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GB 5009.96-2016 English PDF

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GB 5009.96-2016: Determination of ochratoxin A in food -- High performance liquid chromatographic method with immunoaffinity column clean-up
Status: Valid

GB 5009.96: Evolution and historical versions

Standard ID	Contents [version]	USD	STEP2	[PDF] delivered in	Standard Title (Description)	Status	PDF
GB 5009.96-2016	English	519	Add to Cart	5 days [Need to translate]	Determination of ochratoxin A in food -- High performance liquid chromatographic method with immunoaffinity column clean-up	Valid	GB 5009.96-2016
GB/T 5009.96-2003	English	239	Add to Cart	3 days [Need to translate]	Determination of ochratoxin A in cereals and soybeans	Obsolete	GB/T 5009.96-2003

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Basic data

Standard ID	GB 5009.96-2016 (GB5009.96-2016)
Description (Translated English)	Determination of ochratoxin A in food -- High performance liquid chromatographic method with immunoaffinity column clean-up
Sector / Industry	National Standard
Classification of Chinese Standard	X04
Classification of International Standard	67.050
Word Count Estimation	26,227
Date of Issue	2016-12-23
Date of Implementation	2017-06-23
Older Standard (superseded by this standard)	GB/T 23502-2009; GB/T 25220-2010; GB/T 5009.96-2003; SN 0211-1993; SN/T 1746-2006; SN/T 1940-2007
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
Summary	This standard specifies the determination of ochratoxin A in food. The first method of this standard applies to the determination of ochratoxin A in cereals, oils and their products, alcoholic, soy sauce, vinegar, sauces and sauces, raisins, pepper grains / powder. The second method applies to corn, rice (brown rice ), Wheat, wheat flour, soybeans, coffee, wine ochratoxin A determination, the third method for corn, wheat and other food products, pepper and its products, beer and other alcohol, soy sauce and other products, Determination of ochratoxin A in coffee, the fourth method is applicable to the determination of ochratoxin A in maize, wheat, barley, rice, soybean and its products. The fifth method is suitable for the determination of ochratoxin A in wheat, maize and soybean Determination.

GB 5009.96-2016: Determination of ochratoxin A in food -- High performance liquid chromatographic method with immunoaffinity column clean-up

---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.
Determination of ochratoxin A in food-High performance liquid chromatographic method with immunoaffinity column clean-up National Standards of People's Republic of China National standards for food safety Determination of ochratoxin A in food 2016-12-23 released 2017-06-23 Implementation National Health and Family Planning Commission of the People's Republic of China State Food and Drug Administration issued

Foreword

This standard replaces GB/T 23502-2009 "Determination of ochratoxin A in foods Immunoaffinity chromatography Purification High performance liquid chromatography Law ", GB/T 25220-2010" Determination of ochratoxin A in grain and oil inspection food by high performance liquid chromatography and fluorescence spectrophotometry " GB/T 5009.96-2003 "Determination of ochratoxin A in cereals and soybeans", SN/T 1746-2006 "Import and export of soybean, rapeseed and Method for the determination of ochratoxin A in edible vegetable oils ", SN/T 1940-2007" Determination of ochratoxin A in food for import and export Law "and SN0211-1993" Method for the detection of aflatoxin A in export grain ". This standard compared with GB/T 23502-2009, the main changes are as follows. --- standard name changed to "food safety national standard food ochratoxin A determination"; - a third method of immunoaffinity chromatography for the purification of liquid chromatography - tandem mass spectrometry and the fourth method of enzyme - linked immunosorbent assay; --- increase the scope of application and optimize the extraction method; --- Removal of the immunoaffinity column chromatography purification fluorescence spectrophotometry. National standards for food safety Determination of ochratoxin A in food

1 Scope

This standard specifies the determination of ochratoxin A in food. The first method of this standard applies to cereals, oil and its products, alcohol, soy sauce, vinegar, sauce and sauce products, raisins, pepper tablets/powder ochrat Toxin A determination, the second method for corn, rice (brown rice), wheat, wheat flour, soybeans, coffee, wine ochratoxin A test The third method applies to corn, wheat and other food products, pepper and its products, beer and other alcohol, soy sauce and other products, raw coffee, cooked coffee Ochratoxin A determination, the fourth method for corn, wheat, barley, rice, soy and its products in the determination of ochratoxin A, The fifth method is applicable to the determination of ochratoxin A in wheat, maize and soybean. First method immunoaffinity chromatography purification liquid chromatography

2 principle

The ochratoxin A in the sample was extracted with the extract, purified by immunoaffinity column, and then analyzed by high performance liquid chromatography combined with fluorescence detector Determination of ochratoxin A content, external standard method quantitative.

3 reagents and materials

Unless otherwise stated, the reagents used in this method are of analytical grade and water is a primary water specified in GB/T 6682. 3.1 Reagents 3.1.1 Methanol (CH3OH). Chromatographic Purification. 3.1.2 acetonitrile (CH3CN). pure chromatography. 3.1.3 glacial acetic acid (C2H4O2). pure chromatography. 3.1.4 Sodium chloride (NaCl). 3.1.5 Polyethylene glycol [HOCH2 (CH2O · CH2) nCH2OH]. 3.1.6 Tween 20 (C58H114O26). 3.1.7 Sodium bicarbonate (NaHCO3). 3.1.8 Potassium dihydrogen phosphate (KH2PO4). 3.1.9 concentrated hydrochloric acid (HCl). 3.1.10 nitrogen (N2). purity ≥99.9%. 3.2 reagent preparation 3.2.1 Extract I. Methanol - Water (80 20). 3.2.2 Extract II. Weigh 150.0 g of sodium chloride, 20.0 g of sodium bicarbonate dissolved in about 950 mL of water, add water to volume to 1 L. 3.2.3 Extract III. acetonitrile - water (60 40). 3.2.4 Washing solution. Weigh 25.0 g of sodium chloride, 5.0 g of sodium bicarbonate dissolved in about 950 mL of water, add water to volume to 1 L. 3.2.5 Mycotoxin Wash Buffer. Weigh 25.0 g of sodium chloride, 5.0 g of sodium bicarbonate dissolved in water, add 0.1 mL Tween 20, water Diluted to 1L. 3.2.6 phosphate buffer. Weigh 8.0 g of sodium chloride, 1.2 g of sodium hydrogen phosphate, 0.2 g of potassium dihydrogen phosphate, 0.2 g of potassium chloride was dissolved in about 990 mL Water, with concentrated hydrochloric acid to adjust the pH to 7.0, diluted with water to 1L. 3.2.7 sodium bicarbonate solution (10g/L). Weigh 1.0g sodium bicarbonate, dissolved in water and diluted to 100mL. 3.2.8 Leaching buffer. 1.0 mL Tween 20 was added to 1000 mL of phosphate buffer. 3.3 standards Ochratoxin A (C20H18ClNO6, CAS. 303-47-9), purity ≥99%. Or by the national certification and awarded the standard material certificate The standard material of the book. 3.4 standard solution preparation 3.4.1 ochratoxin A standard stock solution. accurately weighed a certain amount of ochratoxin A standard, with methanol - acetonitrile (50 50) dissolved Solution, dubbed 0.1mg/mL standard stock solution, stored at -20 ℃, can be used for 3 months. 3.4.2 ochratoxin A standard working solution. according to the need to accurately remove a certain amount of ochratoxin A standard stock solution (3.4.1), with The mobile phase was diluted with 1 ng/mL, 5 ng/mL, 10 ng/mL, 20 ng/mL, 50 ng/mL standard working solution, Save, can use 7d. 3.5 Materials 3.5.1 ochratoxin A immunoaffinity column. column size 1mL or 3mL, column capacity ≥100ng, or equivalent column. 3.5.2 Quantitative filter paper. 3.5.3 glass fiber filter paper. diameter 11cm, pore size 1.5μm, no fluorescence characteristics.

4 instruments and equipment

4.1 Analysis of balance. sense of 0.001g. 4.2 High Performance Liquid Chromatograph with Fluorescence Detector. 4.3 high-speed homogenizer. ≥ 12000r/min. 4.4 Glass syringe. 10 mL. 4.5 Test sieve. pore size 1mm. 4.6 air pressure pump. 4.7 Ultrasonic generator. power > 180W. 4.8 nitrogen blowing instrument. 4.9 Centrifuge. ≥10000r/min. 4.10 Scroll Mixer. 4.11 reciprocating shaker. ≥250r/min. 4.12 pH meter. accuracy of 0.01.

5 Analysis steps

5.1 Preparation and extraction of samples 5.1.1 Grains, oil and their products 5.1.1.1 Food and food products Granular samples need to be crushed through the test sieve (pore size 1mm), mix and spare. Extraction method 1. Weigh the sample 25.0g (accurate to 0.1g), add 100mL of extract Ⅲ, high-speed homogenization 3min or oscillation 30min, quantitative filter paper filter, remove the 4mL filtrate by adding 26mL phosphate buffer mixture evenly, after mixing in 8000r/min centrifugal 5min, the supernatant as a filtrate A spare. Extraction method 2. Weigh the sample 25.0g (accurate to 0.1g), add 100mL of extract Ⅰ, high-speed homogenization 3min or oscillation 30min, quantitative filter paper filter, remove the 10mL filtrate by adding 40mL phosphate buffer diluted to 50mL, mixed evenly by the glass fiber Filter paper filter, filter B collected in a clean container, spare. 5.1.1.2 Edible vegetable oil Accurately weighed the sample 5.0g (accurate to 0.1g), add 1g of sodium chloride and 25mL extract Ⅰ, shaking 30min, at 6000r/min Centrifuge 10min, remove the 15mL supernatant extract, add 30mL phosphate buffer mixed evenly, filtered by glass fiber filter paper, filtrate C Collected in a clean container, spare. 5.1.1.3 Soybean and rapeseed Accurately weighed the sample 50.0g (accurate to 0.1g) (soybeans need to be ground and particle size ≤ 2mm) in the homogenizer configuration of the mixing cup, plus Into 5g sodium chloride and 100mL methanol (for rapeseed) or 100mL extract Ⅰ, homogenizer high-speed homogeneous extraction 1min. set Filter the filter paper, remove the 10mL filtrate and add 40mL water diluted by the glass fiber filter paper to filter to clarify the filtrate D collected in the Clean containers, spare. 5.1.2 Alcohol Take the sample of the degassed alcohol (the sample containing the carbon dioxide is placed in the refrigerator at 4 ° C for 30 min, filtered or defoamed) Other carbon dioxide-free alcohol sample 20.0g (accurate to 0.1g), placed in a 25mL volumetric flask, add the amount of extract II to volume, mixed Uniform, filtered through the glass fiber filter to the filtrate to clarify, the filtrate E collected in a clean container, spare. 5.1.3 soy sauce, vinegar, sauce and sauce products Weigh 25.0g (accurate to 0.1g) to mix the sample, add the liquid I volume to 50mL, ultrasonic extraction 5min. Quantitative filter paper over Filter, remove the 10mL filtrate in 50mL volumetric flask, add water to constant volume, mix, filter by glass fiber filter to clarify the filtrate, the filtrate F is collected in a clean container. 5.1.4 raisins 50.0 g (accurate to 0.1 g) was pulverized. In a stirrer equipped with a homogenizer, 100 mL of sodium bicarbonate solution was added and stirred Cup placed on the homogenizer to 22000r/min high-speed homogeneous extraction 1min. Quantitative filter paper filter, accurately remove 10mL filtrate and add 40 mL of elution buffer, filtered through a glass fiber filter to clarify the filtrate, and the filtrate G was collected in a clean container and allowed. 5.1.5 pepper tablets/powder Weigh the crushed sample 25.0 g (accurate to 0.1 g) in a homogenizer agitated cup, add 100 mL of sodium bicarbonate solution, stir Cup placed on the homogenizer to 22000r/min high-speed homogeneous extraction 1min. The extract was centrifuged in a centrifugal cup at 4000 r/min 15min. Remove 20 mL of the filtrate and add 30 mL of elution buffer to dilute, filter with glass fiber filter to clarify the filtrate, the filtrate H received Set in a clean container, spare. 5.2 sample purification 5.2.1 Grains, oil and their products 5.2.1.1 Food and food products The immunoaffinity column was attached to a glass syringe and accurately removed from all filtrate A in extraction method 1 or 20 mL of the extraction method 2 Liquid B, into the glass syringe. The air pressure pump is connected to the glass syringe and the pressure is adjusted so that the solution is passed at a flow rate of about 1 drop/s The immunoaffinity column was passed until the air entered the affinity column, washed with 10 mL of mycotoxin wash buffer, 10 mL of water, And the column, the flow rate of 1 drop/s ~ 2 drops/s, discard all the effluent, pumping dry column. 5.2.1.2 edible vegetable oil The immunoaffinity column was attached to a glass syringe and the 30 mL of filtrate C was accurately removed and injected into a glass syringe. Put the air pressure pump Connected to a glass syringe, the pressure was adjusted so that the solution passed through the immunoaffinity column at a flow rate of about 1 drop/s until the air enters the affinity column, Followed by 10mL mycotoxin cleansing buffer, 10mL water elution of immunoaffinity column, the flow rate of 1 drop/s ~ 2 drops/s, abandoned all The effluent is drained. 5.2.1.3 Soybean, rapeseed The immunoaffinity column was attached to a glass syringe and the 10 mL filtrate D was accurately removed and injected into a glass syringe. Put the air pressure pump Connected to a glass syringe, the pressure was adjusted so that the solution passed through the immunoaffinity column at a flow rate of about 1 drop/s until the air enters the affinity column, Followed by 10mL mycotoxin cleansing buffer, 10mL water elution of immunoaffinity column, the flow rate of 1 drop/s ~ 2 drops/s, abandoned all The effluent is drained. 5.2.2 Alcohol The immunoaffinity column was attached to a glass syringe and the 10 mL of filtrate E was accurately removed and injected into a glass syringe. Put the air pressure pump Connected to a glass syringe, the pressure was adjusted so that the solution passed through the immunoaffinity column at a flow rate of about 1 drop/s until the air enters the affinity column, Followed by 10mL rinse solution (3.2.4), 10mL water elution of immunoaffinity column, the flow rate of 1 drop/s ~ 2 drops/s, discard all the effluent, Take a dry pillar. 5.2.3 soy sauce, vinegar, sauce and sauce products The immunoaffinity column was attached to a glass syringe and the 10 mL of filtrate F was accurately removed and injected into a glass syringe. Put the air pressure pump Connected to a glass syringe, the pressure was adjusted so that the solution passed through the immunoaffinity column at a flow rate of about 1 drop/s until the air enters the affinity column, Followed by 10mL mycotoxin cleansing buffer, 10mL water elution of immunoaffinity column, the flow rate of 1 drop/s ~ 2 drops/s, abandoned all The effluent is drained. 5.2.4 raisins The immunoaffinity column was attached to a glass syringe and the 10 mL of filtrate G was accurately removed and injected into a glass syringe. Put the air pressure pump Connected to a glass syringe, the pressure was adjusted so that the solution passed through the immunoaffinity column at a flow rate of about 1 drop/s until the air enters the affinity column, Followed by 10mL rinse buffer, 10mL water elution of immunoaffinity column, flow rate of 1 drop/s ~ 2 drops/s, discard all the effluent, pumping Dry column. 5.2.5 pepper tablets/powder The immunoaffinity column was attached to a glass syringe and the 10 mL of filtrate H was accurately removed and injected into a glass syringe. Put the air pressure pump Connected to a glass syringe, the pressure was adjusted so that the solution passed through the immunoaffinity column at a flow rate of about 1 drop/s until the air enters the affinity column, Followed by 10mL rinse buffer, 10mL water elution of immunoaffinity column, flow rate of 1 drop/s ~ 2 drops/s, discard all the effluent, pumping Dry column. 5.3 elution Accurately add 1.5 mL of methanol or an immunoaffinity column to the recommended eluent for elution at a flow rate of about 1 drop/s to collect all elution The solution was dried in a clean glass tube at 45 ° C. Solvent with the mobile phase to dissolve the residue and set to 500μL for testing purposes. 5.4 Specimen determination 5.4.1 High Performance Liquid Chromatography Reference Conditions High performance liquid chromatography reference conditions are listed below. a) Column. C18 column, column length 150 mm, inner diameter 4.6 mm, particle size 5 μm, or equivalent column; b) mobile phase. acetonitrile - water - glacial acetic acid (96 102 2); c) flow rate. 1.0 mL/min; d) Column temperature. 35 ° C; e) Injection volume. 50 μL; f) Detection wavelength. excitation wavelength 333nm, emission wavelength 460nm. 5.4.2 Chromatographic determination In the 5.4.1 chromatographic conditions, the ochratoxin A standard working solution according to the concentration from low to high into the high performance liquid chromatography, to be After the instrument condition is stable, the concentration of the target substance is the abscissa (x-axis), the peak area of the target substance is the ordinate (y-axis), and the number Strong square linear fitting, the standard working curve according to formula (1) calculation. y = ax b (1) Where. y - the peak area ratio of the target substance; a - the slope of the regression curve; x - the concentration of the target substance; b - the intercept of the regression curve. The response value of the test solution and the sample in the standard solution should be within the linear response range of the instrument. If the sample content exceeds the standard curve Range, need to be diluted and then measured. 5.4.3 blank test Do not take the sample according to 5.1, 5.2 and 5.3 steps to do blank test. It should be confirmed that there is no substance that interferes with the components to be tested.

6 Analysis of the results of the expression

The amount of ochratoxin A in the sample is calculated according to formula (2) X = ρ × V x 1000 m × 1000 × f (2) Where. X - the amount of ochratoxin A in the sample, in micrograms per kilogram (μg/kg); ρ --- the concentration of ochratoxin A in the test solution in nanometer per milliliter (ng/mL); V --- the final volume of the test solution, in milliliters (mL); 1000 --- unit conversion constant; m --- the quality of the sample, in grams (g); f - dilution factor. The blank value should be deducted when the result is calculated. The result of the test is expressed as the mean of the two measured values. The result is two digital.

7 precision

The absolute difference between the two independent test results obtained in the repeatability condition of ochratoxin A in the sample shall not exceed the arithmetic level 15% of the mean.

8 other

Grain and food products, edible vegetable oil, soybean, rapeseed, raisins, pepper tablets/powder detection limit and the limit of quantification were 0.3μg/kg And 1 μg/kg. The detection limits and quantitation limits for alcohol were 0.1 μg/kg and 0.3 μg/kg, respectively. Soy sauce, vinegar, sauce and sauce products and the detection limit The limits were 0.5 μg/kg and 1.5 μg/kg, respectively. Second - stage ion exchange solid - phase extraction column purification high performance liquid chromatography

9 principle

The ochratoxin A in the sample was extracted with the extract and purified by ion-exchange solid-phase extraction (SPE). The mobile phase was purified by high performance liquid chromatography (HPLC) Determination of ochratoxin A by photodetector and quantification by external standard method. 10 reagents and materials Unless otherwise stated, the reagents used in this method are of analytical grade and water is a primary water specified in GB/T 6682. 10.1 Reagents 10.1.1 Acetonitrile (CH3CN). Chromatographic pure. 10.1.2 Methanol (CH3OH). Chromatographic Purification. 10.1.3 glacial acetic acid (C2H4O2). 10.1.4 petroleum ether. analytical grade, 60 ℃ ~ 90 ℃. 10.1.5 Formic acid (CH2O2). 10.1.6 Trichloromethane (CHCl3). 10.1.7 Sodium bicarbonate (NaHCO3). 10.1.8 phosphoric acid (H3PO4). 10.1.9 Potassium hydroxide (KOH). 10.2 Preparation of reagents 10.2.1 potassium hydroxide solution (0.1mol/L). Weigh 0.56g potassium hydroxide, dissolved in 100mL water. 10.2.2 phosphoric acid aqueous solution (0.1mol/L). 0.68mL of phosphoric acid was removed and dissolved in 100mL water. 10.2.3 sodium bicarbonate solution (30g/L). Weigh 30.0 grams of sodium bicarbonate, dissolved in 1000mL of water. 10.2.4 acetic acid aqueous solution (2%). 20 mL of glacial acetic acid was removed and dissolved in 980 mL of water. 10.2.5 Extract. Potassium hydroxide solution (0.1 mol/L) - Methanol - water (2 60 38). 10.2.6 Eluent. Potassium hydroxide solution (0.1 mol/L) - acetonitrile - water (3 50 47). 10.2.7 Eluent. methanol-acetonitrile-formic acid-water (40 50 5 5). 10.2.8 Methanol - Sodium bicarbonate solution (30 g/L) (50 50). 10.2.9 acetonitrile-2% aqueous acetic acid solution (50 50). 10.3 standards Ochratoxin A (C20H18ClNO6, CAS. 303-47-9), purity ≥99%. Or by the national certification and awarded the standard material certificate The standard material of the book. 10.4 standard solution preparation 10.4.1 ochratoxin A standard stock solution. accurately weighed a certain amount of ochratoxin A standard, dissolved in methanol 100μg/mL standard stock solution, stored at -20 ℃ in dark. 10.4.2 ochratoxin A standard working solution. accurate removal of a certain amount of ochratoxin A standard stock solution (10.4.1), dissolved in methanol Preparation of 1μg/mL standard stock solution, stored at 4 ℃ in dark. 10.4.3 ochratoxin A series of standard working fluid. accurate removal of appropriate ochratoxin A standard working solution (10.4.2), diluted with methanol A standard working solution was prepared at 1 ng/mL, 2.5 ng/mL, 5 ng/mL, 10 ng/mL, 50 ng/mL. 11 instruments and equipment 11.1 High Performance Liquid Chromatograph with Fluorescence Detector. 11.2 Analysis of balance. 0.01g and 0.0001g. 11.3 Solid Phase Extraction Column. Polymer Polymer Matrix Anion Exchange Solid Phase Extraction Column, Column Size 3 mL, Column Bed Weight.200 mg, etc. Effect column. 11.4 nitrogen blowing instrument. 11.5 Vortex Oscillator. 11.6 Rotary Evaporator. 11.7 high-speed universal grinder. ≥ 12000r/min. 11.8 20 mesh sieve. 11.9 Organic filter. pore size 0.45μm. 11.10 fast qualitative filter paper. 12 Analysis steps 12.1 Sample preparation Corn, rice (brown rice), wheat, wheat flour, soybeans and coffee beans and other high-speed universal mill crushing the sample, over 20 mesh after mixing spare. 12.2 Sample extraction 12.2.1 Corn Weigh the sample 10.0g (accurate to 0.01g), add 50mL of chloroform and 5mL 0.1mol/L phosphoric acid aqueous solution, Oscillator on the oscillation extraction 3min ~ 5min, extract with qualitative filter paper filter, take 10mL lower filtrate to 100mL flat bottom flask, In 40 ℃ water bath with a rotary evaporator to evaporate to near dry, with 20mL petroleum ether dissolved residue after adding 10mL extract, and then vortex Spin oscillator oscillation extraction 3min ~ 5min, after standing stratified layer of the solution, filter with filter paper, take 5mL filtrate for solid phase extraction Purify. 12.2.2 rice (brown rice), wheat, wheat flour, soybeans Weigh the sample 10.0g (accurate to 0.01g), add 50mL of the extract, in the vortex oscillator on the oscillation extraction 3min ~ 5min, with Qualitative filter paper filter, take 10mL filtrate to 100mL flat bottom flask, add 20mL petroleum ether, vortex oscillator oscillation extraction 3min ~ 5min, after standing stratified layer of the solution, filter with filter paper, take 5mL filtrate for solid phase extraction purification. 12.2.3 Coffee Weigh the sample 2.5g (accurate to 0.01g) in 50mL polypropylene conical tube (with lid), add 25mL methanol - sodium bicarbonate solution Liquid in the vortex oscillator oscillation extraction 3min ~ 5min, 4000r/min centrifugal 10min after the supernatant filter with filter paper, take 10mL filtrate Solid phase extraction purifica...

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