GB 31658.13-2021National food safety standard - Determination of robenidine residues in animal derived food by liquid chromatography-tandem mass spectrometric 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:

1 Scope

This document specifies the sample preparation and liquid chromatography-tandem mass spectrometry detection methods for the detection of chlorpheniramine residues in animal foods:

This document is applicable to the determination of chlorpheniramine residues in chicken muscle, liver, kidney and skin + fat tissue:

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 chlorphenanthrene in the sample was extracted with 0:1% formic acid and acetonitrile, purified with a solid phase extraction column, and measured by liquid chromatography-tandem mass spectrometry, using the internal standard method:

Quantitative:

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 Acetonitrile (CH3CN): chromatographically pure:

5:1:2 Methanol (CH3OH): chromatographically pure:

5:1:3 Formic acid (HCOOH): chromatographically pure:

5:1:4 n-Hexane (C6H14):

5:1:5 Dimethyl sulfoxide (C2H6OS):

5:2 Solution preparation

5:2:1 0:1% formic acid in acetonitrile solution: Take 0:1mL of formic acid and dilute to 100mL with acetonitrile:

5:2:2 85% acetonitrile solution: Take 85mL of acetonitrile and dilute to 100mL with water:

5:2:3 0:1% formic acid aqueous solution: Take 0:1mL of formic acid and dilute to 100mL with water:

5:2:4 0:3% formic acid aqueous solution: Take 0:3 mL of formic acid and dilute to 100 mL with water:

5:2:5 50% acetonitrile formic acid aqueous solution: Take 50 mL of acetonitrile and dilute to 100 mL with 0:3% formic acid aqueous solution:

5:3 Standard products

5:3:2 Robenidine-D8hydrochloride (C15H6Cl3D8N5, CAS number: 1173097-77-2): content >=99:0%:

5:4 Preparation of standard solution

5:4:1 Standard stock solution of chlorpheniramine: Take an appropriate amount of chlorpheniramine hydrochloride standard (equivalent to 10 mg of chlorpheniramine), weigh it accurately, add an appropriate amount of methanol to dissolve and dilute it to a 10mL volumetric flask, and prepare it to a concentration It is a standard stock solution of 1 mg/mL: Stored below -18°C, it is valid for 6 months:

5:4:2 Chlorbenguanide standard working solution: Accurately measure 1 mL of chlorbenguanide standard stock solution, place it in a 100 mL volumetric flask, dilute to the mark with methanol, and prepare a chlorbenguanide standard working solution with a concentration of 10 µg/mL: Store at 2 degrees C~8 degrees C, valid for 3 months:

5:4:3 Chlorbenguanil-D8 internal standard stock solution: Take an appropriate amount of chlorbenguanide-D8 standard (equivalent to 10 mg of chlorbenguanil-D8), add an appropriate amount of dimethyl sulfoxide to dissolve, dilute with methanol to a constant volume: In a 10mL volumetric flask, prepare a chlorpheniramine-D8 internal standard stock solution with a concentration of 1 mg/mL: Store below -18°C and is valid for 6 months:

5:4:4 Chlorbenguanil-D8 internal standard intermediate solution: Accurately measure 1 mL of chlorbenguanil-D8 internal standard stock solution, place it in a 100 mL volumetric flask, dilute it to the mark with methanol, and prepare a chlorine solution with a concentration of 10 µg/mL: Benzene-D8 internal standard intermediate solution: Store at 2 degrees C~8 degrees C, valid for 1 month:

5:4:5 Chlorbenguanil-D8 internal standard working solution: Accurately measure 1 mL of chlorbenguanil-D8 internal standard intermediate solution, place it in a 10 mL volumetric flask, dilute it to the mark with methanol, and prepare a chlorine solution with a concentration of 1 µg/mL: Phenylguanidine-D8 internal standard working solution: Ready for use:

5:5 Materials

1) The HLB solid-phase extraction columns listed here are only for reference and are not for commercial purposes: Standard users are encouraged to try solid-phase extraction columns of different manufacturers or models:

5:5:1 HLB solid phase extraction column 1)::200mg/3mL, or equivalent:

5:5:2 Neutral alumina solid-phase extraction column: 500mg/3mL, or equivalent:

5:5:3 Nylon microporous filter membrane: 0:22µm:

5:5:4 Centrifuge tube: 50mL:

5:5:5 Graduated test tube: 10mL, graduation value 0:1mL:

6 Instruments and equipment

6:1 Liquid chromatography-tandem mass spectrometer: equipped with electrospray ion source:

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

6:3 Low-temperature high-speed centrifuge: the speed can reach 8000r/min:

6:4 Tissue homogenizer:

6:5 Vortex mixer:

6:6 Solid phase extraction device:

6:7 Nitrogen blower:

7: Preparation and preservation of samples

7:1 Preparation of samples

Take 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 Storage of samples

Store below -18 degrees C:

8 Measurement steps

8:1 Extraction

Take 2g of the sample (accurate to +/-0:02g), put it in a 50mL centrifuge tube, add accurately 100µL of chlorpheniramine-D8 internal standard working solution, add 5mL of 0:1% formic acid acetonitrile solution, vortex to mix, and oscillate at medium speed for 10 minutes: Centrifuge at 8000r/min for 10min, take the supernatant, add 1mL of water, mix well, and use it as backup solution 1:

8:2 Purification

Take a neutral alumina solid-phase extraction column, activate it with 2 mL of acetonitrile, pass the standby solution 1 through the column, collect all the effluent in a 10 mL test tube, and use

Elute with 2 mL of 85% acetonitrile solution, combine the effluents, blow with nitrogen at 40°C until the volume is less than 2 mL, add 8 mL of water, mix well, and use it as a backup solution: 2: Take the HLB solid phase extraction column and activate it with 3 mL of methanol and 3 mL of water: Pass the backup solution 2 through the column, rinse with 3 mL of water, drain for 30 s, rinse with 3 mL of n-hexane, drain for 10 min, elute with 5 mL of methanol, collect the eluate, blow dry with nitrogen at 40°C, and use 50% acetonitrile formic acid aqueous solution 1 Dissolve the residue in :0mL, vortex, and filter with a nylon microporous membrane for liquid chromatography-tandem mass spectrometry measurement:

8:3 Preparation of standard curve

Precisely measure an appropriate amount of chlorbenguanide standard working solution and chlorbenguanide-D8 internal standard working solution, dilute it with 50% acetonitrile formic acid aqueous solution, and prepare a chlorbenguanide concentration of 5ng/mL, 10ng/mL, 20ng/mL, and 50ng/mL: mL, 100ng/mL, 20ng/mL, 500ng/mL and chlorpheniramine-D8 internal standard are all 100ng/mL series standard solutions for liquid chromatography-tandem mass spectrometry determination: The characteristic ion mass chromatography peak area is used as the ordinate , the corresponding standard solution concentration is the abscissa, draw the standard curve, and find the regression equation and correlation coefficient:

8:4 Determination

8:4:1 Chromatographic conditions reference conditions

a) Chromatographic column: C18 (100mmx2:1mm, 1:7µm), or equivalent;

b) Column temperature: 30 degrees C;

c) Injection volume: 5µL;

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

e) Mobile phase: A is acetonitrile, B is 0:1% formic acid aqueous solution, and the gradient elution conditions are shown in Table 1:

8:5 Determination method

8:5:1 Qualitative determination

Under the same test conditions, the relative deviation between the retention time of chlorpheniramine in the sample solution and the retention time of chlorpheniramine in the standard working solution is within +/-

Within 2:5%, and the detected relative ion abundance should be consistent with the relative ion abundance of the calibration standard solution with equivalent concentration: The allowable deviation should meet the requirements of Table 3:

8:5:2 Quantitative determination

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

The response values of chlorpheniramine in the quasi-working solution and sample solution should be within the linear range of instrument detection: Under the above chromatography-mass spectrometry conditions, chlorpheniramine

See Appendix A for the characteristic ion mass chromatogram of the standard solution:

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 chlorpheniramine 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 is 5 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 concentration level of 10 µg/kg to:200 µg/kg in muscle, liver and kidney tissues; the recovery rate in skin + fat tissue at the concentration level of 10 µg/kg to 400 µg/kg 70%~120%:

10:3 Precision

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