GB 31658.6-2021National food safety standard - Determination of tetracylines residues in animal derived food 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:

This document is published for the first time:

National food safety standards

Determination of tetracycline drug residues in animal foods

HPLC

1 Scope

This document specifies the sample preparation and high-performance liquid chromatography determination methods for the detection of tetracycline drug residues in animal foods:

This document is applicable to the muscles, livers and kidneys of pigs, cattle, sheep and chickens, the skin + fat of pigs and chickens, eggs, milk, fish skin + meat, and soil in shrimp muscles:

Detection of residual amounts of mycin, tetracycline, chlortetracycline and doxycycline:

2 Normative reference documents

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

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 tetracyclines in the sample were extracted with EDTA.2Na-Mclvaine buffer solution, purified by solid phase extraction column, and analyzed by high performance liquid chromatography:

Determination by spectral UV method and quantification by external standard method:

5 Reagents and materials

The reagents used below are all analytically pure reagents unless otherwise noted; the water is first-grade water that complies with GB/T 6682:

5:1 Reagents

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

5:1:2 Acetonitrile (CH3CN): chromatographically pure:

5:1:3 Trifluoroacetic acid (CF3COOH):

5:1:4 Dichloromethane (CH2Cl2):

5:1:5 Disodium ethylenediaminetetraacetate (C10H14N2Na2O8):

5:1:6 Citric acid (C6H8O7:H2O):

5:1:7 Disodium hydrogen phosphate (NaH2PO4:12H2O):

5:1:8 Oxalic acid (H2C2O4:1:2H2O):

5:1:9 Sulfuric acid (H2SO4):

5:1:10 Sodium tungstate (Na2WO4):

5:2 Standard products

The content of oxytetracycline hydrochloride is >=97:0%, the content of tetracycline hydrochloride is >=97:15%, the content of chlortetracycline hydrochloride is >=93:1%, and the content of doxycycline hydrochloride is

>=98:12%, see Appendix A:

5:3 Solution preparation

5:3:1 Citric acid solution: Take 21:01g of citric acid, dissolve it in water and dilute it to 1000mL:

5:3:2 Disodium hydrogen phosphate solution: Take 71:63g of disodium hydrogen phosphate, dissolve it in water and dilute it to 1000mL:

GB 31658:6-2021

5: Mclvain buffer solution (pH 4:0): Take 1000 mL of citric acid solution and 625 mL of disodium hydrogen phosphate solution, mix well, and add hydrochloric acid or hydrochloric acid to the solution:

Adjust the pH of the sodium hydroxide solution to 4:0+/-0:05:

5:EDTA:3:4 EDTA:2Na-Mclvaine buffer solution: Take 60:5g of disodium ethylenediaminetetraacetate and add Mclvaine buffer solution

1625mL, dissolve and mix:

5:3:5 Oxalic acid solution (0:01mol/L): Take 1:26g of oxalic acid, dissolve it in water and dilute it to 1000mL:

5:3:6 Trifluoroacetic acid solution: Take 0:8mL of trifluoroacetic acid, dissolve it in water and dilute it to 1000mL:

5:3:7 Sulfuric acid solution: Take 1:85mL of sulfuric acid, dissolve it in water and dilute it to 100mL:

5:3:8 Sodium tungstate solution: Take 7g of sodium tungstate, dissolve it in water and dilute it to 100mL:

5:3:9 Oxalic acid solution (1mol/L): Take 12:6g of oxalic acid, dissolve it in water and dilute it to 100mL:

5:3:10 Oxalic acid-acetonitrile solution: Take 20mL of oxalic acid solution (1mol/L), dissolve it in acetonitrile and dilute it to 100mL:

5:4 Preparation of standard solution

5:4:1 Standard stock solution: Take about 10 mg each of oxytetracycline hydrochloride, tetracycline hydrochloride, chlortetracycline hydrochloride and doxycycline hydrochloride standards and weigh them accurately:

Dissolve and dilute with methanol to a 10mL volumetric flask, and prepare oxytetracycline, tetracycline, chlortetracycline and doxycycline at a concentration of 1 mg/mL:

Gluten standard stock solution: Store below -18 degrees C, valid for 1 month:

5:4:2 Mix standard working solution: Precisely measure 1 mL each of oxytetracycline, tetracycline, chlortetracycline and doxycycline standard stock solutions, and put it into a 100 mL volume:

In a measuring flask, dilute with methanol to the mark and prepare a mixed standard working solution with a concentration of 10 µg/mL: Store at 2°C ~ 8°C: Prepare ready for use:

5:5 Materials

5．

Polymer, or equivalent:

5:5:2 LCX solid phase extraction column 1): 500mg/6mL, the filler is phosphorylated polystyrene divinylbenzene polymer, or equivalent:

1) The LCX solid-phase extraction columns listed in this experiment are developed and provided by Agela Company: The solid-phase extraction columns listed here are for reference only and do not involve commercial purposes: Standard users are encouraged

Try using solid phase extraction columns from different manufacturers or models:

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 Tissue homogenizer:

6: Vortex mixer: 3000r/min:

6: Low temperature centrifuge: the speed can reach 8500r/min:

6:6 Solid phase extraction device:

6:7 Nitrogen blower:

6:8 Nylon microporous filter membrane: 0:22µm:

6: Centrifuge tube: 50mL:

7: Preparation and preservation of samples

7:1 Preparation of samples

7:1 Organization

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 a standard solution of appropriate concentration, and add the sample as a blank:

7:1:2 Milk

Take an appropriate amount of fresh or refrigerated blank or test milk and mix evenly:

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 a standard solution of appropriate concentration, and add the sample as a blank:

7:1:3 eggs

Take an appropriate amount of fresh test eggs, peel them, and homogenize them:

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 a standard 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

8:1:1 Skin + Fat

Take 5g of the sample (accurate to +/-0:02g), add 15mL of methylene chloride, vortex for 1 minute, shake for 5 minutes, add EDTA:2Na-Mclvaine

Take 15 mL of buffer solution, vortex for 1 min, shake for 5 min, centrifuge at 8500 r/min for 5 min, and take the supernatant: Use EDTA.2NaＧ for the lower solution:

Repeat the extraction with Mclvaine buffer solution twice, 15 mL each time, combine the supernatants, filter with neutral filter paper, and set aside:

8:1:2 Muscle, liver, kidney, milk, eggs

Weigh 5g of the sample (accurate to +/-0:02g), add 20mL of EDTA:2Na-Mclvaine buffer solution, vortex for 1 minute, and shake

10min, add 5mL of sulfuric acid solution and 5mL of sodium tungstate solution, vortex for 1min, centrifuge at 8500r/min for 5min, take the supernatant and use the residue:

Extract 20 mL and 10 mL of EDTA-2Na-Mclvaine buffer solution twice, combine the supernatants, filter with neutral filter paper, and set aside:

8:2 Purification

The HLB column was activated with 5 mL each of methanol, water and EDTA.2Na-Mclvaine buffer solution in sequence: The backup solution was passed through the column until all the backup solution flowed:

After coming out, rinse with 10 mL of water and 5% methanol solution in sequence, drain for 30 s, elute with 6 mL of methanol, collect the eluate in a graduated test tube, add water

2 mL, mix well, pass through the activated LCX column with 5 mL of methanol and 5 mL of water: After all the liquid flows out, rinse with 5 mL of water and methanol, and drain for 1 min:

Elute with 6 mL of oxalic acid-acetonitrile solution, collect the eluate, blow nitrogen to 0:15 mL~1:0 mL in a water bath at 40°C, add 0:4 mL of methanol, and add oxalic acid

The solution (0:01 mol/L) is diluted to 2:0 mL, filtered through a microporous membrane, and measured by high performance liquid chromatography (the solution on the machine should be measured within 24 hours):

8:3 Preparation of standard curve

Precisely measure an appropriate amount of the mixed standard working solution and dilute it with oxalic acid solution (0:01 mol/L) to a concentration of 0:05 µg/mL, 0:1 µg/mL,

A series of mixed standard solutions of 0:2µg/mL, 0:5µg/mL, 1µg/mL, 2µg/mL and 5µg/mL are used for high performance liquid chromatography determination: To measure

The peak area is the ordinate, the corresponding standard solution concentration is the abscissa, and a standard curve is drawn: Find the regression equation and correlation coefficient:

8: Measurement

8:4:1 Liquid Chromatography Reference Conditions

a) Chromatographic column: C18 (150mmx4:6mm, 5µm), or equivalent;

b) Mobile phase: A is trifluoroacetic acid solution, B is acetonitrile, gradient elution conditions are shown in Table 1;

c) Detection wavelength: 350nm;

d) Injection volume: 50µL;

e) Column temperature: 30 degrees C:

8:4:2 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 tetracyclines in the sample solution should be within the linear range of instrument detection: The retention time of tetracyclines in the sample solution is consistent with the standard

The relative deviation of the retention time of the corresponding peak of the working solution should be within +/-2:5%: Under the above chromatographic conditions, the HPLC chromatogram of the standard solution is

Please see Appendix B:

8:5 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 tetracycline drugs 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 in pig, cow, sheep, chicken muscles, eggs, milk, fish skin + meat, and shrimp muscles is 20 µg/kg, and the quantification limit is

50 µg/kg; in the liver and kidney of pigs, cattle, sheep, and chickens, and the skin + fat of pigs and chickens, the detection limit is 50 µg/kg, and the quantification limit is 100 µg/kg:

10:2 Accuracy

This method has a recovery rate of 60% to 120% in pig, cattle, sheep, and chicken muscles at a concentration of 50 µg/kg to:200 µg/kg:

The recovery rate in the liver at an added concentration of 100 µg/kg~600 µg/kg is 60%~120%, and in the kidney tissue of pigs, cattle, sheep, and chickens, the recovery rate is 100 µg/kg~

The recovery rate of 1200µg/kg added concentration is 60%~120%, and the recovery rate of pig and chicken skin + fat added concentration of 100µg/kg~600µg/kg

The recovery rate is 60% to 120%: The recovery rate of fish skin + meat and shrimp muscle at a concentration of 50 µg/kg to:200 µg/kg is 60% to 120%: In milk

The recovery rate at the added concentration of 50µg/kg~200µg/kg is 60%~120%, and the recovery rate at the added concentration of 50µg/kg~400µg/kg in eggs

The rate is 60%~120%:

10:3 Precision

The intra-batch relative standard deviation of this method is <=15%, and the inter-batch relative standard deviation is <=15%: