GB/T 5009.111-2003 Determination of deoxynivalenol in cereals and their products
Some standard content:
1CS67.040
National Standard of the People's Republic of China
8/T 5009.111—2003
Replaces GB/T14929.5199
Determination of deoxynivalenol in cereals and cereal products
Promulgated on August 11, 2003
Ministry of Health of the People's Republic of China
Administrative Committee of Standardization of the People's Republic of China
Implementation on January 1, 2004
GB/T 5009.111-2003
This standard replaces GB/T14929.5
51994 Determination of deoxynivalenol in cereals and cereal products. Compared with B/T14929.5-1994, this standard is modified as follows: The Chinese name of the standard is modified. The Chinese name of the standard is revised as "Determination of deoxycholate ene in food and its products according to B/T20011.1-2001 Standard Compilation Rules Section 4: Chemical Analysis Methods" The structure of the original standard is modified. This standard was issued by the Ministry of Health of the People's Republic of China and drafted by: Food Hygiene Supervision and Inspection Institute of the Ministry of Health The main drafters of the first method of the whole standard are Yang Chuanhe, Shi Jiayun and Ji Rong. The original standard was first issued in 1954. This standard is GB5009.111-2003. This standard specifies the determination method of deoxycholate enol in contents and base materials and their products (e.g., egg, bread, etc.) and the non-condensing determination method. This standard specifies the detection limit of deoxycholate enol in contents (e.g., millet, rice, cake, blood bag, etc.) and their products (e.g., bread, etc.). The first detection limit of this standard is 0.1%/kg. The second detection limit is 0.1%/kg. The first method is thin layer chromatography determination method. Principle
The olefin alcohol in the grain and its products is extracted and purified, and then the concentrated silica gel 6 thin layer is displayed, such as hot sea carbon energy: when preparing the thin layer, the color can be improved under the spotlight or light, and the amount is much light.
3 Wiping agent
3.1 Methane.
3.2 No permanent ethyl alcohol,
3.3 Methyl alcohol
3.62 Full,
3.7 Propylene glycol:
3.8 Propanol.
3,10 No permanent ethyl alcohol.
3. Aluminum carrier (GF: chemically pure
3.12 Neutral hydrogenation, 3.13 Activated carbon: 20% fluorine, 300% chlorine, 45% fluorine, 3.14 Activated carbon: 20% fluorine, 3.15 mol/l, 10% fluorine, 10% chlorine, 10% fluorine.
3.4 G-phase chromatography.
3.15 Hydrogenated fumarate (hereinafter referred to as DON) standard liquid, weigh 5.0g LX. add acetic acid 2. Lu Chen wake 3! Dissolve in plastic
standard liquid 5a. ethyl acetate-methanol (9+1) release chamber 1 n1. This liquid with bright content g: calibration instrument
4.1 small plastic powder drop machine,
4.2 electric vibrator.
4.3m. or black hair,
GH/T 5009.111—2003
4.4 Chromatography chamber: inner diameter 2cm1, length 1cm. No sample, 4. 510 mL of concentrated solution: diseased part, HC. 2 mL graduated tube, 4.6 Plate. 5cm×2ucm
4.7 Thin layer analyzer n. 5smm.
4.8 Extension tube: inner length 25cm width 6en, commercial 4cn. 4.9 UV lamp: 365nm.
4.10 Volumetric syringe, 10 L, 5J mL
EOml. Flat room,
4.12 Double-layer thin layer scanning electron microscope. 5 Analysis steps
5. 1 Extraction
Weigh 20g of powdered sample and place it in a 230mL bottle with 3ml of water and 10ml of chloroform-anhydrous alcohol + 2ml. Spread the water on a plate, cover tightly, and filter through folded quick-press qualitative elastic paper. Collect 25ml of the filtrate and place in 75ml of gelatin evaporator. Place it in a 90° water bottle and ventilate until dry.
