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Delivery: <= 3 days. True-PDF full-copy in English will be manually translated and delivered via email. SN/T 5116-2019: (Import and export of food animals, feed malachite green, crystal violet determination by liquid chromatography-mass spectrometry/mass spectrometry) Status: Valid
Basic dataStandard ID: SN/T 5116-2019 (SN/T5116-2019)Description (Translated English): (Import and export of food animals, feed malachite green, crystal violet determination by liquid chromatography-mass spectrometry/mass spectrometry) Sector / Industry: Commodity Inspection Standard (Recommended) Classification of Chinese Standard: B46 Word Count Estimation: 12,121 Date of Issue: 2019-09-03 Date of Implementation: 2020-03-01 Regulation (derived from): Natural Resources Department Announcement No. 7 of 2019 Issuing agency(ies): General Administration of Customs SN/T 5116-2019: (Import and export of food animals, feed malachite green, crystal violet determination by liquid chromatography-mass spectrometry/mass spectrometry)---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. (Import and export of food animals, feed malachite green, crystal violet determination by liquid chromatography-mass spectrometry/mass spectrometry) ICS 71.040B46 Liquid chromatography-mass spectrometry/mass spectrometry 1 Scope This standard specifies the malachite green, crystal violet and metabolite malachite green, cryptic crystal violet in food animals and feed for import and export. Liquid chromatography-mass spectrometry/mass spectrometry. This standard applies to fish, shrimp, crab, shellfish, fish meal, plant-based feed, concentrated feed, compound feed, feed additives, concentrate supplements Determination of malachite green, leucomalachite green, crystal violet, leuco crystal violet. 2 Normative referencesThe following documents are essential for the application of text files. For dated references, only the dated version applies to this article Pieces. For undated references, the latest version (including all amendments) applies to this document. GB/T 6682 Analytical laboratory water specifications and test methods.3 Method summaryThe malachite green, leuco malachite green, crystal violet, and leuco crystal violet residues in the sample were extracted with acetonitrile, neutral alumina and cation exchange. Purification by solid-phase extraction column, detection by liquid chromatography-mass spectrometry/mass spectrometry, and quantification by internal standard method.4 reagent materialsUnless otherwise specified, the reagents used are of analytical grade, and the water is deionized water, which meets the requirements of GB/T 6682. 4.1 Acetonitrile. Chromatographic grade. 4.2 Methanol. chromatographic grade. 4.3 Dichloromethane. chromatographic grade. 4.4 Formic acid. chromatographic grade. 4.5 Ammonium acetate. chromatographic grade. 4.6 Hydroxylamine hydrochloride. 4.7 p-Toluenesulfonic acid. 4.8 Ammonia water. 25 to 28%. 4.9 Neutral alumina column. 1g/3mL, activated with 5mL acetonitrile before use. 4.10 Cation exchange column. 60mg/3mL. Before use, activate with 3mL methanol and 3mL 2% formic acid solution. 4.11.25g/mL hydroxylamine hydrochloride solution. Weigh 25g hydroxylamine hydrochloride, dissolve in water and make up to 100mL. 4.12 1mol/L p-toluenesulfonic acid solution. Weigh 17.2g of p-toluenesulfonic acid, dissolve in water and make up to 100mL. 4.13 5mol/L ammonium acetate solution. Accurately weigh 0.435g of ammonium acetate (accurate to 0.01g), dissolve in water and make up to 1L. 4.14 2% formic acid solution. accurately transfer 2mL of formic acid, and make up to 100mL with water. SN/T 5116-201919 ????? ????, ????, ?? 4.15 Methanol solution containing 5% ammonia water. Accurately remove 5mL of ammonia water and make up to 100mL with methanol. 4.16 5mol/L ammonium acetate solution, containing 0.1% formic acid. accurately weigh 0.435g of ammonium acetate (accurate to 0.01g), add 1mL of formic acid, Dissolve in water and make up to 1L. 4.17 Acetonitrile containing 0.1% formic acid; accurately transfer 1mL of formic acid and make up to 1L with acetonitrile. 4.18 Standards. Malachite Green (MalachiteGreen CAS Number. 2437-29-8), D5-Malachite Green (D5-Malachite Green), Hidden Color Malachite Green (LeucomalaciteGreenCAS No .. 129-73-7), D6-LeucomacalGreen (D6-LeucomalaciteGreen), Crystal Violet (CAS Number. 548-62-9), D6-Crystal Violet (D6-Crystal Violet), Leuco Crystal Staple Violette CAS number. 603-48-5), D6-leucocrystal Violet, with a purity greater than 98%. 4.19 Preparation of standard working solution. Weigh 0.01g (accurate to 0.0001g) malachite green, leucomalachite green, crystal violet, cryptic respectively Color crystal violet standard, dissolved with acetonitrile and made up to 10mL, each standard stock solution concentration is 1mg/mL; standard stock solution Store in a refrigerator at -18 ° C, protected from light, with a validity of 6 months. Take appropriate amount of the above standard stock solution, dilute with acetonitrile solution step by step, prepare It becomes a standard working solution of 100ng/mL and is stored in a refrigerator at 4 ° C, protected from light, with a validity of one month. 4.20 Preparation of internal standard standard working solution. Weigh 0.01g (accurate to 0.0001g) D5-malachite green, D6-cryptocachite green, D6-Crystal Violet and D6-Crystalline Violet Crystal Violet Standards were dissolved in acetonitrile and made up to 10 mL. 1mg/mL; The standard stock solution is stored in a refrigerator at -18 ° C, protected from light, and has a validity of 6 months. Transfer the appropriate amount of the internal standard storage It is ready to dissolve. It is diluted with acetonitrile solution step by step to prepare an internal standard working solution of 100ng/mL. It is stored in a refrigerator at 4 ° C, protected from light. 1 month. 4.21 Drawing of standard curve. Absorb a certain amount of standard working solution and internal standard working solution as needed, and prepare with blank solution It becomes a standard curve, and it is equipped immediately before use. 4.22 Filter. 0.22 μm organic phase filter.5 instruments and equipment5.1 Liquid chromatography-mass spectrometry/mass spectrometer. equipped with electrospray ionization source (ESI). 5.2 Analytical balance. accurate to 0.0001 g and 0.01 g. 5.3 Homogenizer. 5.4 Centrifuge. The speed can reach 10,000r/min... 5.5 Ultrasonic generator. 5.6 Rotary evaporator. 5.7 Solid-phase extraction device.6 Sample preparation and storageTake a representative part of the sample about 500g, divided into two parts, and put them into clean containers as samples, sealed, and marked. Store the sample at -18 ° C, protected from light. Note. During sample preparation, sample contamination or changes in residue content should be prevented. 7 pre-processing steps 7.1 Extraction and purification of fish, shrimp, crab and shellfish samples 7.1.1 Extraction Weigh 5g sample (accurate to 0.01g) into a 50mL plastic centrifuge tube, add 20μL internal standard working solution, and add 10mL B SN/T 5116-201919 ????? ????, ????, ?? Nitrile, homogenate for 2 min (1000r/min), ultrasonic extraction for 10min, centrifugation for 3min (3,000r/min), take the supernatant, and add 10mL of ethyl acetate Nitrile, repeated extraction once, make up to 25mL with acetonitrile to be purified. 7.1.2 Purification Take 5mL of the extraction solution onto a neutral alumina column, rinse the solid-phase extraction column with 4mL of acetonitrile, collect the effluent, and blow at 45 ° C under nitrogen. About 0.5mL, the residual solution was made up to 1mL with acetonitrile, and 1mL of ammonium acetate solution was added to mix, passed through a 0.22μm microporous filter membrane, and analyzed on the machine. 7.2 Extraction and purification of other samples 7.2.1 Extraction Weigh 2g sample (accurate to 0.01g) into a 50mL plastic centrifuge tube, add 20μL internal standard working solution, and then add 1mL hydroxylamine hydrochloride solution, 2mL p-toluenesulfonic acid solution, 2mL ammonium acetate buffer solution, 40mL Acetonitrile solution, homogenate 2 min (1000r/min), ultrasonically extract 10min, centrifuge 3min (3,000r/min), transfer the supernatant to a 250mL separatory funnel, The residue was repeatedly extracted with 20 mL of acetonitrile, and the supernatants were combined. Add 30 mL of dichloromethane and 35 mL of water to the separatory funnel and shake for 2 min. The layers were allowed to stand still, and the organic phase layer was collected in a pear-shaped bottle, and extracted once with 20 mL of dichloromethane. The organic layers were combined, and evaporated at 45 ° C to near Dry and dissolve the extract with 6 mL of acetonitrile three times (2 mL each). 7.2.2 Purification Connect the neutral alumina column in series above the cation exchange column, take 3mL of the extraction solution onto the neutral alumina column, pass through the column in turn, and use 2mL Rinse with methanol. Discard the neutral alumina column. Rinse the cation exchange column with 3 mL of 2% formic acid solution and 3 mL of methanol. Discard the flow. liquid. Eluted with 10mL methanol solution containing 5% ammonia water, collected the eluent, blown to dryness with nitrogen, 1mL acetonitrile. water 1. 9 (v/v) volume, and 0.22μm filter membrane, analysis on the machine.8 determination8.1 Liquid chromatography conditions 8.1.1 Chromatographic column. C184.6mm × 50mm, particle size 1.8μm, or equivalent performance; 8.1.2 Mobile phase. A. 5 mmol/L ammonium acetate + 0.1% formic acid, B. acetonitrile + 0.1% formic acid; 8.1.3 Flow rate. 400 μL/min; gradient elution procedure is shown in Table 1; 8.1.4 injection volume. 20 μL; 8.1.5 column temperature. normal temperature. Table 1 Gradient elution procedure Time (min) 5 mmol/L ammonium acetate with 0.1% formic acid (mobile phase A) Acetonitrile with 0.1% formic acid (Mobile phase B) 0.0 95 5 5 1.5 40 60 3.0 0 100 10.0 0 100 10.5 95 5 5 15.0 95 5 SN/T 5116-201919 ????? ????, ????, ?? 8.2 Mass spectrometry conditions 8.2.1 Ion source. Electrospray ESI positive ion mode; 8.2.2 Scanning mode. multiple response monitoring MRM; 8.2.3 See Appendix A for other reference mass spectrometry conditions. 8.3 Quantitative determination According to the content of the test substance in the sample, select a standard working solution with a suitable response value for chromatographic analysis. The standard curve working solution should have 5 concentration levels. The response values of malachite green, leucomalachite green, crystal violet, and cryptic crystal violet in the matrix standard working solution and the test solution are all It should be within the linear response range of the instrument. Reference retention of chert green, leuco malachite green, crystal violet, leuco crystal violet under the above chromatographic conditions See Appendix A for time. See Appendix B for the multiple reaction monitoring chromatograms of the analytes in the standard solution. 8.4 Qualitative determination Determine the sample and standard working solution according to liquid chromatography-mass spectrometry/mass spectrometry conditions. The relative abundance of the qualitative ion pair is expressed by the intensity percentage relative to the abundance of the strongest ion. It should be equivalent to the standard concentration. The relative abundance of the solution is consistent, and the allowable deviation of the relative abundance does not exceed the range specified in Table 2. Table 2 Maximum allowable deviation of relative ion abundance during qualitative confirmation Relative ion abundance > 50% > 20% to 50% > 10% to 20% ≤ 10% Allowable relative deviation ± 20% ± 25% ± 30% ± 50% 8.5 Blank test Except for not adding samples, all operations are carried out according to the above operation steps. 8.6 Calculation and presentation of results Use a chromatographic data processor or formula (1) to calculate the residual content of malachite green, leucomalachite green, crystal violet, and cryptic crystal violet in the sample. Amount, the calculation result needs to deduct the blank value. In the formula. X --- the content of the component to be measured in the sample, the unit is micrograms per kilogram (μg/kg); 犮 --- the concentration of the component in the standard working solution, the unit is nanograms per milliliter (ng/mL) ; A 狊 --- the peak area of the component to be measured in the standard working solution; A --- the peak area of the component to be measured in the test solution; 犞 --- total sample dilution volume, unit is ml; SN/T 5116-201919 ????? ????, ????, ?? 九 Lower limit of detection and recovery rate 9.1 Low limit of determination (LOQ) The lower limit of determination of malachite green, leucomalachite green, crystal violet, and crystal violet in fish, shrimp, crab, and shellfish was 0.5 μg/kg. The method applies malachite green, leucomalachite green, crystal violet, and cryptic crystal violet to fish meal, plant-derived feed, compound feed, and concentrated feed. The lower limit of determination is 1 μg/kg. The lower limit of determination of malachite green, leucomalachite green, crystal violet and crystal violet in feed additives and concentrate supplements by this method is 10μg/kg. 9.2 Recovery rate Using this method, fish, shrimp, crab, shellfish, fish meal, plant-based feed, concentrated feed, compound feed, feed additives, concentrate supplements, etc. The matrix was subjected to an additional recovery test. See Appendix C for the recovery rates of malachite green, leucomalachite green, crystal violet, and leuco crystal violet. SN/T 5116-201919 ????? ????, ????, ??Appendix A(Informative appendix) Parameters of tandem quadrupole mass spectrometer (1) Mass spectrometer parameters. a) Electrospray voltage (IS). positive mode. 5500V. b) Atomizing gas pressure (GSI); 380kPa (50PSi). c) Curtain gas pressure (CUR). 172 kPa (25 Psi). d) Auxiliary gas flow rate (GS2). 310kPa (45PSi). e) Ion source temperature (TEM). 500 ° C. f) Monitor ion pairs, collision voltage (CE), and de-clustering voltage (DP) as shown in Table A. 1 shown. Table A. 1 Qualitative, quantitative ion pair, collision energy (CE), malachite green, leucomalachite green, crystal violet, crystal violet De-clustering voltage (DP) reference value and retention time Qualitative ion pairs of compounds (m/ｚ) De-clustering voltage (DP) (V) Bang gas energy (CE) (V) Reference retention time (Min) Malachite green 329.4/311.3/3329.4/208.2 47 2.88 Hidden Malachite Green 331.2/239.2/2331.2/2315.3 37 4.65 Crystal Violet 372.4/356.3. 377.23.4/328.3 50 3.22 Leuco crystal violet 374.3/33588.4374.3/3233.3. 44 4.50 D5-malachite green 334.4/318.3 3110 50 2.86 D6-Hidden malachite green 337.4/240.3 3104 45 4.61 D6-Crystal Violet 378.4/362.2 2 115 51 3.21 D6- leuco crystal violet 380.4/364.5 5130 45 4.41 (1) Non-commercial statement. The reference mass spectrometry conditions listed in Appendix A were completed on an ABAPI 5500 mass spectrometer. The test instrument types listed here are only It is provided for reference and does not involve commercial purposes. Standard users are encouraged to try different manufacturers or models of instruments. SN/T 5116-201919 ????? ????, ????, ??Appendix B(Informative appendix) Multiple reaction monitoring (MRM) chromatograms of malachite green, leucomalachite green, crystal violet, and leuco crystal violet Figure B. 1 Multi-reaction monitoring (MRM) chromatograms of malachite green, leucomalachite green, crystal violet, and leuco crystal violet SN/T 5116-201919 ????? ????, ????, ??Appendix C(Informative appendix) Additive recovery range of malachite green, leucomalachite green, crystal violet, cryptic crystal violet in food animals and feed Table C. 1 The range of recovery rates of malachite green, leucomalachite green, crystal violet, and leuco crystal violet in food animals and feed Matrix Compound Malachite green cryptic malachite green crystal violet cryptic crystal violet Add concentration (Μg/kg) Recovery rate range Add concentration (Μg/kg) Recovery rate range Add concentration (Μg/kg) Recovery rate range Add concentration (Μg/kg) Recovery rate range 0.5 84.6 to 115 0.5 5 86.4 to 1 17 0.5 5 84.2 to 112 0.5 5 84.4 to 116 1 29.2 to 112 1 900.6 to 1119 1 91.2 to 112 1 19.6 to 112 2 100 ～ 112 2 97.5 ～ 112 2 97.5 ～ 110 2 97.5 ～ 11 2 5 98.0 to 112 5 98.2. To 103 5 98.4 to 105 5 98. 8 to 105 0.5 84.2 to 116 0.5 5 84.6 to 112 0.5 54.2 to 115 0.5 5 82.6 to 116 1 87.7 to 113 1 88.2 to 112 1 89.4 to 109 1 900.2 to 110 2 91.5 to 103 2 94.0 to 111 2 92.5 to 107 2 92.0 to 106 5 97.4 to 105 5 94.0 to 102 5 98. 8 to 104 5 97. 2 to 107 0.5 83.4 to 106 0.5 5 84.2 to 112 0.5 50.8 to 108 0.5 5 84.2 to 115 1 89.9 to 112 1 29.2 to 1109 1 91.1 to 112 1 192.3 to 112 2 97.5 to 112 2 93.5 to 106 2 96.0 to 107 2 97.0 to 107 5 97.8 to 104 5 98. 8 to 103 5 97.8 to 102 5 96.2 to 103 0.5 86.8 ~ 117 0.5 0.5 88.6 ~ 112 0.5 84.2 ~ 112 0.5 84.2 ~ 108 196.11 ~ 112 1 95.4 ~ 115 1 92.1 ~ 110 1 195.4 ~ 112 2 96.5 to 108 2 95.5 to 106 2 89.5 to 102 2 94.5 to 111 5 98.2 to 104 5 98.0 to 104 5 97.4 to 105 5 5 97.2. To 103 Fish meal 1 87.5 to 114 1 87.1 to 112 1 88.4 to 112 1 88.1 to 111 2 92.5 to 111 2 92.0 to 108 2 92.0 to 108 2 94.0 to 111 10 99.6 to 103 10 98.8 to 103 10 98.4 to 102 10 98.6 to 100 Plant source Sex feed 1 85.0 to 111 1 84.3 to 11 1 1 84.3 to 112 1 89.5 to 11 1 2 900.0 to 107 2 91.5 to 11 2 2 87.0 to 106 2 89.5 to 100 10 99.1 to 104 10 99.9 to 102 10 10 99.1 to 102 10 99.4 to 111 Compound feed 1 84.5 to 113 1 87.9 to 112 1 88.4 to 112 1 88.1 to 111 2 89.0 to 111 2 92.0 to 111 2 94.0 to 113 2 91.5 to 111 10 98.0 to 103 10 99.8 to 103 10 99.2 to 102 10 97.3 to 103 range Concentrated feed 1 83.2 to 112 1 85.6 to 112 1 82.3 to 111 1 89.2 to 112 2 92.0 to 111 2 91.0 to 105 2 93.0 to 111 2 92.5 to 105 10 98.4 to 102 10 99.2 to 103 10 10 99.6 to 104 10 98.9 to 102 feed additive 10 82.3 to 106 10 10 98.4 to 103 10 99.8 to 104 10 96.8 to 104 20 97.6 to 102 20 99.7 to 106 20 10 100 to 108 20 99.6 to 106 100 99.0 ～ 101 100 98.4 ～ 101 100 96.4 ～ 103 100 98.2 ～ 102 Concentrate Supplement 10 81.2 to 114 10 10 81.3 to 113 10 10 82.1 to 116 10 81.2 to 110 20 88.0 to 11 20 20 87.5 to 11 20 20 89.5 to 11 20 20 96. 7 to 112 100 96.7 to 104 100 98.5 to 103 100 98.3 to 104 100 98.1 to 104 SN/T 5116-201919 ????? ????, ????, ?? ......Tips & Frequently Asked Questions:Question 1: How long will the true-PDF of SN/T 5116-2019_English be delivered?Answer: Upon your order, we will start to translate SN/T 5116-2019_English as soon as possible, and keep you informed of the progress. 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