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National food safety standard - Determination of ceftiofur residues in animal tissues by high performance liquid chromatography method
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Basic data
| Standard ID | GB 31658.1-2021 (GB31658.1-2021) |
| Description (Translated English) | National food safety standard - Determination of ceftiofur residues in animal tissues by high performance liquid chromatography method |
| Sector / Industry | National Standard |
| Classification of Chinese Standard | X04 |
| Word Count Estimation | 12,187 |
| Issuing agency(ies) | National Health Commission of the People's Republic of China, State Administration for Market Regulation |
GB 31658.1-2021: National food safety standard - Determination of ceftiofur residues in animal tissues by high performance liquid chromatography method
---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.
National food safety standards
Determination of ceftiofur residues in animal foods
HPLC
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:
National food safety standards
Determination of ceftiofur residues in animal foods by high performance liquid chromatography
Method 1 Determination of ceftiofur residues in edible animal tissues
HPLC
1 Scope
This document specifies the sample preparation and HPLC determination methods for the detection of ceftiofur residues in edible animal tissues:
This document is applicable to the determination of ceftiofur residues in muscle, fat, liver and kidney of pigs and cattle:
2 Normative application documents
The contents of the following documents constitute essential provisions of this document through normative citations in the text: Among them, the dated citations
For undated referenced documents, only the version corresponding to that date applies to this document; for undated referenced documents, the latest version (including all amendments) applies to
this 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 residual ceftiofur in the sample is related to ceftiofur and desfuranoylceftiofur (DFC) through the action of dithioerythritol (DTE):
Metabolites are separated from proteins or sulfur-containing compounds and react with iodoacetamide to generate stable acetamide derivatives (DCA), which are extracted, purified and purified:
chemical, high performance liquid chromatography, and external standard method for quantitation:
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):
5:1:3 Dithioerythritol (C4H10O2S2, DTE):
5:1:4 Iodoacetamide (C2H4INO):
5:1:5 Anhydrous calcium chloride (CaCl2):
5:1:6 Glacial acetic acid (CH3COOH):
5:1:7 Phosphoric acid (H3PO4): 85%:
5:1:8 Potassium chloride (KCl):
5:1:9 Potassium hydroxide (NaOH):
5:1:10 Potassium dihydrogen phosphate (KH2PO4):
5:1:1 Sodium tetraborate (Na2B4O7:10H2O):
5:1:12 Sodium chloride (NaCl):
5:1:13 Sodium hydroxide (NaOH):
5:1:14 Trifluoroacetic acid (CF3COOH, TFA):
5:2 Standard products
or equivalent:
5:3 Solution preparation
5:3:1 Boric acid buffer (0:05mol/L): Take 19g sodium tetraborate and 3:7g potassium chloride, add water to dissolve and dilute to 1000mL:
5:3:2 Phosphate buffer (0:025mol/L): Take 3:04g of potassium dihydrogen phosphate, add water to dissolve and dilute to 1000mL, and oxidize with 45% hydrogen phosphate:
Adjust the pH of the potassium solution to 7:0:
5:3 Extraction solution (0:4% DTE): Take 1g of DTE, add an appropriate amount of boric acid buffer to dissolve it and dilute it to 250mL: Prepare it now:
5:3:4 Iodoacetamide solution (14%): Dissolve 7g of iodoacetamide in 50mL of phosphate buffer and prepare it immediately:
5:3:5 Sodium hydroxide solution (0:01mol/L): Take 0:4g of sodium hydroxide, add water to dissolve and dilute to 1000mL:
5:3:6 Sodium chloride solution (0:1mol/L): Take 5:9g of sodium chloride, dissolve it in water and dilute it to 1000mL:
5:3:7 Calcium chloride solution (0:1mol/L): Take 11:1g of anhydrous calcium chloride, dissolve it in water and dilute it to 1000mL:
5:3:8 Phosphoric acid solution (25%): Take 25mL of phosphoric acid, add water to dissolve and dilute to 100mL:
5:3:9 Acetic acid solution (5%): Take 5mL of glacial acetic acid, add water to dissolve and dilute to 100mL:
5:3:10 C18 eluent: acetonitrile-water (15:85)
5:3:11 SAX pre-wash solution: methanol-sodium chloride solution (25:75):
5:3:12 SAX eluent: acetonitrile-acetic acid solution (5:95):
5:3:13 SCX pre-wash solution: methanol-calcium chloride solution (25:75):
5:3:14 SCX eluent: acetonitrile-sodium chloride solution (5:95):
5:3:15 Mobile phase:
A (0:1% TFA aqueous solution): Take 1mL of trifluoroacetic acid and add water to make it 1000mL:
B (0:1% TFA acetonitrile solution): Take 1mL of trifluoroacetic acid and add acetonitrile to 1000mL:
5:4 Preparation of standard solution
5:4:1 Standard stock solution (100μg/mL): Take about 11mg of ceftiofur hydrochloride standard, weigh it accurately, add methanol to dissolve and dilute to volume
into a 100mL volumetric flask, shake well, and you have it: Store at -18°C and is valid for 6 months:
5:4:2 Standard working solution (10 μg/mL): Precisely measure 5 mL of the standard stock solution into a 50 mL volumetric flask, and add phosphate buffer solution to the mark:
Shake well and get it: Store at 2℃~8℃, valid for 1 month:
5:5 Materials
5:5:1 C18 solid phase extraction column: 1g/6mL, or equivalent:
5:5:2 SAX solid phase extraction column: 500mg/10mL, or equivalent:
5:5:3 SCX solid phase extraction column: 100mg/10mL, or equivalent:
6 Instruments and equipment
6:1 High performance liquid chromatograph: equipped with UV detector:
6:2 Analytical balance: sensitivity 0:00001g and 0:01g:
6:3 Constant temperature water bath oscillator:
6: Homogenizer:
6: Vortex mixer:
6: Refrigerated centrifuge
6:7 Solid phase extraction device:
6:8 pH meter:
6: Centrifuge tube: 50mL:
7 Preparation and preservation of specimens
7:1 Preparation of specimens
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 specimens
Store below -18℃:
8 Measurement steps
8:1 Extraction
Take 2g of the sample (accurate to ±0:02g), add 30:0mL of extraction solution, shake at medium speed for 5min, and centrifuge at 4000r/min for 5min: Take
Put 15:0mL into another 50mL centrifuge tube, shake at 20r/min~30r/min speed and 50℃ constant temperature water bath for 15min, set aside:
8:2 Derivatives
Add 3 mL of iodoacetamide solution to the above reserve solution, mix well, and place at room temperature for 30 min: Centrifuge at 4°C and 10,000 r/min for 20 min: Take the
Supernatant, set aside:
8:3 Purification
8:3:1 Take a C18 solid-phase extraction column and prewash the column with 4 mL of methanol and 5 mL of phosphate buffer in sequence: Pass the derivatized stock solution in 8:2 through the column:
Wash the column with 5 mL of phosphate buffer and 3 mL of sodium hydroxide solution in sequence, squeeze dry: Add 3 mL of C18 eluent, elute, and collect the eluent: Add
Dilute 15 mL of water to a total volume of 18 mL, mix well, and set aside:
8:3:2 Take the SAX solid-phase extraction column and prewash the column with 2 mL each of methanol, SAX pre-wash solution and water: Pass 8:3:1 reserve solution through the column and add water:
Wash the column with 1 mL and squeeze it dry: Add 3 mL of SAX eluent, elute, and collect the eluate: Add 10 mL of water to make the total volume 13 mL, mix well, and set aside:
Note: After the adipose tissue diluted extract is purified by the SAX solid-phase extraction column, wash it with 1 mL of water and squeeze it dry: Add 2:0 mL of the SAX solid-phase extraction column eluent and collect the washed solution:
Deliquidate, mix, and prepare for high performance liquid chromatography analysis:
8:3:3 Take the SCX solid phase extraction column and prewash the column with 1mL of methanol, 2mL of SCX pre-wash solution and 2mL of water: Take 8:3:2 reserve solution:
Pass through the column, rinse with 1 mL of water, squeeze dry or vacuum dry: Add 2:0 mL of SCX eluent, elute, squeeze dry, and collect the eluent for high-performance liquid chromatography:
Spectrum measurement:
8:4 Preparation of standard curve
Accurately measure an appropriate amount of the standard stock solution and dilute it with phosphate buffer to make the concentrations 0:1 μg/mL, 0:2 μg/mL, 0:4 μg/mL,
For solutions of 2:0 μg/mL, 10:0 μg/mL and 20:0 μg/mL, accurately measure 0:5 mL of each solution, and follow the same derivatization and purification steps to prepare the solutions:
The standard ceftiofur concentrations are 25ng/mL, 50ng/mL, 100ng/mL, 500ng/mL, 2500ng/mL and 5000ng/mL:
Solution for high performance liquid chromatography determination: With the peak area as the ordinate and the corresponding standard solution concentration as the abscissa, draw a standard curve: Find the regression
Equations and correlation coefficients:
Note: Preparation of the standard curve for adipose tissue detection: After purification with the SAX solid-phase extraction column, add 1 mL of water to wash the column, squeeze it dry: Add SAX eluent
2:0 mL, collect the eluate, mix well, and use for high performance liquid chromatography measurement:
8:5 Measurement
8:5:1 Chromatographic conditions
a) Chromatographic column: C18 column (250mm×4:6mm, 5μm), or equivalent;
b) Detection wavelength: 266nm;
c) Column temperature: 30℃;
d) Injection volume: 40μL;
e) Flow rate: 1:0mL/min;
f) Mobile phase: A is 0:1% trifluoroacetic acid aqueous solution, B is 0:1% trifluoroacetic acid acetonitrile solution;
GB 31658:1-2021
g) The mobile phase gradient elution conditions are shown in Table 1:
8:5:2 Measurement method
Take the sample solution and the corresponding standard solution, perform single-point or multi-point calibration, quantify it based on the chromatographic peak area, and calculate it according to the external standard method: The standard solution and
The response value of DCA in the sample solution should be within the linear range of the instrument detection: Under the above chromatographic conditions, the values of the standard solution and the sample solution
Please see Appendix A for high performance liquid chromatography:
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 ceftiofur in the sample is calculated according to formula (1):
10 Sensitivity, accuracy and precision of detection methods
10:1 Sensitivity
The quantitative limit of this method in cattle, pig muscle, fat, kidney and pig liver is 100 μg/kg, and the quantitative limit in cattle liver is 500 μg/kg:
10:2 Accuracy
The recovery rate of this method at the added concentration of 100 μg/kg~6000 μg/kg is 80%~110%:
10:3 Precision
The intra-batch relative standard deviation of this method is ≤15%, and the inter-batch relative standard deviation is ≤15%:
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