This standard specifies the thin layer chromatography method for the determination of ochratoxin A in cereals and soybeans. This standard is applicable to the determination of ochratoxin A in wheat, corn and soybeans. The detection limit of this method is 10μg/kg. GB/T 5009.96-2003 Determination of ochratoxin A in cereals and soybeans GB/T5009.96-2003 Standard download decompression password: www.bzxz.net

ICS67.040
National Standard of the People's Republic of China
GB/T5009.96—2003
Replaces GB/T13111--1991
Determination of ochratoxin A in cereals and soybeans
Determination of ochratoxin A in cereals and soybeansIssued on August 11, 2003
Ministry of Health of the People's Republic of China
China National Standardization Administration
Implemented on January 1, 2004
GB/T5009.96—2003
This standard replaces GB/T13111—1991 "Determination of ochratoxin A in cereals and soybeans". Compared with GB/T13111-1991, this standard has been modified as follows: The Chinese name of the standard has been modified and changed to "Determination of ochratoxin A in cereals and soybeans". The structure of the original standard has been modified according to GB/T20001.4-2001 "Standard Preparation Rules Part 4: Chemical Analysis Methods".
This standard is proposed and managed by the Ministry of Health of the People's Republic of China. The drafting units of this standard are: Food Hygiene Supervision and Inspection Institute of the Ministry of Health, Liaoning Provincial Food Hygiene Supervision and Inspection Institute, Heilongjiang Provincial Food Hygiene Supervision and Inspection Institute, Beijing Municipal Health and Epidemic Prevention Station, Shanxi Provincial Health and Epidemic Prevention Station. The main drafters of this standard are: Wei Runyun, Bao Fengzhen, Fang Xiaoming, Yang Maoduan, Gao Xiaolan. The original standard was first issued in 1991, and this is the first revision. 682
1 Scope
Determination of ochratoxin A in cereals and soybeans This standard specifies the thin layer chromatography method for the determination of ochratoxin A in cereals and soybeans. This standard applies to the determination of ochratoxin A in wheat, corn and soybean. The detection limit of this method is 10μg/kg. Www.bzxZ.net
2 Normative references
GB/T5009.96—2003
The clauses in the following documents become the clauses of this standard through reference in this standard. For all dated referenced documents, all subsequent amendments (excluding errata) or revisions are not applicable to this standard. However, the parties who reach an agreement based on this standard are encouraged to study whether the latest versions of these documents can be used. For all undated referenced documents, the latest versions are applicable to this standard. GB/T5009.22-2003 Determination of aflatoxin B in food 3 Principle
Use triazine-0.1mol/L phosphoric acid or petroleum ether-methanol/water to extract ochratoxin A in the sample. After liquid-liquid partitioning, the sample extract is compared with the standard on a thin layer chromatography plate based on the yellow-green fluorescence produced under a 365nm ultraviolet lamp. 4 Reagents
4.1 Petroleum ether (60℃~90℃ or 30℃～60℃). 4.2 Methanol.
4.3 Triazine.
4.4 Toluene.
4.5 Ethyl acetate.
4.6 Formic acid.
4.7 Glacial acetic acid.
4.8 Ether.
4.9 Benzene-acetonitrile (98+2).
4.10 0.1mol/L phosphoric acid Lc(H,PO,)=0.1mol/L]: Weigh 11.5g phosphoric acid (85%) and dilute to 1000mL with water. 4.112mol/L hydrochloric acid solution Lc(HCI)=2mol/L]: Measure 20mL hydrochloric acid and dilute to 120mL with water. 4.12 Sodium chloride solution (40g/L)).
4.13 0.1mol/L sodium bicarbonate solution Lc(NaHCO)=0.1mol/L: Weigh 8.4g sodium bicarbonate, dissolve in appropriate amount of water, and dilute to 1000mL with water.
4.14 Silica gel G: For thin layer chromatography.
4.15 Ochratoxin A standard (hereinafter referred to as OA). 4.16 Ochratoxin A standard solution:
4.16.1 Ochratoxin A standard stock solution: Prepare 40ug/mL ochratoxin A standard stock solution with benzene-glacial acetic acid (99+1), and determine its concentration with a UV spectrophotometer. The concentration is determined in accordance with 3.14 of GB/T5009.22-2003 (the maximum absorption peak wavelength of ochratoxin A is 333nm, the molecular weight is 403, and the gram molecular extinction coefficient value is 5550). Store in a refrigerator away from light. 4.16.2 Ochratoxin A standard working solution: Accurately pipette the stock solution, dilute it with benzene to 0.5μg of ochratoxin A per milliliter, and store in a refrigerator away from light.
GB/T5009.96—2003
5 Instruments
All glass instruments must be soaked in dilute hydrochloric acid and rinsed with tap water and distilled water. 5.1 Small crusher.
5.2 Electric oscillator.
5.3 Glass plate: 5cmX20cm.
5.4 Thin layer applicator.
5.5 Development tank: inner length 25cm, width 6cm, height 4cm. 5.6 Ultraviolet lamp: 365nm.
5.7 Micro syringe: 10uL, 50μL. 5.8 10mL small concentration bottle with 0.2mL tail tube. 6 Analysis steps
6.1 Preparation of sample
Weigh 250g sample, crush it and pass it through a 20-mesh sieve for later use. 6.2 Extraction
6.2.1 Method A
Weigh about 20g of sample, accurate to 0.001g, and place it in a 200mL stoppered conical flask. Add 100mL of chloroform and 10mL of 0.1mol/L phosphoric acid. Shake for 30min and filter through rapid qualitative filter paper. Take 20mL of the filtrate and place it in a 250mL separatory funnel. Add 50mL of 0.1mol/L sodium bicarbonate solution and shake for 2min. After standing and stratification, place the chloroform layer in another 100mL separatory funnel (a small amount of emulsified layer, or even if the chloroform layer is completely emulsified, it can be placed in a separatory funnel), add 50mL of 0.1mol/L sodium bicarbonate solution to repeatedly extract the chloroform layer. After standing and stratification, discard the chloroform layer (if the chloroform layer is still emulsified, discard it and it will not affect the result). Put the sodium bicarbonate water layer into the first separatory funnel, add about 5.5mL 2mol/L hydrochloric acid solution to adjust pH 2~3 (test with pH test paper), add 25mL chloroform and shake for 2min, let stand to separate, put the chloroform layer into another 250mL separatory funnel containing 100mL water, shake the acid water layer with 10mL chloroform, extract, let stand, put the chloroform layer into the same separatory funnel. Shake, let stand to separate, wipe the lower end of the separatory funnel with absorbent cotton, put the chloroform layer into 75mL evaporating blood, put the evaporating blood on a steam bath to ventilate and evaporate. Dissolve the residue in the evaporated blood with about 8 mL of trifluoromethane in portions, transfer to a 10 mL concentration bottle with a tail tube, place in a 80°C water bath, heat with steam and blow nitrogen (N2), concentrate to dryness, add 0.2 mL of benzene-acetonitrile (98+2) to dissolve the residue, shake and use for thin layer chromatography. 6.2.2 Method B
Weigh 20 g of the sample that has been crushed and passed through a 20-mesh sieve and add it to a 200 mL stoppered conical bottle, add 30 mL of petroleum ether and 100 mL of methanol-water (55+45), apply a layer of water on the stopper and cover tightly to prevent leakage. After shaking for 30 minutes, filter through a rapid qualitative filter paper into a separatory funnel. After the lower methanol-water layer is separated, take out 20 mL of the filtrate and place it in a 100 mL separatory funnel. Test with pH test paper, generally pH 5-6. Add 25mL of chloroform and shake for 2 minutes. After standing and separating, release the chloroform layer into another separatory funnel. Repeat the shaking with 10mL of chloroform to extract the methanol-water layer (when shaking and extracting with chloroform, if emulsification occurs, methanol can be added dropwise to promote separation). Combine the chloroform layers in the same separatory funnel and add 50mL~100mL of sodium chloride solution (4.12) (the amount added varies depending on the variety, 100mL for soybeans and 100mL for small For wheat and corn, add about 50mL), shake and place (for soybean sample extract, gently invert the separatory funnel repeatedly to make the emulsified layer rise gradually. If the emulsification is serious, add a little methanol). After the cyanomethane layer is clarified, wipe the lower end of the separatory funnel with absorbent cotton, place the chloroform layer in a 75mL evaporating dish (for soybean sample, add 10mL of chloroform and shake, and combine the chloroform layer in the same evaporator III), and place the evaporator III on a steam bath for ventilation and drying. The following operations are performed according to method A starting from "dissolve the residue in the evaporated blood with about 8mL of chloroform in batches".
6.3 Determination
6.3.1 Preparation of thin layer plate
Weigh 4g of silica gel G, add about 10mL of water and grind in a mortar until it becomes a paste. Immediately pour into the applicator to make 5cm×20cm, thickness 684
GB/T5009.96—2003
0.Three 3mm thin layer plates were dried in air, activated at 105℃～110℃ for 1h, taken out and stored in a desiccator. 6.3.2 Spotting
Take two thin layer plates and use a micro syringe to drop two points on the baseline 2.5cm from the bottom of the thin layer plates: drop 8μL of OA standard solution (concentration 0.5μg/mL) at 1.7cm from the left edge of the plate, drop 25μL of sample solution at 2.5cm from the left edge of the plate, and then drop 8μL of OA standard solution (concentration 0.5μg/mL) on the sample solution point of the second plate. When spotting, blow dry with a hair dryer while dropping, using hot and cold air alternately.
6.3.3 Development
6.3.3.1 Development agent
Developing agent: ether or ether-methanol-water (94+5+1). Longitudinal spreading agent:
a) Toluene-ethyl acetate-formic acid-water (6+3+1.2+0.06) or toluene-ethyl acetate-formic acid (6+3+1.4); b) Benzene-glacial acetic acid (9+1).
6.3.3.2 Development
Horizontal development Pour 10mL of horizontal spreading agent into the development tank, first vertically develop the thin layer plate to 2cm~3cm from the origin, take out the solvent for ventilation and volatilization for 1min~2min, then place the long side of the thin layer plate near the standard point in the solvent in the same development tank for horizontal development. If the horizontal spreading agent is insufficient, add an appropriate amount, develop to the end of the plate for 1min, take out the solvent for ventilation and volatilization for 2.min~3min. Vertical development: Pour 10mL of vertical spreading agent into another development tank, and vertically develop the thin layer plate after horizontal development to 13cm~15cm from the origin at the leading edge. Take out and ventilate and evaporate until there is no sour smell on the plate surface (about 5min~10min). 6.3.4 Observation and evaluation
Place the thin layer chromatography plate under 365nm wavelength ultraviolet light for observation. a) Compare the two plates under ultraviolet light. If the sample solution of the second plate shows the minimum detection amount at the corresponding position of the OA standard point, and no fluorescent spot appears at the same position of the first plate, the OA content in the sample is below the minimum detection amount of 10μg/kg in this determination method.
b) If the sample solution of the first plate shows a fluorescent spot at the same position as the sample solution of the second plate, check whether the fluorescent spot of the sample solution of the second plate overlaps with the added standard fluorescent spot, and then perform the following quantitative and confirmation tests. 6.3.5 Dilution and determination
Compare the fluorescence intensity of OA in the sample solution and the standard OA spot, and estimate the dilution multiple. After the thin layer plate is developed in both directions, when the OA content in the positive sample is high, the fluorescent spot of OA will be horizontally elongated, making the spot flat, or divided into full yellow-green fluorescent spots. This is because the amount of OA at the origin exceeds the adsorption capacity of silica gel during the horizontal expansion process. The impurities and residual solvents at the origin extend the OA point horizontally during the horizontal expansion process. At this time, the total intensity of the yellow-green fluorescence of OA can be compared with the standard fluorescence intensity to estimate the number of microliters to be added or the required dilution multiple. When determining the content after dilution, two standard points can be added to the left baseline of the sample point. The amount of OA can be 4ng and 8ng. Compare the fluorescence intensity of the sample solution and the two standard 0A fluorescence points to roughly quantify. 6.3.6 Confirmation test
Spray the chromatographic plate with sodium bicarbonate ethanol solution (dissolve 6.0g of sodium bicarbonate in 100mL of water and add 20mL of ethanol), dry at room temperature, and observe under a long-wave ultraviolet light. At this time, the OA fluorescence point should change from yellow-green to blue, and the fluorescence intensity will increase, which can make the method detection limit reach 5ug/kg, but the rough quantification is still based on the yellow-green fluorescence before spraying. 7 Calculation of results
XD×1000
Wherein:
X—the content of ochratoxin A in the sample, in micrograms per kilogram (μg/kg); mi—the amount of OA measured on the sample solution spot on the thin layer plate, in micrograms (μg);685
GB/T5009.96—2003
D Total dilution multiple of the sample solution:
V, the volume of the benzene-acetonitrile mixture, in milliliters (mL) V. The volume of the sample solution added when the minimum fluorescence point appears, in milliliters (mL) - the mass of the equivalent sample when the benzene-acetonitrile is dissolved, in grams (g). m
8 Precision
The absolute difference between two independent determination results obtained under repeatability conditions shall not exceed 20% of the arithmetic mean. 686
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