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GB 5009.111-2016 English PDF

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GB 5009.111-2016: [Including 2025XG1] Determination of deoxynivalenol in food -- High performance liquid chromatographic method with immunoaffinity column clean-up
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GB 5009.111: Evolution and historical versions

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GB 5009.111-2016English489 Add to Cart 4 days [Need to translate] [Including 2025XG1] Determination of deoxynivalenol in food -- High performance liquid chromatographic method with immunoaffinity column clean-up Valid GB 5009.111-2016
GB/T 5009.111-2003English319 Add to Cart 3 days [Need to translate] Determination of deoxynivalenol in cereal and cereal products Obsolete GB/T 5009.111-2003

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Basic data

Standard ID GB 5009.111-2016 (GB5009.111-2016)
Description (Translated English) [Including 2025XG1] Determination of deoxynivalenol in food -- High performance liquid chromatographic method with immunoaffinity column clean-up
Sector / Industry National Standard
Classification of Chinese Standard X04
Word Count Estimation 23,211
Date of Issue 2016-12-23
Date of Implementation 2017-06-23
Older Standard (superseded by this standard) GB/T 23503-2009; GB/T 5009.111-2003; SN/T 1571-2005
Regulation (derived from) National Health and Family Planning Commission Notice No.17 of 2016
Issuing agency(ies) National Health and Family Planning Commission of the People's Republic of China, State Food and Drug Administration

GB 5009.111-2016: [Including 2025XG1] Determination of deoxynivalenol in food -- High performance liquid chromatographic method with immunoaffinity column clean-up


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Determination of deoxynivalenol in food - High performance liquid chromatographic method with immunoaffinity column clean-up National Standards of People's Republic of China National Food Safety Standard Foods deoxynivalenol bacteria enolase Determination and acetylated derivatives Issued on. 2016-12-23 2017-06-23 implementation National Health and Family Planning Commission People's Republic of China China Food and Drug Administration released

Foreword

This standard replaces GB/T 5009.111-2003 "cereal and cereal products deoxynivalenol bacteria enol determination", GB/T 23503- 2009 "Food in deoxynivalenol bacteria enolase measurement immunoaffinity chromatography HPLC purification", SN/T 1571-2005 "Importers and deoxynivalenol in cereals in liquid chromatography test method." This standard compared with GB/T 5009.111-2003, the main changes are as follows. --- The name changed to the standard test "national food safety standards in food deoxynivalenol bacteria enolase and acetylated derivatives set"; --- Increasing the scope of application of the method; --- Increased DETERMINATION deoxynivalenol bacteria enol acetylated derivatives; --- Increased isotope dilution liquid chromatography - tandem mass spectrometry; --- Increasing the solid-phase extraction (SPE) of the pre-treatment; --- Increased immunoaffinity column clean - high performance liquid chromatography; --- Increased commercialization immunoaffinity column evaluate technical parameters required. National Food Safety Standard Foods deoxynivalenol bacteria enolase Determination and acetylated derivatives

1 Scope

This standard specifies the method for determination of deoxynivalenol in food fungus alcohol and acetylated derivatives. The first method is by isotope dilution liquid chromatography - tandem mass spectrometry for cereal and cereal products, wine, soy sauce, vinegar, soy sauce and butter products off Oxygen snow rot Fusarium enol, 3-acetyl deoxynivalenol and 15-acetyl alcohol bacteria deoxynivalenol fungi alcohol measurement. The second method is immunoaffinity chromatography and HPLC purification, suitable for grain and its products, wine, soy sauce, vinegar, soy sauce and butter products Determination of deoxynivalenol bacteria enolase. The third method of thin layer chromatography assay, a fourth method of ELISA screening method for cereals and products deoxynivalenol bacteria Determination of alcohol. The first method isotope dilution liquid chromatography - tandem mass spectrometry Principle 2 Sample of deoxynivalenol fungi alcohol, 3-acetyl deoxynivalenol and 15-acetyl alcohol bacteria deoxynivalenol fungi with water and alcohol A mixed solution of acetonitrile extraction, the supernatant after solid phase extraction columns or immuno-affinity column purification, concentration, volume, and after filtration, high pressure liquid chromatography Spectral separation, tandem mass spectrometry, isotope internal standard.

3 Reagents and materials

Unless otherwise indicated, the methods used were of analytical grade reagents and water as a water GB/T 6682 regulations. 3.1 Reagents 3.1.1 acetonitrile (CH3CN). chromatography. 3.1.2 Methanol (CH3OH). chromatography. 3.1.3 n-hexane (C6H14). 3.1.4 Ammonia (NH3 · H2O). 3.1.5 formic acid (HCOOH). 3.1.6 Nitrogen (N2). purity ≥99.9%. 3.2 reagent preparation 3.2.1 acetonitrile - water (8416). amount of 160mL of water was added to 840mL of acetonitrile and mix. 3.2.2 acetonitrile saturated solution of hexane. amount of n-hexane in 200mL 250mL separatory funnel, adding a small amount of acetonitrile, shake vigorously A few minutes, standing layer, discarding the lower acetonitrile layer, that is, too. 3.2.3 Methanol - water (595). amount of 5mL of methanol was added to 95mL of water, and mix. 3.2.4 0.01% aqueous ammonia solution. Take 100μL aqueous ammonia was added to 1000mL of water, and mix (for ion source ESI- mode is used). 3.2.5 0.1% formic acid solution. Take 1mL formic acid is added to 1000mL of water, and mix (for ESI ion source mode is used). 3.3 Standard 3.3.1 deoxynivalenol bacteria enolase (DON, C15H20O6, CAS number. 51481-10-8). purity ≥99%, or certified by the state and granted Standards Standards certificate. 3.3.2 3-acetyl deoxynivalenol bacteria enolase (3-ADON, C17H22O6, CAS number. 50722-38-8). purity ≥99%, or by the State Certified and standard substance substance certificate. 3.3.3 15- acetyl deoxynivalenol bacteria enolase (15-ADON, C17H22O6, CAS number. 88337-96-6). purity ≥99%, or by State Hotels certified and reference material reference material certificate. 3.3.4 13C15- deoxynivalenol fungi alcohol standard solution isotopes (13C-DON, 13C15H20O6). 25μg/mL, the purity of ≥99%. 3.3.5 13C17-3- acetyl - deoxynivalenol fungi alcohol standard solution isotopes (13C-3-ADON, 13C17H22O6). 25μg/mL, purity ≥99%. 3.4 Standard Solution 3.4.1 Standard stock solution (100μg/mL). Weigh DON, 3-ADON and 15-ADON1mg (accurate to 0.01mg), points Do not dissolved in acetonitrile and set the volume to 10mL. The solution was transferred to a vial, sealed and stored at -20 ℃, valid for 1 year. 3.4.2 mixed standard working solution (10μg/mL). Imbibe 100μg/mLDON, 3-ADON and 15-ADON stock standard solution Each 1.0mL in the same 10mL volumetric flask, add acetonitrile to volume. Sealed and stored at -20 ℃, valid for six months. 3.4.3 Mixed isotope internal standard working solution (1μg/mL). Imbibe 13C15-DON and 13C17-3-ADON isotope internal standard (25μg/mL) each 1mL to 25mL same flask, add acetonitrile to volume. Sealed and stored at -20 ℃, valid Six months. 3.4.4 standard series working solution. accurate Pipette the appropriate amount of mixed standard working solution and mixed isotope internal standard working solution with the initial mobile phase Formulated as 10ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 160ng/mL, 320ng/mL, mixed standard 640ng/mL of Series, in which the isotope internal standard at a concentration of 100ng/mL. Standard series solution at 4 ℃ preservation, valid 7d.

4 instruments and equipment

4.1 Liquid chromatography - tandem mass spectrometry. a charged ion source. 4.2 Electronic balance. a sense of the amount of 0.01g and 0.00001g. 4.3 high-speed grinder. Speed 10000r/min. 4.4 homogenizer. 4.5 Screen. 0.5mm ~ 1mm aperture. 4.6 Ultrasound/or a vortex shaker. 4.7 nitrogen blowing instrument. 4.8 high-speed centrifuge. speed of not less than 12000r/min. 4.9 Pipette. Range 10μL ~ 100μL and 100μL ~ 1000μL. 4.10 solid phase extraction device. 4.11 Universal SPE. both hydrophilic group (group pyrrolidone) and hydrophobic groups (divinyl benzene) packing solid phase adsorbents Extraction column, 200mg, 6mL, or equivalent person. 4.12 DONs special solid phase clean-up columns, or equivalent person. 4.13 deoxynivalenol bacteria enol immunoaffinity column. Column capacity ≥1000ng (column size and column recovery verification method, see A.2). 4.14 aqueous phase porous membrane. 0.22μm. Step 5 Analysis 5.1 Sample Preparation 5.1.1 cereals and cereal products. taking at least 1kg sample was pulverized with a high-speed grinder and sieved to a particle size of less than 0.5mm ~ 1mm Aperture test sieve, mixed uniformly reduced to 100g, stored in a vial, sealed and stored for detection. 5.1.2 Alcohol. Take bulk wine at least 1L, for bags, bottles and other packaging samples taken at least three packaging (or the same batch number), all the liquid Body with a sample container after homogenous mixing machine, reduced to 100g (mL) stored in the vial, sealed and stored for detection. Including two Wine samples of carbon monoxide before you use the refrigerator at 4 ℃ for 30min, filtered before use or ultrasonic degassing. 5.1.3 soy sauce, vinegar, soy sauce and butter products. taking at least 1L samples for bags, bottles and other packaging samples taken at least three packaging (or the same batch Number), after all the liquid sample in a container with a homogenizer mix, reduced to 100g (mL) stored in the vial, sealed for Detection. 5.2 Sample Extraction 5.2.1 cereal and its products. Weigh 2g (accurate to 0.01g) sample was 50mL centrifuge tube was added 400μL mixed isotope internal standard After shaking the working fluid mixed stand 30min. Added 20.0mL of acetonitrile - water (8416), placed in an ultrasonic/or a vortex shaker Ultrasonic or oscillation 20min. 10000r/min centrifugal 5min, the supernatant was collected A spare in a clean container. 5.2.2 Alcohol. Weigh 5g (accurate to 0.01g) sample was 50mL centrifuge tube was added 200μL mixed isotope internal standard working solution vibrator After mixing swing standing 30min, with acetonitrile dilute to 10mL, mix, placed in an ultrasonic/Vortex shaker or ultrasonic or oscillation 20min. 10000r/min centrifugal 5min, the supernatant was collected B alternate in a clean container. 5.2.3 soy sauce, vinegar, soy sauce and butter products. Weigh 2g (accurate to 0.01g) sample was 50mL centrifuge tube, mixed with bits added 400μL Su internal standard working solution After shaking the mixing stand 30min. Added 20.0mL of acetonitrile - water (8416), placed in an ultrasonic/Vortex Or shaker or ultrasonic oscillations 20min. 10000r/min centrifugal 5min, the supernatant was collected C alternate in a clean container. 5.3 Sample purification NOTE. The following sample purification process, according to the actual situation, choose one of the methods can be. 5.3.1 Universal solid-phase extraction (SPE) A take 5mL supernatant or supernatant or supernatant B C a 50mL centrifuge tube, 10mL of acetonitrile was added a saturated solution of n-hexane Liquid vortex mixing 2min, after 5000r/min centrifugal 2min, discard the layer of n-hexane at 40 ℃ ~ 50 ℃ under dry nitrogen, was added 4mL The residue was dissolved sufficient water to be purified. The SPE is connected to the solid phase extraction, washed with methanol and 3mL 3mL water activated balance. The above-mentioned water 4mL reconstituted The liquid column to control the flow rate drops 1 to 2 drops per second. With 3mL water, 1mL5% methanol - aqueous solution followed by rinsing thoroughly drained after the column. use 4mL methanol, after collecting all the eluent at 40 ℃ ~ 50 ℃ under dry nitrogen. The initial mobile phase was added 1.0mL residue, Vortex 10s, with 0.22μm filter membrane in an injection vial until injection. 5.3.2 DONs special solid phase purification column purification A glass tube take 8mL supernatant or supernatant or supernatant B C DONs dedicated to solid phase clean-up columns will fill the purification column Feeding tube inserted into a glass tube and slowly push the filler tube to purify liquid precipitation, pipette 5mL decontamination liquid at 40 ℃ ~ 50 ℃ under dry nitrogen. plus The initial mobile phase into 1.0mL residue was vortexed 10s, with 0.22μm filter membrane in an injection vial, until injection. Note. The operational aspects of the use of different vendors DONs special type of solid-phase purification column, in the sample, purification may be slightly different, can be carried out in accordance with the specification requirements operating. 5.3.3 immunoaffinity column purification In advance of recovering cryopreserved immunoaffinity column to room temperature. Pipette accurately 5mL supernatant A or B supernatant or supernatant C, at 40 ℃ ~ 50 ℃ under dry nitrogen, was added 2mL water sufficient The residue was dissolved, to be immunoaffinity column within the original liquid flow after doing the above sample solution was transferred to a glass syringe barrel. The air pressure injection pump and glass Emitter connected to adjust the dripping speed, control sample solution with 1 drop per second flow rate through the immunoaffinity column, until the air into the affinity column. use 5mLPBS buffered saline solution and 5mL water leaching has immunoaffinity column at a flow rate of approximately 1 drop to 2 drops per second, until the air and into the pro-Note The discarded all effluent, drained cartridge. Accurate added 2mL methanol affinity column, under the control of one drop per second, the speed drops to collect all the eluent to a test tube, at 50 ℃ Firmly eluent blown with nitrogen to nearly dry, add 1.0mL initial phase flow, vortex 30s dissolve the residue, 0.22μm filter membrane, The filtrate was collected in an injection vial to prepare injections. Note. Use different vendors immunoaffinity column, the sample in the sample, operational leaching and elution may be slightly different, should be carried out in accordance with specification requirements operating. 5.4 Liquid chromatography - tandem mass spectrometry reference conditions (refer to one of these methods can be based on the actual situation) 5.4.1 Ion Source mode. ESI Liquid chromatography - mass spectrometry reference conditions are listed below. a) LC Column. C18 column (column length 100mm, column diameter 2.1mm; particle size 1.7μm), or equivalent person; b) Mobile phase. A phase. 0.1% formic acid; B phase. 0.1% formic acid - acetonitrile; c) gradient. 2% B (0min ~ 0.8min), 24% B (3.0min ~ 4.0min), 100% B (6.0min ~ 6.9min), 2% B (6.9min ~ 7.0min); d) flow rate. 0.35mL/min; e) Column temperature. 40 ℃; f) Injection volume. 10μL; g) Capillary voltage. 3.5kV; cone voltage. 30V; desolvation temperature. 