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GB 31658.5-2021: (National Food Safety Standard Determination of Florfenicol and Florfenicol Residues in Animal Foods Liquid Chromatography-Tandem Mass Spectrometry)
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(National Food Safety Standard Determination of Florfenicol and Florfenicol Residues in Animal Foods Liquid Chromatography-Tandem Mass Spectrometry)
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GB 31658.5-2021
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Basic data | Standard ID | GB 31658.5-2021 (GB31658.5-2021) | | Description (Translated English) | (National Food Safety Standard Determination of Florfenicol and Florfenicol Residues in Animal Foods Liquid Chromatography-Tandem Mass Spectrometry) | | Sector / Industry | National Standard | | Classification of Chinese Standard | X04 | | Word Count Estimation | 8,858 | | Issuing agency(ies) | National Health Commission of the People's Republic of China, State Administration for Market Regulation | GB 31658.5-2021: (National Food Safety Standard Determination of Florfenicol and Florfenicol Residues in Animal Foods Liquid Chromatography-Tandem Mass Spectrometry)
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National food safety standards
Florfenicol and florfenicolamide residues in animal foods
Quantity determination liquid chromatography-tandem 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
Determination of florfenicol and florfenicolamide residues in animal foods
Liquid chromatography-tandem mass spectrometry
1 Scope
This document specifies sample preparation and liquid chromatography-tandem mass spectrometry for the detection of florfenicol and florfenicolamine residues in animal foods:
method:
This document is applicable to the determination of florfenicol and florfenicol amide residues in muscle, skin + fat, liver and kidney tissue of chicken, pig, cattle and sheep:
Determination:
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 florfenicol and florfenicolamide in the sample were extracted with alkalized ethyl acetate, degreased with n-hexane, purified with a solid phase extraction column, and liquid chromatographed:
Determination by spectrum-G tandem mass spectrometry and quantification by internal 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 Formic acid (HCOOH): chromatographically pure:
5:1:4 Ethyl acetate (CH3COOC2H5):
5:1:5 Ammonia (NH3:H2O):
5:1:6 Acetic acid (CH3COOH):
5:2 Solution preparation
5:2:1 5% acetic acid aqueous solution: Take 5mL of acetic acid, dissolve it in water and dilute it to 100mL:
5: 2: 10% ammonia-methanol solution: Take 5 mL of ammonia water, dissolve it in methanol and dilute it to 50 mL: Prepare it now:
5:2:3 30% acetonitrile aqueous solution: Take 30mL of acetonitrile, dissolve it in water and dilute it to 100mL:
5:2:4 Ethyl acetate-ammonia solution: Take 98mL of ethyl acetate, add 2mL of ammonia, and mix:
5:3 Standard products
5:3:1 Florfenicol (Florfenicol, C12H14Cl2FNO4S, CAS number: 73231-34-2): content ≥99:0%:
5: Florfenicolamine (C10H14FNO3S, CAS number: 76639-93-5): Content ≥ 97:6%:
GB 31658:5-2021
5: Deuterated chloramphenicol (Chloramaphenicol D5, d5-CAP, CAS number: 202480-68-0): content is 100ng/mL:
5:4 Preparation of standard solution
5:4:1 Florfenicol and florfenicol amine standard stock solutions: Take 25 mg of florfenicol and florfenicol amine standard solutions, weigh them accurately, and add acetonitrile appropriately:
Dissolve and dilute to a 25mL volumetric flask to prepare standard stock solutions of florfenicol and florfenicolamine respectively: Store below -18°C:
Valid for 6 months:
5:4:2 10 μg/mL mixed standard working solution: Precisely measure 1 mL each of florfenicol and florfenicol amine standard stock solutions, and place in 100 mL
Dilute the volumetric flask with acetonitrile to the mark to make a mixed standard working solution with a concentration of 10 μg/mL: Store it at 2°C ~ 8°C and is valid for one month:
5:4:3 1μg/mL mixed standard working solution: Precisely measure 1mL of 10μg/mL mixed standard working solution, put it in a 10mL volumetric flask, and add acetonitrile to it:
Dilute to the mark to make a mixed standard working solution with a concentration of 1 μg/mL: Store at 2°C ~ 8°C and is valid for 1 month:
5:5 Materials
MCX solid phase extraction column 60mg/3mL, or equivalent:
6 Instruments and equipment
6:1 Ultra-high performance liquid chromatography-tandem mass spectrometer: equipped with electrospray ion source:
6:2 Analytical balance: sensitivity 0:00001g and 0:01g:
6: High speed centrifuge:
6: Vortex mixer:
6:5 horizontal oscillator:
6: Homogenizer:
6:7 Solid phase extraction device:
6: 8 Nitrogen blowing instrument:
6:9 Filter membrane: organic phase, 0:22μm:
6:10 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 a homogeneous test sample as a test material;
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 Preservation of samples
Store below -18℃:
8 Measurement steps
8:1 Extraction
Weigh 2g of the sample (accurate to ±0:02g), put it into a 50mL centrifuge tube, add 100μL of deuterated chloramphenicol internal standard solution, and vortex to mix:
Mix well and let stand for 15 minutes: Add 10 mL of ethyl acetate-ammonia solution, vortex for 1 minute, centrifuge at 3000 r/min for 5 minutes, and take the supernatant:
In a 100 mL chicken heart bottle, the residue was repeatedly extracted twice, and the three extracts were combined: Add 2 mL of 5% acetic acid to the chicken heart bottle, shake and mix, and
Concentrate to 1:5 mL in a 40°C water bath: Transfer to another 50 mL centrifuge tube, wash the chicken heart bottle with 2 mL of 5% acetic acid, and transfer the washing solution to
In the same centrifuge tube, add 5 mL of n-hexane for defatting, vortex for 1 min, and centrifuge at 3000 r/min for 5 min: Discard the upper layer and repeat the degreasing of the lower layer extract:
Once, spare:
8:2 Purification
The MCX solid-phase extraction column was activated with 2 mL of methanol and 2 mL of water in sequence: Pass the backup solution through the column, elute with 2 mL of 5% acetic acid, and rinse with 10% ammonia:
Elute with 5 mL of methanol: Collect the eluate and blow dry with nitrogen at 40°C: Dissolve the residue with 500 μL of 30% acetonitrile aqueous solution, filter with a membrane, and provide
Ultra-high performance liquid chromatography-tandem mass spectrometry:
8:3 Preparation of matrix matching standard curve
Precisely measure 5 μL, 25 μL and 50 μL of the 1 μg/mL mixed standard working solution, and 10 μL, 25 μL of the 10 μg/mL mixed standard working solution:
and 50 μL, and sequentially added to 6 portions of the extracted and purified blank sample concentrate, and at the same time, 100 ng/mL deuterated chloramphenicol was added:
Take 100 μL of the standard solution, add 30% acetonitrile aqueous solution to dissolve and dilute to 0:5 mL, and prepare the concentrations to 10 ng/mL, 50 ng/mL,
Matrix matching series standard solutions of 100ng/mL,:200ng/mL, 500ng/mL, and 1000ng/mL were filtered and used for liquid chromatography-tandem mass spectrometry:
Spectrum determination: Using the measured characteristic ion mass chromatographic peak area ratio as the ordinate and the corresponding standard solution concentration as the abscissa, draw the matrix matching standard:
Quasi-curve: Find regression equation and correlation coefficient:
8: Measurement
8:4:1 Chromatographic conditions
a) Chromatographic column: C18 (100mm×2:1mm, 1:7μm), or equivalent;
b) Mobile phase: A: aqueous solution, B: acetonitrile solution;
c) Gradient elution: see Table 1 for gradient elution conditions;
d) Flow rate: 0:25mL/min;
e) Column temperature: 30℃;
f) Injection volume: 10μL:
8:4:2 Mass spectrometry conditions
a) Ion source: electrospray ion source;
b) Scanning method: positive ion scanning (florfenicol amide) and negative ion scanning (florfenicol and deuterated chloramphenicol);
c) Detection method: multiple reaction monitoring;
d) Ionization voltage: 2:8kV;
e) Source temperature: 80℃;
f) Atomization temperature: 300℃;
g) Cone air flow rate: 30L/h;
h) Atomizing gas flow rate: 600L/h;
i) The qualitative ion pairs, quantitative ion pairs and corresponding cone voltages and collision energies of the test drugs are shown in Table 2:
8: 4: Determination method
Take the sample solution and matrix-matching standard solution, perform single-point or multi-point calibration, and quantify the sample solution and matrix by chromatographic peak area according to the internal standard method:
The characteristic ion mass chromatographic peak areas of florfenicol and florfenicolamide in the matching standard solution should be within the linear range of instrument detection:
Compared with the relative ion abundance in the matrix-matched standard solution, the relative ion abundance in the sample solution meets the requirements of Table 3: The standard solution characteristics
See Appendix B for the characteristic ion mass chromatogram:
10 Sensitivity, accuracy and precision of detection methods
10:1 Sensitivity
The detection limit of this method is 3 μg/kg, and the quantification limit is 10 μg/kg:
10:2 Accuracy
The recovery rate of this method is 70% to 120% at the added concentration level of 10μg/kg~300μg/kg:
10:3 Precision
The intra-batch relative standard deviation of this method is ≤20%, and the inter-batch relative standard deviation is ≤20%:
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