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GB 5009.240-2023 English PDF (GB 5009.240-2016)

GB 5009.240-2023_English: PDF (GB5009.240-2023)
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GB 5009.240-2023English410 Add to Cart 0--9 seconds. Auto-delivery National food safety standard - Determination of fumonisin in foods Valid GB 5009.240-2023
GB 5009.240-2016EnglishRFQ ASK 7 days [Need to translate] Inspection of grain and oils -- Determination of fumonisin in corn and its products by high liquid chromatography and fluorometer with immunoaffinity column cleanup Obsolete GB 5009.240-2016


BASIC DATA
Standard ID GB 5009.240-2023 (GB5009.240-2023)
Description (Translated English) (National food safety standards Determination of cadmium in food)
Sector / Industry National Standard
Classification of Chinese Standard X09
Word Count Estimation 7,780
Date of Issue 2023-09-06
Date of Implementation 2024-03-06
Issuing agency(ies) National Health Commission of the People's Republic of China, State Administration for Market Regulation
Summary This standard specifies the method for the determination of cadmium in food by graphite furnace atomic absorption spectrometry and inductively coupled plasma mass spectrometry. This standard applies to the determination of cadmium in food.

BASIC DATA
Standard ID GB 5009.240-2016 (GB5009.240-2016)
Description (Translated English) Inspection of grain and oils -- Determination of fumonisin in corn and its products by high liquid chromatography and fluorometer with immunoaffinity column cleanup
Sector / Industry National Standard
Classification of Chinese Standard X09
Word Count Estimation 42,456
Date of Issue 2016-08-31
Date of Implementation 2017-03-01
Older Standard (superseded by this standard) SN/T 1958-2007; SN/T 1572-2005; GB/T 25228-2010
Regulation (derived from) Announcement of the State Administration of Public Health and Family Planning 2016 No.11


GB 5009.240-2023 GB NATIONAL STANDARD OF THE PEOPLE’S REPUBLIC OF CHINA National Food Safety Standard - Determination of Fumonisin in Foods ISSUED ON. SEPTEMBER 6, 2023 IMPLEMENTED ON. MARCH 6, 2024 Issued by. National Health Commission of the People’s Republic of China; State Administration for Market Regulation. Table of Contents Foreword... 3 1 Scope... 4 Method I - Immunoaffinity Column Purification - Post-column Derivatization High- performance Liquid Chromatography... 4 2 Principle... 4 3 Reagents and Materials... 4 4 Instruments and Equipment... 7 5 Analytical Procedures... 7 6 Expression of Analysis Results... 9 7 Precision... 10 8 Others... 10 Method II - High-performance Liquid Chromatography - Tandem Mass Spectrometry ... 10 9 Principle... 10 10 Reagents and Materials... 11 11 Instruments and Equipment... 13 12 Analytical Procedures... 14 13 Expression of Analysis Results... 18 14 Precision... 19 15 Others... 19 Method III - Immunoaffinity Column Purification - Pre-column Derivatization High- performance Liquid Chromatography... 19 16 Principle... 19 17 Reagents and Materials... 19 18 Instruments and Equipment... 22 19 Analytical Procedures... 22 20 Expression of Analysis Results... 25 21 Precision... 25 22 Others... 25 Appendix A Post-column Derivatization - High-performance Liquid Chromatogram 27 Appendix B MRM Mass Chromatogram... 28 Appendix C Pre-column Derivatization - High-performance Liquid Chromatogram. 31 Appendix D Column Capacity and Column Recovery Verification Methods... 32 National Food Safety Standard - Determination of Fumonisin in Foods 1 Scope This Standard specifies the immunoaffinity column purification - post-column derivatization high-performance liquid chromatography, high-performance liquid chromatography - tandem mass spectrometry and immunoaffinity column purification - pre-column derivatization high- performance liquid chromatography for the determination of fumonisin in foods. This Standard is applicable to the determination of fumonisin B1, fumonisin B2 and fumonisin B3 (hereinafter referred to as FB1, FB2 and FB3) in cereals and products, cereal supplementary foods for infants and young children, and vegetable oils. Method I - Immunoaffinity Column Purification - Post- column Derivatization High-performance Liquid Chromatography 2 Principle The specimen is extracted, purified by an immunoaffinity column, separated by C18 reversed- phase chromatography column, derivatized by o-phthalaldehyde, detected by fluorescence detector, and quantified by the external standard method. 3 Reagents and Materials Unless it is otherwise specified, the reagents used in this Method are all analytically pure, and the water is Grade-1 water specified in GB/T 6682. 3.1 Reagents 3.1.1 Methanol (CH3OH). chromatographically pure. 3.1.2 Acetonitrile (CH3CN). chromatographically pure. 3.1.3 Formic acid (HCOOH). chromatographically pure. 3.1.4 Acetic acid (CH3COOH). 3.1.5 Sodium hydroxide (NaOH). 3.1.6 Sodium chloride (NaCl). 3.1.7 Disodium hydrogen phosphate (Na2HPO4). 3.1.8 Potassium dihydrogen phosphate (KH2PO4). 3.1.9 Potassium chloride (KCl). 3.1.10 Borax (Na2B4O7  10H2O). 3.1.11 2-mercaptoethanol (C2H6OS). 3.1.12 o-phthalaldehyde (C8H6O, OPA). 3.1.13 Tween-20 (C58H114O26). 3.1.14 Hydrochloric acid (HCl). 3.2 Preparation of Reagents 3.2.1 Formic acid - water solution (0.1%). accurately transfer-take 1 mL of formic acid, use water to dilute to 1,000 mL and evenly mix it. 3.2.2 Acetonitrile - water solution (50 + 50). respectively measure-take 500 mL of acetonitrile and 500 mL of water, and evenly mix them. 3.2.3 Acetonitrile - water solution (20 + 80). respectively measure-take 20 mL of acetonitrile and 80 mL of water, and evenly mix them. 3.2.4 Methanol - acetic acid solution (98 + 2). accurately transfer-take 2 mL of acetic acid, use methanol to dilute to 100 mL and evenly mix it. 3.2.5 Sodium hydroxide solution (2 mol/L). accurately weigh-take 8.0 g of sodium hydroxide, add 50 mL of water to dissolve it; after cooling, use water to dilute it to 100 mL and evenly mix it. 3.2.6 Phosphate buffer solution (PBS). weigh-take 8.0 g of sodium chloride, 1.2 g of disodium hydrogen phosphate, 0.2 g of potassium dihydrogen phosphate and 0.2 g of potassium chloride; use 980 mL of water to dissolve it, use hydrochloric acid to adjust pH to 7.4, use water to dilute to 1,000 mL and evenly mix it. 3.2.7 Tween-20/PBS solution (0.1%). weigh-take 1.0 g of Tween-20, add phosphate buffer solution, dilute to 1,000 mL and evenly mix it. 3.2.8 Borax solution (0.05 mol/L, pH 10.5). weigh-take 19.1 g of borax, dissolve it in 980 mL of water, use sodium hydroxide solution to adjust pH to 10.5, use water to dilute to 1,000 mL and evenly mix it. 3.2.9 Derivatization solution. weigh-take 0.5 g of o-phthalaldehyde, dissolve it in 20 mL of methanol, use borax solution (0.05 mol/L, pH 10.5) to dilute to 500 mL, add 500 L of 2- mercaptoethanol and evenly mix it. After filtration, put it into a brown bottle; store it at room temperature away from light. It shall remain valid for 1 week. 3.3 Reference Materials 3.3.1 Fumonisin B1 (C34H59NO15, FB1, CAS No.. 116355-83-0), purity  95%, or a standard substance certified by the state and awarded a standard substance certificate. 3.3.2 Fumonisin B2 (C34H59NO14, FB2, CAS No.. 116355-84-1), purity  95%, or a standard substance certified by the state and awarded a standard substance certificate. 3.3.3 Fumonisin B3 (C34H59NO14, FB3, CAS No.. 136379-59-4), purity  95%, or a standard substance certified by the state and awarded a standard substance certificate. 3.4 Preparation of Standard Solutions 3.4.1 Standard stock solutions (100 g/mL). respectively and accurately weigh-take 10 mg (accurate to 0.01 mg) of FB1, FB2 and FB3 into small beakers, use acetonitrile - water solution (50 + 50) to dissolve them, and respectively transfer to 100 mL volumetric flasks. Use acetonitrile - water solution (50 + 50) to reach a constant volume to the scale. Store them at 18 C away from light. They shall remain valid for 6 months. 3.4.2 Mixed standard stock solution. accurately transfer-take 1.0 mL of FB1 standard stock solution, 0.5 mL of FB2 standard stock solution and 0.5 mL of FB3 standard stock solution into the same 10 mL volumetric flask; add acetonitrile - water solution (50 + 50) to dilute to the scale. The mass concentration of FB1 is 10 g/mL, and the mass concentration of FB2 and FB3 is 5 g/mL. Store it at 18 C away from light. It shall remain valid for 6 months. 3.4.3 Mixed standard working solution. accurately transfer-take 1.0 mL of the mixed standard stock solution into a 10.0 mL volumetric flask, add acetonitrile - water solution (50 + 50) to dilute it and reach a constant volume to the scale. The mass concentration of FB1 is 1 g/mL, and the mass concentration of FB2 and FB3 is 0.5 g/mL. Store it at 4 C away from light. It shall remain valid for 6 months. 3.4.4 Mixed standard series of working solutions. accurately transfer-take the mixed standard working solution, use acetonitrile - water solution (20 + 80) to dilute it, and prepare mixed standard series of working solutions respectively with a FB1 mass concentration of 20.0 ng/mL, 80.0 ng/mL, 160 ng/mL, 240 ng/mL, 320 ng/mL, 400 ng/mL and 480 ng/mL, and a FB2 and FB3 mass concentration of 10.0 ng/mL, 40.0 ng/mL, 80.0 ng/mL, 120 ng/mL, 160 ng/mL, 200 ng/mL and 240 ng/mL. Prepare them right before use. 3.5 Materials 3.5.1 Immunoaffinity column (see Appendix D for column capacity and column recovery verification methods). NOTE. before use, the column capacity and column recovery rate of each batch of affinity columns 5.2.2 Vegetable oils The extraction of vegetable oils shall be performed in accordance with the steps in 5.2.1.The extracting solution is the lower layer. 5.3 Specimen Purification 5.3.1 Cereal supplementary foods for infants and young children Accurately transfer-take 5.0 mL of the extracting solution, add 45.0 mL of Tween-20/PBS solution for dilution and evenly mix it. At 4,000 r/min, centrifuge it for 10 minutes, take the supernatant and pass it all through the immunoaffinity column, and control the flow rate to 1 mL/min ~ 2 mL/min. Then, use 10 mL of PBS buffer solution and 10 mL of water to successively rinse the immunoaffinity column. Then, use 3 mL of methanol - acetic acid solution to elute the immunoaffinity column in three times; collect and combine the eluent. At 55 C, use nitrogen to blow-dry it; add 1 mL of acetonitrile - water solution (20 + 80) to dissolve the residue and vortex for 30 s. After passing it through a microporous filter membrane, collect it in a sampling bottle and reserve it for testing. 5.3.2 Cereals and products, and vegetable oils Accurately transfer-take 0.5 mL of the extracting solution, add 4.5 mL of Tween-20/PBS solution for dilution and evenly mix it. At 4,000 r/min, centrifuge it for 10 minutes, take the supernatant and pass it all through the immunoaffinity column, and control the flow rate to 1 mL/min ~ 2 mL/min. Then, use 10 mL of PBS buffer solution and 10 mL of water to successively rinse the immunoaffinity column. Then, use 3 mL of methanol - acetic acid solution to elute the immunoaffinity column in three times; collect and combine the eluent. At 55 C, use nitrogen to blow-dry it; add 1 mL of acetonitrile - water solution (20 + 80) to dissolve the residue and vortex for 30 s. After passing it through a microporous filter membrane, collect it in a sampling bottle and reserve it for testing. NOTE. since the immunoaffinity column operating procedures provided by different manufacturers may be different, during actual operation, please refer to the operating instructions and procedures provided by the manufacturer. 5.4 Reference Conditions of Instrument 5.4.1 Chromatographic column. C18, particle size 5 m, 4.6  250 mm, or equivalent. 5.4.2 Detection wavelength. excitation wavelength 335 nm; emission wavelength 440 nm. 5.4.3 Mobile phase. A. formic acid - water solution (0.1%); B. methanol. Gradient elution. The elution procedure is shown in Table 1. 5.4.4 Mobile phase flow rate. 0.8 mL/min. 5.4.5 Derivative solution flow rate. 0.4 mL/min. 10.5 Preparation of Isotope Internal Standard Solution 10.5.1 Mixed isotope standard stock solution. accurately transfer-take 1 mL of 13C34-FB1 (25 g/mL), 13C34-FB2 (10 g/mL) and 13C34-FB3 (10 g/mL) into the same 10 mL volumetric flask. Add acetonitrile - water solution (50 + 50) to dilute it and reach a constant volume to the scale. The mass concentration of 13C34-FB1 is 2.5 g/mL, and the mass concentration of 13C34-FB2 and 13C34-FB3 is respectively 1 g/mL. Store it at 18 C away from light. It shall remain valid for 6 months. 10.5.2 Mixed isotope standard working solution. accurately transfer-take 1.0 mL of the mixed isotope standard stock solution into a 10 mL volumetric flask, add acetonitrile - water solution (50 + 50) to dilute it and reach a constant volume to the scale. The mass concentration of 13C34- FB1 is 250 ng/mL, and the mass concentration of 13C34-FB2 and 13C34-FB3 is respectively 100 ng/mL. Store it at 4 C away from light. It shall remain valid for 6 months. 10.6 Preparation of Mixed Standard Series of Working Solutions Accurately transfer-take the mixed standard working solution, use acetonitrile - water solution (20 + 80) to dilute it, and add the mixed isotope standard working solution. Thus, mixed standard series of working solutions with a FB1 mass concentration of 20.0 ng/mL, 80.0 ng/mL, 160 ng/mL, 240 ng/mL, 320 ng/mL, 400 ng/mL and 480 ng/mL, and a FB2 and FB3 mass concentration of 10.0 ng/mL, 40.0 ng/mL, 80.0 ng/mL, 120 ng/mL, 160 ng/mL, 200 ng/mL and 240 ng/mL are prepared. In each standard working solution, the mass concentration of 13C34- FB1, 13C34-FB2 and 13C34-FB3 is respectively 50.0 ng/mL, 20.0 ng/mL and 20.0 ng/mL. Prepare them right before use. 10.7 Materials 10.7.1 Immunoaffinity column (see Appendix D for column capacity and column recovery verification methods). NOTE. before use, the column capacity and column recovery rate of each batch of affinity columns need to be verified. 10.7.2 Strong anion exchange solid-phase extraction column (6 mL, 500 mg). 10.7.3 Microporous filter membrane. 0.22 m, organic type. 11 Instruments and Equipment 11.1 High-performance liquid chromatograph - tandem mass spectrometer. equipped with electrospray ion source. 11.2 Balance. with a division value of 0.01 g and 0.01 mg. 11.3 Homogenizer. with a rotation speed  2,000 r/min. 11.4 Oscillator (with a rotation speed  1,000 r/min) or ultrasonic extraction instrument (with a power  500 W). 11.5 Centrifuge. with a rotation speed  4,000 r/min. 11.6 Nitrogen blower. 12 Analytical Procedures 12.1 Specimen Preparation For cereals and products, cereal supplementary foods for infants and young children, the sampling size shall be not lower than 1 kg; when the mass of sample is less than 1 kg, all specimens shall be taken. Use a high-speed pulverizer to pulverize it, and the fineness of pulverization shall be less than 1 mm. After evenly mix it, store in a clean container, seal and store at 4 C away from light. For vegetable oils, the sampling size shall be not lower than 1 kg; when the mass of sample is less than 1 kg, all specimens shall be taken. After evenly mix it, store in a clean container, seal and store at 4 C away from light. During the specimen preparation, sample contamination or changes in fumonisin content shall be prevented. 12.2 Specimen Extraction 12.2.1 Cereals and products, cereal supplementary foods for infants and young children Accurately weigh-take 20 g (accurate to 0.01 g) of specimen into a 250 mL conical flask, accurately add 100 mL of acetonitrile - water (50 + 50) extracting solution, conduct ultrasonic or oscillating extraction for 20 minutes, transfer 20 mL of the extracting solution into a 50 mL centrifuge tube. At 4,000 r/min, centrifuge it for 5 minutes and reserve it for purification. 12.2.2 Vegetable oils The extraction of vegetable oils shall be the same as 12.2.1.The extracting solution is the lower layer. 12.3 Specimen Purification 12.3.1 Immunoaffinity column purification 12.3.1.1 Cereal supplementary foods for infants and young children Accurately transfer-take 5.0 mL of the extracting solution, add 200 L of the mixed isotope standard working solution and 45.0 mL of Tween-20/PBS solution for dilution, evenly mix it. At 4,000 r/min, centrifuge it for 10 minutes, take all the supernatant and pass it through the immunoaffinity column, and control the flow rate to 1 mL/min ~ 2 mL/min. Then, use 10 mL of PBS buffer solution and 10 mL of water to successively rinse the immunoaffinity column. Then, use 3 mL of methanol - acetic acid solution (98 + 2) to elute the immunoaffinity column in three times; combine the eluent. At 55 C, use nitrogen to blow-dry it; add 1 mL of acetonitrile - water solution (20 + 80) to dissolve the residue and vortex for 30 s. After passing it through a microporous filter membrane, collect it in a sampling bottle and reserve it for testing. 12.3.1.2 Cereals and products, and vegetable oils Accurately transfer-take 0.5 mL of the extracting solution, add 200 L of the mixed isotope standard working solution and 4.5 mL of Tween-20/PBS solution for dilution and evenly mix it. At 4,000 r/min, centrifuge it for 10 minutes, take all the supernatant and pass it through the immunoaffinity column, and control the flow rate to 1 mL/min ~ 2 mL/min. Then, use 10 mL of PBS buffer solution and 10 mL of water to successively rinse the immunoaffinity column. Then, use 3 mL of methanol - acetic acid solution to elute the immunoaffinity column in three times; collect and combine the eluent. At 55 C, use nitrogen to blow-dry it; add 1 mL of acetonitrile - water solution (20 + 80) to dissolve the residue and vortex for 30 s. After passing it through a microporous filter membrane, collect it in a sampling bottle and reserve it for testing. 12.3.2 Strong anion exchange solid-phase extraction purification column 12.3.2.1 Cereal supplementary foods for infants and young children Accurately transfer-take 5.0 mL of the extracting solution, add 200 L of the mixed isotope standard working solution and 15.0 mL of methanol - water solution (60 + 20) for dilution, and evenly mix it. At 4,000 r/min, centrifuge it for 10 minutes. Take all the supernatant and pass it through a strong anion exchange solid-phase extraction column (before use, successively use 6.0 mL of methanol and 6.0 mL of water to activate it), and control the flow rate to 1 mL/min ~ 2 mL/min. Use 8 mL of methanol - water solution (60 + 20) and 3 mL of methanol to successively rinse it, and use 10 mL of methanol - acetic acid solution (99 + 1) to elute it; collect the eluent. At 55 C, use nitrogen to blow-dry it. Add 1 mL of acetonitrile - water solution (20 + 80) to dissolve the residue and vortex for 30 s. After passing it through a microporous filter membrane, collect it in a sampling bottle and reserve it for testing. 12.3.2.2 Cereals and products, and vegetable oils Accurately transfer-take 0.5 mL of the extracting solution, add 200 L of the mixed isotope standard working solution and 5.0 mL of methanol - water solution (60 + 20) for dilution, and evenly mix it. At 4,000 r/min, centrifuge it for 5 minutes. Take all the supernatant and pass it through a strong anion exchange solid-phase extraction column (before use, successively use 6.0 mL of methanol and 6.0 mL of water to activate it), and control the flow rate to 1 mL/min ~ 2 mL/min. Use 8 mL of methanol - water solution (60 + 20) and 3 mL of methanol to successively rinse it, and use 10 mL of methanol - acetic acid solution (99 + 1) to elute it; collect the eluent. At 55 C, use nitrogen to blow-dry it. Add 1 mL of acetonitrile - water solution (20 + 80) to dissolve the residue and vortex for 30 s. After passing it through a microporous filter membrane, collect it in a sampling bottle and reserve it for testing. hydrogen phosphate, 0.2 g of potassium dihydrogen phosphate and 0.2 g of potassium chloride; use 980 mL of water to dissolve it, then, use hydrochloric acid to adjust pH to 7.4.Finally, use water to dilute to 1,000 mL and evenly mix it. 17.2.7 Tween-20/PBS solution (0.1%). weigh-take 1.0 g of Tween-20, add phosphate buffer solution, dilute to 1,000 mL and evenly mix it. 17.2.8 Borax solution (0.1 mol/L). weigh-take 3.8 g of borax, use water to dissolve it and dilute to 100 mL, and evenly mix it. 17.2.9 Derivatization solution. weigh-take 25 mg of o-phthalaldehyde, dissolve it in 1 mL of methanol, use borax solution (0.1 mol/L) to dilute to 50 mL, add 50 L of 2-mercaptoethanol and evenly mix it. After filtration, put it into a brown bottle; store it at room temperature away from light. It shall remain valid for 1 week. 17.3 Reference Materials 17.3.1 Fumonisin B1 (C34H59NO15, FB1, CAS No.. 116355-83-0), purity  95%, or a standard substance certified by the state and awarded a standard substance certificate. 17.3.2 Fumonisin B2 (C34H59NO14, FB2, CAS No.. 116355-84-1), purity  95%, or a standard substance certified by the state and awarded a standard substance certificate. 17.3.3 Fumonisin B3 (C34H59NO14, FB3, CAS No.. 136379-59-4), purity  95%, or a standard substance certified by the state and awarded a standard substance certificate. 17.4 Preparation of Standard Solutions 17.4.1 Standard stock solutions (100 g/mL). respectively and accurately weigh-take 10 mg (accurate to 0.01 mg) of FB1, FB2 and FB3 into small beakers, use acetonitrile - water solution (50 + 50) to dissolve them, and respectively transfer to 100 mL volumetric flasks. Use acetonitrile - water solution (50 + 50) to reach a constant volume to the scale. Store them at 18 C away from light. They shall remain valid for 6 months. 17.4.2 Mixed standard stock solution. accurately transfer-take 1.0 mL of FB1 standard stock solution, 0.5 mL of FB2 standard stock solution and 0.5 mL of FB3 standard stock solution into the same 10 mL volumetric flask; add acetonitrile - water solution (50 + 50) to dilute to the scale. The mass concentration of FB1 is 10 g/mL, and the mass concentration of FB2 and FB3 is 5 g/mL. Store it at 18 C away from light. It shall remain valid for 6 months. 17.4.3 Mixed standard working solution. accurately transfer-take 1.0 mL of the mixed standard stock solution into a 10.0 mL volumetric flask, add acetonitrile - water solution (50 + 50) to dilute it and reach a constant volume to the scale. The mass concentration of FB1 is 1.0 g/mL, and the mass concentration of FB2 and FB3 is 0.5 g/mL. Store it at 4 C away from light. It shall remain valid for 6 months. 17.4.4 Mixed standard series of working solutions. accurately transfer-take the mixed standard During the specimen preparation, sample contamination or changes in fumonisin content shall be prevented. 19.2 Specimen Extraction 19.2.1 Cereals and products, cereal supplementary foods for infants and young children Accurately weigh-take 20 g (accurate to 0.01 g) of specimen into a 250 mL conical flask, accurately add 100 mL of acetonitrile - water (50 + 50) extracting solution, conduct ultrasonic or oscillating extraction for 20 minutes, transfer 20 mL of the extracting solution into a 50 mL centrifuge tube. At 4,000 r/min, centrifuge it for 10 minutes and reserve it for purification. 19.2.2 Vegetable oils The extraction of vegetable oils shall be performed in accordance with the steps in 19.2.1.The extracting solution is the lower layer. 19.3 Specimen Purification 19.3.1 Cereal supplementary foods for infants and young children Accurately transfer-take 5.0 mL of the extracting solution, add 45.0 mL of Tween-20/PBS solution for dilution and evenly mix it. At 4,000 r/min, centrifuge it for 10 minutes, take all the supernatant and pass it through the immunoaffinity column, and control the flow rate to 1 mL/min ~ 2 mL/min. Then, use 10 mL of PBS buffer solution and 10 mL of water to successively rinse the immunoaffinity column. Then, use 3 mL of methanol - acetic acid solution (98 + 2) to elute the immunoaffinity column in three times; collect the eluent. At 55 C, use nitrogen to blow-dry it; add 1 mL of acetonitrile - water solution (20 + 80) to dissolve the residue and vortex for 30 s. After passing it through a microporous filter membrane, collect it in a sampling bottle and reserve it for testing. 19.3.2 Cereals and products, and vegetable oils Accurately transfer-take 0.5 mL of the extracting solution, add 4.5 mL of Tween-20/PBS solution for dilution and evenly mix it. At 4,000 r/min, centrifuge it for 10 minutes, take all the supernatant and pass it through the immunoaffinity column, and control the flow rate to 1 mL/min ~ 2 mL/min. Then, use 10 mL of PBS buffer solution and 10 mL of water to successively rinse the immunoaffinity column. Then, use 3 mL of methanol - acetic acid solution (98 + 2) to elute the immunoaffinity column in three times; collect the eluent. At 55 C, use nitrogen to blow-dry it; add 1 mL of acetonitrile - water solution (20 + 80) to dissolve the residue and vortex for 30 s. After passing it through a microporous filter membrane, collect it in a sampling bottle and reserve it for testing. NOTE. since the immunoaffinity column operating procedures provided by different manufacturers may be different, during actual operation, please refer to the operating instructions and procedures provided by the manufacturer. ......


GB 5009.240-2016 (Food safety national standard - Determination of fumonisin in foods) National Standards of People's Republic of China National Food Safety Standard Determination of fumonisins Issued on.2016-08-31 2017-03-01 implementation People's Republic of China National Health and Family Planning Commission released Foreword This standard replaces GB/T 25228-2010 "Determination Inspection of grain and corn products fumonisins content immunoaffinity column net High Performance Liquid Chromatography and Fluorescence spectrophotometry ", SN/T 1958-2007" import and export food fumonisins B1 Residue Detection enzyme Linked immunosorbent assay ", SN/T 1572-2005" Importers fumonisins in cereals HPLC testing method. " This standard is above standard integration, major amendments are as follows. --- Standard name was changed to "national food safety standards of food fumonisins determination"; --- Fumonisin species increased to fumonisin B1, B2, B3 three kinds; --- Increased immunoaffinity column clean - after the High Performance Liquid Chromatography method as the first method; --- Increased performance liquid chromatography - tandem mass spectrometry as the second method; --- Cancellation fluorescence spectrometry; --- Change the sample extraction solution; --- Change immunoaffinity column clean - pre-column derivatization HPLC mobile phase consisting of. National Food Safety Standard Determination of fumonisins 1 Scope This standard specifies the corn and its products fumonisins B1, fumonisin B2, fumonisin B3 (hereinafter abbreviated as FB1, FB2, FB3) Measurement method. The standard method for the first immunoaffinity column clean - post column derivatization high performance liquid chromatography method for the second liquid chromatography - tandem mass spectrometry Usage, the third method is immunoaffinity column clean - precolumn derivatization HPLC method for the determination of corn and its products of fumonisins. The first method with immunoaffinity column clean - after the High Performance Liquid Chromatography Method Principle 2 Samples with acetonitrile - aqueous extract, after diluted immunoaffinity column purification, removal of fat, protein, carbohydrates and other dry pigment and Interference substances. By high performance liquid chromatography column after PHTHALALDEHYDS derivative, fluorescence detection, external standard. 3 Reagents and materials Unless otherwise indicated, the reagents used in this method are analytically pure water as a water GB/T 6682 regulations. 3.1 Reagents 3.1.1 Methanol (CH3OH). chromatography. 3.1.2 acetonitrile (CH3CN). chromatography. 3.1.3 acetic acid (CH3COOH). 3.1.4 sodium hydroxide (NaOH). 3.1.5 Sodium chloride (NaCl). 3.1.6 disodium hydrogen phosphate (Na2HPO4). 3.1.7 potassium dihydrogen phosphate (KH2PO4). 3.1.8 Potassium chloride (KCl). 3.1.9 borax (Na2B4O7 · 10H2O). 3.1.10 2-mercaptoethanol (C2H6OS). 3.1.11 o-phthalaldehyde (OPA, C8H6O2). 3.1.12 Tween -20 (C58H114O26). 3.2 solution preparation 3.2.1 formic acid (0.1%). lessons 1mL of formic acid, was added to 999mL water, and mix. 3.2.2 acetonitrile - water (5050). amount of acetonitrile and 500mL, respectively 500mL water, mix well. 3.2.3 acetonitrile - water (2080). amount of acetonitrile and 800mL, respectively 200mL water, mix well. 3.2.4 methanol - acetic acid (982). lessons 2mL acetic acid, 98mL of methanol was added to and mixed uniformly. 3.2.5 Sodium hydroxide solution (1mol/L). Weigh accurately sodium hydroxide 4.0g, dissolved in 100mL of water, mix well. 3.2.6 phosphate buffered saline (PBS). Weigh 8.0g of sodium chloride, 1.2 g of disodium hydrogen phosphate, potassium dihydrogen phosphate 0.2g, 0.2g potassium chloride, with 980mL dissolved in water, adjusted to pH 7.4 with hydrochloric acid, diluted with water to 1000mL, and mix well. 3.2.7 Tween -20/PBS solution (0.1%). lessons 1mL Tween-20, was added phosphate buffer (3.2.6) and diluted to 1000mL, well mixed. 3.2.8 borax solution (0.05mol/L, pH10.5). borax weighed 19.1g, dissolved in 980mL water, neutralized with sodium hydroxide solution to adjust the pH 10.5, diluted with water to 1000mL, and mix well. 3.2.9 Derivatives solution. Weigh 2.0g PHTHALALDEHYDS, dissolved in 20mL methanol, treated with borax solution (0.05mol/L, pH10.5) (3.2.8) Dilute Diluted to 500mL, was added 2-mercaptoethanol 500μL, homogenized and filled brown bottle using now. 3.3 Standard 3.3.1 fumonisin B1 (FB1, C34H59NO15), purity ≥95%, or certified reference solution. 3.3.2 fumonisin B2 (FB2, C34H59NO14), purity ≥95%, or certified reference solution. 3.3.3 fumonisin B3 (FB3, C34H59NO14), purity ≥95%, or certified reference solution. 3.4 Standard Solution 3.4.1 Standard stock solution (0.1mg/mL). Weigh accurately respectively FB1, FB2, FB3 each 0.01g (accurate to 0.0001g) to a small beaker With acetonitrile - water (3.2.2) was dissolved and transferred to 100mL volumetric flask, dilute to the mark. This solution was sealed dark -20 ℃ insurance Deposit. Valid for six months. 3.4.2 mixed standard solution. Imbibe FB1 standard stock solution 1mL, FB2 and FB3 same standard stock solution 0.5mL to 10mL Flask, add acetonitrile - water (3.2.2) diluted to the mark, give FB1 at a concentration of 10μg/mL, FB2 and FB3 concentration of 5μg/mL The mixed standard solution. A factor of 10-fold to give a concentration of FB1 of 1μg/mL, FB2 and FB3 concentration of 0.5μg/mL of mixed standard Solution. This solution was sealed dark at 4 ℃, valid for six months. 3.4.3 mixed standard working solution. Imbibe mixed standard solution with acetonitrile - a dilute aqueous solution (3.2.3), followed by concentration formulated as FB1 Of 20ng/mL, 80ng/mL, 160ng/mL, 240ng/mL, 320ng/mL, 400ng/mL, FB2 and FB3 concentration was followed 10ng/series of mixed standard working solution mL, 40ng/mL, 80ng/mL, 120ng/mL, 160ng/mL, 200ng/mL of. 4 instruments and equipment 4.1 high performance liquid chromatography with fluorescence detector. After 4.2 column derivatization system. 4.3 Balance. a sense of volume and 0.01g 0.0001g. 4.4 homogenizer. 4.5 oscillator. 4.6 nitrogen blowing instrument. 4.7 centrifuge. speed ≥4000r/min. 4.8 immunoaffinity column (column capacity ≥5000ng, FB1 column recovery ≥80%) (column column capacity and recovery verification method, see Appendix B). NOTE. For each batch of affinity columns should be inspected before use. 4.9 microporous membrane. 0.45μm, there are models. Step 5 Analysis 5.1 Sample Preparation The solid sample was reduced by quartering to 1kg, with all the grinding mill and grain to fine particle size less than 1mm, mixed into 2 parts for The samples were loaded into a clean container, sealed and placed in 4 ℃ after identification stored in the dark. Corn oil samples taken directly as a sample 2 parts were loaded into a clean container, sealed and placed in 4 ℃ after identification stored in the dark. In the sample preparation procedure, it should prevent sample contamination or the occurrence of the change of residue content. 5.2 Sample Extraction Accurately weighed solid sample 5g (accurate to 0.01g) sample in 50mL centrifuge tube, add 20mL of acetonitrile - water (3.2.2), Vortex or shaking extraction 20min, after removing the centrifugal 5min at 4000r/min, the supernatant was transferred to another tube. Corn oil sample handling with solid samples, extract the lower layer. 5.3 Sample purification Of 2mL extract was added 47mL Tween -20/PBS solution (3.2.7), mix evenly over the immunoaffinity column, flow rate control 1mL/min ~ 3mL/min, rinsed with 10mLPBS buffer immunoaffinity column, respectively 1mL methanol - acetic acid solution (3.2.4) wash De immunoaffinity column three times, collecting the eluent at 55 ℃ nitrogen blow, added 1mL of acetonitrile - water (3.2.3) to dissolve the residue. vortex 30s, had 0.45μm microporous membrane was collected in an injection vial, test. NOTE. Because of different vendors immunoaffinity column operating procedures may differ, the actual operation, refer to the instructions and procedures provided by the manufacturer to use. 5.4 Instrument Reference conditions Column. C18 column. 250mm × 4.6mm, 5μm, or equivalent person. Detection wavelength. excitation wavelength of 335nm; emission wavelength of 440nm. Mobile phase. A. aqueous formic acid (3.2.1); B. methanol. Gradient elution shown in Table 1. Mobile phase flow rate. 0.8mL/min. Derived liquid flow rate. 0.4mL/min. Column temperature. 40 ℃. Reactor temperature. 40 ℃. Injection volume. 50μL. Table 1 mobile phase elution time min Mobile phase A Mobile Phase B 0.00 45.0 55.0 2.00 45.0 55.0 9.00 30.0 70.0 14.00 10.0 90.0 14.50 10.0 90.0 15.00 45.0 55.0 22.00 45.0 55.0 5.5 Determination of the sample solution Under 5.4 chromatographic conditions, 50.0μL series fumonisin mixed standard working solution (3.4.3) at a concentration of from low to high note Into the high performance liquid chromatography; stable condition after the instrument to the concentration of the target substance as the horizontal (x-axis), the peak area of the target substance is ordinate Standard (y-axis) for each data point least squares linear fit standard curve according to equation (1). y = ax b (1) Where. Y --- peak area ratio of the target substance; --- A slope of the regression curve; X --- concentration of the target substance; b --- intercept of the regression line. Response standard solution and sample solution analyte should be within the linear response range of the instrument, if the content of the sample exceeds the standard curve Scope, be diluted before measurement. 5.6 blank test Not weighed sample, follow the steps 5.2 and 5.3 of the blank experiment. Confirm contain interfering substances should not be tested components. 6 expression analysis Test sample FB1, FB2, FB3 content according to formula (2). X = ci × V × f (2) Where. X---- test sample FB1, FB2, FB3 content, in micrograms per kilogram (μg/kg); CI --- analyte into the sample liquid FB1, FB2, FB3 concentration unit nanograms per milliliter (ng/mL); V --- volume by volume, in milliliters (mL); f --- test solution dilution factor; M --- sample weight of sample in grams (g). Note. The blank value should be subtracted from the calculation results of the measurement results by the arithmetic mean of the parallel determination expressed to two significant figures. 7 precision Two independent determination results fumonisins content in the sample under the same condition shall not exceed the arithmetic mean of the absolute difference 20%. 8 Other When sample weight is 5g time, FB1, FB2, FB3 detection limits were 17μg/kg, 8μg/kg, 8μg/kg; quantification limits were 50μg/kg, 25μg/kg, 25μg/kg. The second method performance liquid chromatography - tandem mass spectrometry Principle 9 Samples were added isotope internal standard acetonitrile - aqueous extract, after diluted immunoaffinity column or strong anion exchange solid-phase extraction (SPE), Removal of fat, protein, carbohydrates and pigments and other interfering substances. Purifying liquid fumonisin through High Performance Liquid Chromatography tandem Mass spectrometry, isotope internal standard. 10 Reagents and materials Unless otherwise indicated, the reagents used in this method are analytically pure 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 acetic acid (CH3COOH). 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 Tween -20 (C58H114O26). 10.2 solution preparation 10.2.1 formic acid (0.1%). lessons 1mL formic acid was added to 999mL water and mix well. 10.2.2 acetonitrile - methanol solution (5050). amount of 500mL of methanol and 500mL of acetonitrile were mixed uniformly. 10.2.3 acetonitrile - water (5050). amount of acetonitrile and 500mL, respectively 500mL water, mix well. 10.2.4 acetonitrile - water (2080). amount of acetonitrile and 800mL, respectively 200mL water, mix well. 10.2.5 methanol - water (6020). amount of each 600mL of methanol and 200mL water, mix well. 10.2.6 methanol - acetic acid (991). lessons 1mL acetic acid was added to 99mL of methanol, mixed uniformly. 10.2.7 methanol - acetic acid (982). lessons 2mL acetic acid, 98mL of methanol was added to and mixed uniformly. 10.2.8 phosphate buffered saline (PBS). Weigh 8.0g of sodium chloride, 1.2 g of disodium hydrogen phosphate, potassium dihydrogen phosphate 0.2g, 0.2g potassium chloride, with 980mL dissolved in water, adjusted to pH 7.4 with hydrochloric acid, diluted with water to 1000mL, and mix well. 10.2.9 Tween -20/PBS solution (0.1%). lessons 1mL Tween-20 was added phosphate buffer (10.2.8) and diluted to 1000mL, mixed Together evenly. 10.3 Standards 10.3.1 FB1, FB2, FB3 Standard Fumonisin B1 (FB1, C34H59NO15), purity ≥95%, or certified reference solution. Fumonisin B2 (FB2, C34H59NO14), purity ≥95%, or certified reference solution. Fumonisin B3 (FB3, C34H59NO14), purity ≥95%, or certified reference solution. 10.3.2 13C34- fumonisin B1, B2, B3 isotope internal standard 13C34- fumonisin B1 (13C34-FB1, C34H59NO15), purity ≥95%, or certified reference solution. 13C34- fumonisin B2 (13C34-FB2, C34H59NO14), purity ≥95%, or certified reference solution. 13C34- fumonisin B3 (13C34-FB3, C34H59NO14), purity ≥95%, or certified reference solution. Note. You can only use in the detection 13C34-FB1 isotope internal standard, but needs to be measured sample matrix spiking experiments conducted to evaluate and determine the 13C34-FB1 And other substrates tested effects of fumonisin. Or matrix-matched calibration curve. 10.4 Standard Solution 10.4.1 Standard stock solution (0.1mg/mL). Weigh accurately respectively FB1, FB2, FB3 each 0.01g (accurate to 0.0001g) to a small burn Cup with acetonitrile - water (10.2.3) was dissolved and transferred to 100mL volumetric flask, dilute to the mark. This solution was sealed after dark -20 ℃ to save. Valid for six months. 10.4.2 mixed standard solution. Imbibe FB1 standard stock solution 1mL, FB2 and FB3 same standard stock solution 0.5mL to 10mL Flask, add acetonitrile - water (10.2.3) diluted to the mark, give FB1 at a concentration of 10μg/mL, FB2 and FB3 concentrations Mixed standard solution 5μg/mL of. A factor of 10-fold to give a concentration of FB1 of 1μg/mL, FB2 and FB3 concentration of 0.5μg/mL The mixed standard solution. This solution was sealed dark at 4 ℃. Valid for six months. 10.4.3 mixed isotope standard solution. Imbibe 13C34-FB1 (25μg/mL), 13C34-FB2 (10μg/mL), 13C34-FB3 (10μg/mL) each 1mL to 10mL same volumetric flask, add acetonitrile - water (10.2.3) diluted to the mark, having obtained 13C34-FB1 2.5μg/mL, 13C34-FB2 and 13C34-FB31μg/mL standard solution of mixed isotopes. A factor of 10-fold, to give 13C34-FB1 containing 250ng/mL, 13C34-FB2 and 13C34-FB3100ng/mL mixed standard working solution of the isotope. Valid for six months. 10.4.4 mixed standard working solution. Imbibe mixed standard solution with acetonitrile - a dilute aqueous solution (10.2.4), into the mixing Isotope Standard Quasi working solution (10.4.3), were formulated as a concentration of FB1 20ng/mL, 80ng/mL, 160ng/mL, 240ng/mL, 320ng/mL, 400ng/mL, FB2 and FB3 concentrations were 10ng/mL, 40ng/mL, 80ng/mL, 120ng/mL, 160ng/mL, 200ng/mL A series of mixed standard working solution, a standard working solution contains 13C34-FB125ng/mL, 13C34-FB2 and 13C34-FB3 10ng/mL. 11 instruments and equipment 11.1 High performance liquid chromatography - tandem mass spectrometry. equipped with electrospray ionization source. 11.2 balance. a sense of volume and 0.01g 0.0001g. 11.3 homogenizer. 11.4 oscillator. 11.5 nitrogen blowing instrument. 11.6 Centrifuge. Speed ≥4000r/min. 11.7 strong anion exchange solid phase extraction column (silica, 6mL, 500mg). 11.8 immunoaffinity column (column capacity ≥5000ng, FB1 column recovery ≥80%). (Capacity of the column and the column recovery verification method, see Appendix B) NOTE. For each batch of affinity columns should be inspected before use. 11.9 microporous membrane. 0.22μm, there are models. 12 analysis steps 12.1 Sample Preparation The solid sample was reduced by quartering to 1kg, with all the grinding mill and grain to fine particle size less than 1mm, mixed into 2 parts for The samples were loaded into a clean container, sealed and placed in 4 ℃ after identification stored in the dark. Corn oil samples taken directly as a sample 2 parts were loaded into a clean container, sealed and placed in 4 ℃ after identification stored in the dark. In the sample preparation procedure, it should prevent sample contamination or the occurrence of the change of residue content. 12.2 Sample Extraction Accurately weighed solid sample 5g (accurate to 0.01g) in 50mL centrifuge tube samples, adding a mixed isotope standard working solution (10.4.3) 400μL, was added 20mL of acetonitrile - water (10.2.3), or vortex shaking extraction 20min, after removing at 4000r/min Centrifugal 5min, the supernatant was transferred to another tube. Corn oil sample handling with solid samples, extract the lower layer. 12.3 sample purification 12.3.1 immunoaffinity column purification Of 2mL extract was added 47mL Tween -20/PBS solution (10.2.9), mixed evenly over the immunoaffinity column, flow rate control 1mL/min ~ 3mL/min, with 10mLPBS buffer (10.2.8) immunoaffinity column leaching, respectively 1mL methanol - acetic acid solution (10.2.7) immunoaffinity column was eluted three times, collecting the eluent, 55 ℃ under nitrogen blow, added 1mL of acetonitrile - water (10.2.4) was dissolved residues Slag. Vortex 30s, had 0.22μm microporous membrane was collected in an injection vial, test. NOTE. Because of different vendors immunoaffinity column operating procedures may differ, the actual operation, refer to the instructions and procedures provided by the manufacturer to use. 12.3.2 strong anion exchange solid-phase extraction (SPE) Take 3mL extract was added 8mL of methanol - water (10.2.5), after mixing too strong anion exchange solid phase extraction column (before use 3mL methanol aqueous solution and rinsed (10.2.5) with 10mL of methanol - - wash acetic acid solution (10.2.6) required activation), respectively, with 8mL of methanol Off, at 55 ℃ nitrogen blow, added 1mL of acetonitrile - water (10.2.4) to dissolve the residue. Vortex 30s, after over 0.22μm microporous membrane, to close Set in an injection vial, test. 12.4 Instrument Reference conditions 12.4.1 LC Conditions Column. C18 column, 100mm × 2.1mm, 1.7μm, or equivalent person. Mobile phase. A. aqueous formic acid (0.1%); B. acetonitrile - methanol solution (5050). Gradient elution gradient shown in Table 2. Flow rate. 0.35mL/min. Column temperature. 30 ℃. Injection volume. 10μL. Table 2 gradient elution program time min Mobile phase A Mobile Phase B 0.00 70.0 30.0 2.30 30.0 70.0 4.00 30.0 70.0 4.20 0100 4.80 0100 5.00 70.0 30.0 12.4.2 MS parameters Ionization mode. positive ion mode electrospray ionization (ESI). MS scan mode. multiple reaction monitoring (MRM). Ion monitoring information in Table 3, other instruments reference conditions given in Appendix C. Table 3 MS parameters The name of the parent ion toxin Quantitative Ions Collision energy eV Qualitative Ions Collision energy eV FB1 722 352 25 334 35 FB2 706 336 35 354 30 FB3 706 336 35 354 30 13C34-FB1 756 374 35 356 40 13C34-FB2 740 358 35 376 30 13C34-FB3 740 358 35 376 30 12.5 qualitative determination High performance liquid chromatography - tandem mass spectrometry for qualitative determination of the samples, under the same test conditions, the sample should be presented from the quantitative Son and qualitative peaks of ion pairs, mass chromatogram peak retention time of the standard solution of the test substance in the corresponding mass peaks Paul substance Stay consistent time; the relative abundance of sample chromatogram selected ion monitoring of relative abundance rather than the ion concentration of the standard ratio The deviation does not exceed the range specified in Table 4, it can be determined corresponding to the target substance present in the sample. The maximum permissible relative ion abundances Table 4 Qualitative confirmation bias when Relative ion abundances (k) k≥50% 50% > k≥20% 20% > k≥10% k≤10% The maximum allowable deviation of ± 20% ± 25% ± 30% ± 50% Quantitative Determination 12.6 5.4 high performance liquid chromatography - tandem mass spectrometry analysis under the conditions, 10.0μL series fumonisin mixed standard solution (10.4.4) Press From low to high concentration of high performance liquid chromatography - tandem mass spectrometry; stable condition after the instrument to the target substance and the concentration of the internal standard Ratio of the horizontal (x-axis), the target substance and the internal standard peak area ratio of the vertical axis (y-axis), the value of each point of least squares linear fit Together, the standard curve according to equation (3) Calculated. y = ax b (3) Where. y --- Target substance/internal standard peak area ratio; --- A slope of the regression curve; x --- Target substance/internal standard concentration ratio; b --- intercept of the regression line. Standard solution and the sample solution in response to the analyte values should be within the linear response range of the instrument, if the content exceeds the range of the standard curve, Re-sampling, a corresponding increase in the amount of added internal standard, so that the concentration of the internal standard and analyte concentration to match, and then diluted to ... ......

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