350 ℃; desolvation gas flow rate. 900L/h; h) deoxynivalenol bacteria enolase and acetylated derivatives thereof MS conditions refer to Table 1. Table 1 deoxynivalenol bacteria enolase and Acetyl Derivatives Mass reference conditions (ESI) Compound name Parent ion (M/z) Cone voltage eV Quantitative ion (M/z) Collision voltage eV Qualifier ion (M/z) Collision voltage eV DON 297 20 249 10 203 16 3-ADON 339 17 231 13 203 13 15-ADON 339 18 137 9 321 7 13C-DON 312 20 263 10 245 16 13C-3-ADON 356 17 245 13 - - Note. 3-ADON and 15-ADON calculated as isomers, 15-ADON optional 13C-3-ADON isotope internal standard as appropriate quantification. 5.4.2 Ion Source mode. ESI- Liquid chromatography - mass spectrometry reference conditions are listed below. a) LC Column. C18 column (column length 100mm, column diameter 2.1mm; particle size 1.7μm), or equivalent person; b) Mobile phase. A phase. 0.01% aqueous ammonia solution; phase B. acetonitrile; c) gradient. 2% B (0min ~ 0.8min), 24% B (3.0min ~ 4.0min), 100% B (6.0min ~ 6.9min), 2% B (6.9min ~ 7.0min); d) flow rate. 0.35mL/min; e) Column temperature. 40 ℃; f) Injection volume. 10μL; g) capillary voltage. 2.5kV; cone voltage. 45V; desolvation temperature. 500 ℃; desolvation gas flow rate. 900L/h; h) deoxynivalenol bacteria enolase and acetylated derivatives thereof MS conditions refer to Table 2. Table 2 deoxynivalenol bacteria enolase and Acetyl Derivatives Mass reference conditions (ESI-) Compound name Parent ion (M/z) Cone voltage eV Quantitative ion (M/z) Collision voltage eV Qualifier ion (M/z) Collision voltage eV DON 295 14 265 12 138 18 3-ADON 337 12 307 10 173 12 15-ADON 337 12 150 12 219 12 13C-DON 310 16 279 12 145 14 13C-3-ADON 354 18 323 14 230 18 Note. 3-ADON and 15-ADON calculated as isomers, 15-ADON optional 13C-3-ADON isotope internal standard as appropriate quantification. Ion scan of each compound, multiple reaction monitoring (MRM) ion channel FIG see Figure B.1 ~ Figure B.8. 5.5 Qualitative Determination The target compound in the sample peak retention time compared with the corresponding standard chromatographic retention time, the range of variation should be ± 2.5% within. MS qualifier ion for each compound should appear, including at least a parent ion and two ions, and detect the same batch of The same compound, the relative abundance of the two sub-target in the sample ion compound concentration equivalent ratio compared to the standard solution, which is not allowable deviation Table 3 exceeds the range specified. The maximum permissible relative ion abundances Table 3 Qualitative deviations Relative ion abundances > 50% 20% ~ 50% 10% ~ 20% ≤10% Permissible relative deviation of ± 20% ± 25% ± 30% ± 50% 5.6 standard curve Under 5.4 liquid chromatography tandem mass spectrometry analysis conditions, the standard solution series from low to high concentration injection testing to DON, 3- Peak area ratio ADON and 15-ADON peaks corresponding to each of the internal standard peak - concentration plotted standard curve regression equation, which Linear correlation coefficient should be greater than 0.99. 5.7 Determination of the sample solution Take the test solution injection treatment was 5.2, 5.3, calculate the mass concentration of the target substance in the test solution of the internal standard method, the sample is calculated by 6 Content of the analyte. Analyte response test solution should be within the linear range of the standard curve over the linear range should be appropriate to reduce the amount of sampling After the re-determination. 5.8 blank test Except that no sample addition, according to the steps 5.3 and 5.4 of the blank experiment. Confirm contain interfering substances should not be tested components.

