GB 31658.12-2021National food safety standard - Determination of cyromazine in animal derived food by high performance liquid chromatographic method (English PDF)

Foreword

This document is drafted in accordance with the provisions of GB/T 1:1-2020 "Standardization Work Guidelines Part 1: Structure and Drafting Rules of Standardization Documents":

This document is published for the first time:

National food safety standards

Determination of cypromethine residues in animal foods

HPLC

1 Scope

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

This document is applicable to the detection of cipropromazine residues in sheep and poultry muscles, fat, liver and sheep kidney tissues:

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 cypromethazine in the sample was extracted with trichloroacetic acid/acetonitrile solution, purified with a mixed cation exchange solid phase extraction column, and subjected to high performance liquid chromatography:

Determination, external standard method quantification:

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

5:1:1 Methanol (CH3OH):

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

5:1:3 Hydrochloric acid (HCl):

5:1:4 Ammonium acetate (CH3CONH3):

5:1:5 Acetic acid (CH3COOH):

5:1:6 n-Hexane (C6H14):

5:1:7 Ammonia (NH3H2O):

5:1:8 Trichloroacetic acid (CCl3COOH):

5:2 Solution preparation

5:2:1 25mmol/L ammonium acetate solution: Take 0:19g of ammonium acetate, add 950mL of water to dissolve, adjust the pH to 5:0 with acetic acid, and add water to dilute to 1000mL:

5:2:2 1% trichloroacetic acid solution: Take 1g of trichloroacetic acid, dissolve it in water and dilute it to 100mL:

5:2:3 Extraction solution: Take 15 mL of 1% trichloroacetic acid solution and dilute to 100 mL with acetonitrile:

5:2:4 0:1mol/L hydrochloric acid solution: Take 9mL of hydrochloric acid and dilute it to 1000mL:

5:2:5 5% ammonia water and methanol solution: Take 5 mL of ammonia water and dilute it with methanol to 100 mL:

5:2:6 Mobile phase: Take 40:0 mL of 25 mmol/L ammonium acetate solution and adjust the volume to 1000 mL with acetonitrile:

5:3 Standard products

Cyromazine (Cyromazine, C6H10N6, CAS number: 66215-27-8), content >= 97:0%:

5:4 Preparation of standard solutions

5:4:1 Standard stock solution (1 mg/mL): Accurately weigh the cipropromazine standard equivalent to 25 mg of cipropromazine, dissolve it with methanol and dilute it to a 25 mL volumetric flask, and prepare a concentration of 1 mg/mL: mL standard stock solution: Store below -18 degrees C, valid for 1 year:

5:4:2 Standard working solution (10 mg/L): Precisely measure 1 mL of the standard stock solution, put it in a 100 mL volumetric flask, dilute it to the mark with methanol, and prepare a standard working solution of ciprofloxacin with a concentration of 10 mg/L: - Stored below 18 degrees C, the validity period is 6 months:

5:5 Materials

5:5:1 Mixed cation exchange solid phase extraction column: 60mg/3mL, or equivalent:

5:5:2 Universal filter membrane for syringe filter: nylon material, pore size 0:45µm or equivalent performance:

6 Instruments and equipment

6:1 High performance liquid chromatograph: equipped with UV detector:

6:2 Analytical balance: sensitivity 0:00001g and sensitivity 0:01g:

6:3 Nitrogen blowing instrument:

6:4 Solid phase extraction device:

6:5 Homogenizer:

6:6 Vortex mixer:

6:7 Centrifuge: the maximum speed is not less than 10000r/min:

6:8 pH meter:

6:9 Rotary evaporator:

6:10 Separating funnel: 100mL:

6:11 Chicken heart bottle: 100mL:

6:12 Graduated centrifuge tube: graduation value is 0:1mL:

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 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 Storage of samples

Store below -18 degrees C:

8 Measurement steps

8:1 Extraction

Take 5g of the sample (accurate to +/-0:02g), put it in a 50mL centrifuge tube, add 15mL of the extraction solution, homogenize at high speed to evenly disperse, centrifuge at 5000r/min for 5min, take the supernatant into a 100mL separatory funnel, and add the residue to Extract 10 mL, repeat the extraction once, combine the supernatants, add 30 mL of n-hexane, shake for 2 minutes, and let stand for stratification: Collect the lower liquid in a 100 mL chicken heart bottle, and rotary evaporate it in a 50°C water bath to 1 mL (the vacuum needs to be controlled (to prevent bumping), transfer to a 10 mL graduated centrifuge tube, rinse the chicken heart bottle with the extract twice, 2 mL each time, combine the extracts, centrifuge at 10,000 r/min for 5 min, take the supernatant and set aside:

8:2 Purification

The solid-phase extraction column was activated with 3 mL of methanol and 3 mL of water in sequence: Pass the reserve solution through the column and control the flow rate to 1 mL/min: Elute with 3 mL of methanol, 3 mL of 0:1 mol/L hydrochloric acid solution, 3 mL of water, and 3 mL of methanol in sequence, and drain: Elute with 5 mL of ammonia and methanol solution: Collect the eluate, blow dry with nitrogen at 50°C, dissolve the residue with 1:0 mL of mobile phase, vortex for 30 s, and filter with a membrane for measurement by high performance liquid chromatography:

8:3 Preparation of standard curve

Precisely measure an appropriate amount of cipropromazine standard working solution (10 mg/L), dilute it with mobile phase, and prepare a concentration of 0:05 µg/mL:

A series of standard solutions of 0:1µg/mL, 0:2µg/mL, 0:4µg/mL, 1µg/mL and 2µg/mL are used for high performance liquid chromatography determination: The measured peak area is used as the ordinate, corresponding to the standard solution Concentration is the abscissa, draw a standard curve, and find the regression equation and correlation coefficient:

8:4 Determination

8:4:1 Chromatographic conditions reference conditions

a) Chromatographic column: HILIC (hydrophilic reaction ion exchange chromatographic column, 2:1mmx150mm, 3:5µm), or equivalent;

b) Flow rate: 0:3mL/min;

c) Detection wavelength: 214nm;

d) Injection volume: 20µL;

e) Column temperature: 30 degrees C:

8:4:2 Determination method

Take the sample solution and the corresponding standard solution, perform single-point or multi-point calibration, and quantify the chromatographic peak area according to the external standard method: Standard working solution and sample

The response value of the substance to be tested in the solution should be within the linear range of the instrument detection: The relative deviation between the retention time of the peak of the substance to be tested in the sample and the retention time of the corresponding peak of the standard working solution should be within +/-2:5%: Cyclopropylamine See Appendix A for the chromatogram of azine standard solution:

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 cipromethine in the test material 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 10 µg/kg, and the quantitation limit is 25 µg/kg:

10:2 Accuracy

The recovery rate of this method is 60% to 110% at the added concentration level of 25µg/kg~600µg/kg:

10:3 Precision

The intra-batch relative standard deviation of this method is <=15%, and the inter-batch relative standard deviation is <=20%: