GB 31658.14-2021National food safety standard - Determination of o-trenbolone, B-trenbolone 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 determination of alpha-trenbolone and beta-trenbolone residues in animal foods:

This document is applicable to the determination of alpha-trenbolone and beta-trenbolone residues in muscle, fat, liver and kidney tissues of pigs, cattle, sheep and chickens, as well as rabbit meat, eggs, milk and goat milk:

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 alpha-trenbolone and beta-trenbolone in the sample were enzymatically hydrolyzed under weakly acidic conditions, extracted with tert-butyl methyl ether, degreased with n-hexane, HLB and ammonia:

Based on solid phase extraction column purification, liquid chromatography-tandem mass spectrometry determination, and external standard method for 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 Acetonitrile (CH3CN): chromatographically pure:

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

5:1:3 Tert-butyl methyl ether (C5H12O):

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

5:1:5 Glacial acetic acid (CH3COOH):

5:1:6 Ammonia (NH3:H2O): content (NH3) is about 25%:

5:1:7 Sodium acetate (C2H3NaO2):

5:1:8 Acetone (C3H6O):

5:1:9 beta-glucuronidase/arylsulfatase: >100000U/mL:

5:2 Solution preparation

5:2:1 0:04mol/L sodium acetate solution: Take 3:28g of sodium acetate, dissolve it in water and dilute it to 1000mL:

5:2:2 80% methanol aqueous solution: Take 80mL of methanol and dilute to 100mL with water:

5:2:3 10% methanol aqueous solution: Take 10 mL of methanol and dilute to 100 mL with water:

5:2:4 2% ammonia solution: Take 8 mL of ammonia water and dilute it with water to 100 mL:

5:2:5 Methanol-2% ammonia solution (5:95): Take 5 mL of methanol, add 95 mL of 2% ammonia solution (5:2:4), and mix well:

5:2:6 Methanol-2% ammonia solution (40:60): Take 40 mL of methanol, add 60 mL of 2% ammonia solution (5:2:4), and mix:

5:2:7 Acetone-methanol solution (80:20): Take 80 mL of acetone, add 20 mL of methanol, and mix:

5:3 Standard products

5:3:1 alpha-Trenbolone (17alpha-Trenbolone, C18H22O2, CAS number: 80657-17-6): content >= 98:5%:

5:3:2 beta-Trenbolone (17beta-Trenbolone, C18H22O2, CAS number: 10161-33-8): content >= 98:0%:

5:4 Preparation of standard solution

5:4:1 Standard stock solution: Take about 10 mg each of alpha-trenbolone and beta-trenbolone standard products, weigh them accurately, dissolve them with an appropriate amount of methanol and dilute them to a 10 mL volumetric flask, and prepare them to a concentration of 1mg/mL alpha-trenbolone and beta-trenbolone standard stock solution: Store below -18 degrees C and is valid for 6 months:

5:4:2 Mix standard intermediate solution: Accurately measure 1 mL each of alpha-trenbolone and beta-trenbolone standard stock solutions in a 10 mL volumetric flask, and dilute with methanol

Release to the mark and prepare a mixed standard intermediate solution with a concentration of 100 µg/mL: Store below -18°C and has a validity period of 3 months:

5:4:3 Mixed standard working solution: Precisely measure appropriate amounts of mixed standard intermediate solutions, dilute with methanol, and prepare concentrations of 20ng/mL, 50ng/mL, 100ng/mL,:200ng/mL, 500ng/mL and 1000ng respectively: /mL mixed standard working solution: Ready for use:

5:5 Materials

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

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:

5:5:2 Amino solid-phase extraction column: 500mg/6mL, or equivalent:

6 Instruments and equipment

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

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

6:3 pH meter:

6:4 Centrifuge: the speed can reach 5000r/min:

6:5 Vortex mixer:

6:6 Constant temperature shaker:

6:7 Horizontal oscillator:

6:8 Solid phase extraction device:

6:9 Nitrogen blower:

6:10 Nylon microporous filter membrane: 0:22µm:

7: Preparation and preservation of samples

7:1 Preparation of samples

7:1:1 Muscle, fat, liver and kidney tissue

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:

7:1:2 Milk

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

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:

7:1:3 Eggs

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

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 Enzymatic hydrolysis

Take 5g of the sample (accurate to +/-0:02g), place it in a 50mL centrifuge tube, add 10mL of 0:04mol/L sodium acetate solution, vortex to mix, and adjust the pH to 4:3~4:8 with glacial acetic acid: Add beta- Add 20 µL of glucuronidase/arylsulfatase, vortex to mix, and shake in a constant temperature water bath shaker at 37°C for more than 14 hours:

8:2 Extraction

Cool the enzymatic hydrolyzate to room temperature, add 10 mL of tert-butyl methyl ether (for milk and goat milk samples, add 5 mL of acetonitrile first), vortex and mix, and shake for 10 minutes:

Centrifuge at 5000 r/min for 10 minutes, and put the upper liquid into another centrifuge tube: Repeat the extraction of the lower liquid once with 10 mL of tert-butyl methyl ether, combine the two upper liquids, and blow dry with nitrogen in a water bath at 50°C: Add 4 mL of 80% methanol aqueous solution and sonicate for 2 minutes: , dissolve the residue: Add 5 mL of n-hexane (add 10 mL for fat sample), shake for 5 min, centrifuge at 5000 r/min for 10 min, discard the upper layer of n-hexane: Add 5 mL of n-hexane (add 10 mL for fat sample) and repeat the above operation: Remove the water layer phase, blow nitrogen in a 50°C water bath to about 0:5 mL, add 3 mL of 10% methanol aqueous solution, sonicate for 5 min, dissolve the residue and set aside:

8:3 Purification

The HLB 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 use methanol-2% ammonia solution (5:95) in sequence:

3 mL, 3 mL of methanol-2% ammonia solution (40:60) and 3 mL of water, rinse and drain: Take 5 mL of acetone-methanol solution (80:20) and activate the amino solid-phase extraction column in series with the HLB solid-phase extraction column: Below, use 5 mL of acetone-methanol (80:20) to elute, collect the eluate, and blow dry with nitrogen in a water bath at 50°C: Dissolve the residue with 1:0 mL of 80% methanol solution, sonicate for 5 min, vortex for 1 min, and pass 0: 22µm nylon microporous filter membrane for liquid chromatography-tandem mass spectrometry:

8:4 Preparation of matrix addition standard curve

Accurately measure an appropriate amount of the mixed standard working solution and add it to 6 blank samples respectively: The added concentrations are 0:4 µg/kg, 1 µg/kg, and

2µg/kg, 4µg/kg, 10µg/kg and 20µg/kg, mix thoroughly: Follow the enzymatic hydrolysis, extraction and purification steps to make the concentrations of 2ng/mL, 5ng/mL, 10ng/mL and 20ng/mL respectively: Standard solutions were added to a series of matrices of , 50ng/mL and 100ng/mL for liquid chromatography-tandem mass spectrometry measurement: Using the quantitative ion pair mass chromatography peak area as the ordinate and the corresponding standard solution concentration as the abscissa, draw a standard curve , find the regression equation and correlation coefficient:

8:5 Determination

8:5:1 Chromatographic reference conditions

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

b) Mobile phase: A is water, B is acetonitrile;

c) Gradient elution: see Table 1 for elution conditions;

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

e) Column temperature: 40 degrees C;

f) Injection volume: 5µL:

8:6 Determination method

8:6:1 Qualitative determination

Under the same test conditions, the retention times of alpha-trenbolone and beta-trenbolone chromatographic peaks in the sample solution correspond to those in the matrix-added standard solution:

The relative deviation of the retention time of the peak is within +/-2:5%, and the relative ion abundance detected should be consistent with the relative ion abundance of the matrix-added standard solution of equivalent concentration: The allowable deviation should meet the requirements of Table 3:

8:6:2 Quantitative determination

Take the sample solution and the corresponding matrix, add the standard solution, perform single-point or multi-point calibration, and determine the characteristic ion mass chromatographic peak area according to the external standard method:

The response values of alpha-trenbolone and beta-trenbolone in the matrix-added standard solution and sample solution should be within the linear range of the instrument detection: In the above

Under the above chromatography-mass spectrometry conditions, the characteristic ion mass chromatograms of alpha-trenbolone and beta-trenbolone in the matrix-added standard solution are shown in Appendix A:

8:7 Blank test

Take a blank sample and perform parallel operations using the same measurement steps except that no standard solution is added:

9 Result calculation and expression

The residual amount of alpha-trenbolone or beta-trenbolone 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 liver is 1:0 µg/kg, and the limit of quantification is 2:0 µg/kg; the detection limit in muscle, kidney, fat, eggs, and milk is 0:5 µg/kg, and the limit of quantification is 1: 0µg/kg:

10:2 Accuracy

This method has a recovery rate of 60% to 120% at a concentration level of 2 µg/kg to 10 µg/kg in the liver, and a recovery rate of 60% at a concentration level of 1 µg/kg to 10 µg/kg in muscles, kidneys, fat, eggs, and milk: ~120%:

10:3 Precision

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