6 expression analysis

Sample DON, 3-ADON or 15-ADON content according to formula (1). X = ρ × V1 × V3 × 1000 V2 × m × 1000 (1) Where. X --- sample DON, 3-ADON or 15-ADON content in micrograms per kilogram (μg/kg); ρ --- sample DON, 3-ADON or 15-ADON internal standard method in accordance with the corresponding concentration of the standard curve, the unit is Nanograms per milliliter (ng/mL); Vl --- sample extract volume in milliliters (mL of); V3 --- the final volume of the sample, in milliliters (mL); 1000 --- conversion factor; --- For purifying V2 of the dispensing volume, in milliliters (mL of); Sample weight m --- sample in grams (g). The results to three significant figures.

7 precision

Two independent determination results under the absolute difference in repeatability condition must not exceed 23% of the arithmetic mean.

8 Other

When weighed and cereal products, wine, soy sauce, vinegar, soy sauce and butter products sample 2g, methods deoxynivalenol bacteria enolase, 3-B Acyl deoxynivalenol fungi alcohol, 15-acetyl deoxynivalenol bacteria enolase detection limit of 10μg/kg, the limit of quantification of 20μg/kg. When the sample liquor weighed 5g, methods deoxynivalenol fungi alcohol, 3-acetyl deoxynivalenol fungi alcohol, 15-acetyl-deoxy Snow rot Fusarium enol detection limit of 5μg/kg, the limit of quantification of 10μg/kg. The second method of immune affinity chromatography and HPLC purification Principle 9 Sample of deoxynivalenol fungi alcohol extracted with water, after immunoaffinity column purified by high performance liquid chromatography - ultraviolet detector measurement, External standard. 10 Reagents and materials Unless otherwise indicated, the methods used were of analytical grade reagents and water as a water GB/T 6682 regulations. 10.1 Reagents 10.1.1 methanol (CH3OH). chromatography. 10.1.2 acetonitrile (CH3CN). chromatography. 10.1.3 polyethylene glycol [molecular weight of 8000, HO (CH2CH2O) nH]. 10.1.4 Sodium chloride (NaCl). 10.1.5 disodium hydrogen phosphate (Na2HPO4). 10.1.6 potassium dihydrogen phosphate (KH2PO4). 10.1.7 potassium chloride (KCl). 10.1.8 hydrochloric acid (HCl). 10.2 reagent preparation 10.2.1 phosphate buffer solution (hereinafter referred to as PBS). Weigh 8.00g of sodium chloride, 1.20 g of disodium hydrogenphosphate, potassium dihydrogenphosphate 0.20 g, 0.20g potassium chloride, dissolved with 900mL of water, adjusted to pH 7.0 with hydrochloric acid, water volume to 1000mL. 10.2.2 methanol - water (2080). amount of 200mL of methanol was added to 800mL of water, and mix. 10.2.3 acetonitrile - water (1090). Measure 100mL acetonitrile was added to 900mL of water, and mix. 10.3 Standards Deoxynivalenol bacteria enolase (C15H20O6, CAS number. 51481-10-8). purity ≥99%, or granted by the National Certification and Standards Standard Substance certificate. 10.4 Standard Solution 10.4.1 Standard stock solution (100μg/mL). Weigh deoxynivalenol bacteria enol 1mg (accurate to 0.01mg), dissolved in acetonitrile and Volume to 10mL. The solution was transferred to a vial, sealed and stored at -20 ℃, valid for 1 year. 10.4.2 series of standard working solution. accurate Pipette the appropriate amount deoxynivalenol fungi alcohol standard stock solution was diluted with an initial phase flow, preparation To 100ng/mL, 200ng/mL, 500ng/mL, 1000ng/mL, 2000ng/mL, 5000ng/mL working solution of the standard series, 4 ℃ preservation, valid 7d. 11 instruments and equipment 11.1 High performance liquid chromatograph. equipped with an ultraviolet detector or diode array detector. 11.2 Electronic balance. a sense of the amount of 0.01g and 0.00001g. 11.3 High-speed grinder. Speed 10000r/min. 11.4 Screen. 1mm ~ 2mm aperture. 11.5 Ultrasound/or a vortex shaker. 11.6 nitrogen blowing instrument. 11.7 high-speed centrifuge. speed ≥12000r/min. 11.8 pipettes. Range 10μL ~ 100μL and 100μL ~ 1000μL. 11.9 deoxynivalenol bacteria enol immunoaffinity column. Column capacity ≥1000ng. (Column size and column recovery verification method, see A.2). Note. For different batches affinity column in quality verification before use. 11.10 Glass fiber filter. diameter 11cm, pore size 1.5μm. 11.11 aqueous phase microporous membrane. 0.45μm. 11.12 Polypropylene graduated centrifuge tube. a plug, 50mL. 11.13 glass syringes. 10mL. 11.14 air pressure pump. 12 analysis steps Use different vendors immunoaffinity column, the sample in the sample, operational leaching and elution may be slightly different, should be in accordance with the instructions Requested operation. 12.1 Sample Preparation With 5.1. 12.2 Sample Extraction 12.2.1 cereal and its products. Weigh 25g (accurate to...

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