mass spectrometry
1 Scope
This document stipulates the steroid hormone drugs (17β-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 17β-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 17β-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
5:17β-Estradiol (17β-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 17β-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℃ or below
Save, valid for 6 months:
5:4:2 Mix standard working solution (1μg/mL): Precisely measure each standard stock solution of 17β-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℃ 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 17β-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 (30m×0:25mm, 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℃;
b) EM voltage::200V higher than the tuning voltage;
c) Electron energy: 70eV;
d) GC/MS transfer line temperature: 280℃;
e) Quadrupole temperature: 150℃;
f) Selected ion monitoring (SIM): The monitoring ions of 17β-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%:
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