GB 31658.1-2021National food safety standard - Determination of ceftiofur residues in animal tissues by high performance liquid chromatography 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:

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 degrees C~8 degrees C, 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 degrees C:

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 degrees C 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 (250mmx4:6mm, 5µm), or equivalent;

b) Detection wavelength: 266nm;

c) Column temperature: 30 degrees C;

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%: