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Delivery: <= 3 days. True-PDF full-copy in English will be manually translated and delivered via email. GB 31658.14-2021: National food safety standard - Determination of o-trenbolone, B-trenbolone residues in animal derived food by liquid chromatography-tandem mass spectrometric method Status: Valid
Basic dataStandard ID: GB 31658.14-2021 (GB31658.14-2021)Description (Translated English): National food safety standard - Determination of o-trenbolone, B-trenbolone residues in animal derived food by liquid chromatography-tandem mass spectrometric method Sector / Industry: National Standard Classification of Chinese Standard: X04 Word Count Estimation: 8,847 Issuing agency(ies): National Health Commission of the People's Republic of China, State Administration for Market Regulation GB 31658.14-2021: National food safety standard - Determination of o-trenbolone, B-trenbolone residues in animal derived food by liquid chromatography-tandem mass spectrometric method---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order. National food safety standards Alpha-trenbolone and beta-trenbolone in animal foods Determination of residual amounts liquid chromatography-tandem mass spectrometry National Standards of People's Republic of China Released by the National Health Commission of the People's Republic of China State Administration for Market Regulation Ministry of Agriculture and Rural Affairs of the People's Republic of China ForewordThis 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 ScopeThis document specifies the sample preparation and liquid chromatography-tandem mass spectrometry detection methods for the determination of α-trenbolone and β-trenbolone residues in animal foods: This document is applicable to the determination of α-trenbolone and β-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 documentsThe 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 laboratories3 Terms and definitionsThere are no terms or definitions that need to be defined in this document:4 PrinciplesThe remaining α-trenbolone and β-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 materialsUnless 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 β-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 α-Trenbolone (17α-Trenbolone, C18H22O2, CAS number: 80657-17-6): content ≥ 98:5%: 5:3:2 β-Trenbolone (17β-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 α-trenbolone and β-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 α-trenbolone and β-trenbolone standard stock solution: Store below -18℃ and is valid for 6 months: 5:4:2 Mix standard intermediate solution: Accurately measure 1 mL each of α-trenbolone and β-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 equipment6:1 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℃:8 Measurement steps8:1 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 β- 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 (100mm×2:1mm, 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℃; f) Injection volume: 5μL: 8:6 Determination method 8:6:1 Qualitative determination Under the same test conditions, the retention times of α-trenbolone and β-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 α-trenbolone and β-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 α-trenbolone and β-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 expressionThe residual amount of α-trenbolone or β-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%: ......Tips & Frequently Asked Questions:Question 1: How long will the true-PDF of GB 31658.14-2021_English be delivered?Answer: Upon your order, we will start to translate GB 31658.14-2021_English as soon as possible, and keep you informed of the progress. 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