5.2 Purification
5.2.1 Liquid-liquid distribution
5.2.1.1 Cereals: Use 50mL petroleum ether to concentrate the residues in the evaporated solution, add 100mL separatory tube, wash with 20mL (4-1 mL) methanol-water in batches, transfer to the separatory tube 5.2.1.2 Cereal products (egg lattice, cake, noodles, etc.), use 100=1% petroleum ether in batches to dissolve the residues in the evaporated solution, wash with 250mL separatory tube, add 1 mL methanol-water (1 mL) in batches to evaporate, transfer to the separatory tube. 1.5m1n was obtained by digging and separating, and the static content was about 15m:n. After the layers were separated, the alcohol in the lower layer was collected and purified by injection. The colored objects at the junction of the two phases were put into the column:
5.2.2 Column purification
5.2.2.F small table and its products: about 0.1g of absorbent cotton was plugged at the reverse junction of the lower layer and the small tube. The whole layer was filled with 0.5g of light-sensitive chemical, and the surface was flat. Then 0.4g of active chemical was added and it was tightened. Insert a leather stopper into the small kidney at the lower end of the special layer column and plug it on the push filter bottle. Put a flat-bottomed tube in the axial flow bottle to connect the suction filter bottle to the water system or vacuum system when passing through the column. Slightly open the pump to make the activated carbon ballasted and the alcohol-water extract in the funnel be carefully and evenly added into the column through the active tube wall. Control the flow rate to be 11~1% per full (3mlia~2). When the methanol-water extraction is almost finished passing through the column, add 10L methanol-water (4+1) to precipitate and filter until no more liquid flows out of the column. Control the flow rate at 2ml./min~3m1./min. If the speed is too slow, the purification result will be too bad; if it is too slow, the time will be too long. 5.2.2.2 is the same as 5.2.2.1 except that the amount of activated carbon is changed to 0.3g. 5.3 Prepare the sample for analysis. Pour the eluate after passing through the column into 75mL glass evaporator III, wash it with a small amount of methanol-water (1+1), and put it in a water bath to dry. 5.3.1 Add 31mL ethyl acetate to the hot water, heat to 400℃, and gently rotate it in a water bath. Heat and degas for several times to make it fully boil and remove the V in the residue. Then add ethyl acetate and heat until it reaches T. Then add 3 mL of ethyl acetate and treat it in the same way. Heat the last 3 mL of ethyl acetate to boiling, cool to room temperature and transfer to a bottle. Wash the evaporating liquid with three portions of 1.5 mL of ethyl acetate and put it into a concentration bottle. 5.3.2. Add 3 mL of ethyl acetate to the whole heat and heat to boiling. Gently invert the heat plate on a water bath for several times: quickly boil and dissolve the TDCN in the residue. Cool to room temperature and transfer to a concentration bottle. Add about 1.5 mL of methanol-1-2 to the evaporating liquid and dissolve it in a glass shaker. After the solution was evaporated on the water tower, 3 mL of ethyl acetate was added, heated to boiling, and then stirred to make it fully saturated. After being placed in the room temperature, it was transferred to the same concentration medium, and 0.5 mL of 112% ethanol and 3 mL of Z. pyrogenic bacteria were treated in the same way, and the ethyl acetate extraction method was used. 54
GB/T 5009.111—2003
The concentrated solution was concentrated to dryness in water at about 95°C with sufficient heating air, cooled to room temperature, and then 2 mL of trimethylolethane (type 10) was added to the solution, leaving it for thin layer analysis. 5. Adjustment
5.4.1 Preparation of thin plate: Silicon stock G about S1 photoaluminum fluoride (Al: 6H: 0) water solution, about 2min sticky, pinned into a 5×20cm thin layer, placed in a room for drying, activated at 105% for 1h, and stored in a desiccator for later use. 5. Rate.2 Spotting: Spot the sample on the line 2.5cm below each thin layer. For each sample, spot the first thin layer plate first. Spot the sample at 8n near the left edge, and spot 2TION standard liquid (n2) on the horizontal line 1.5m from the top of the required plate corresponding to the base short Beijing point. According to 5.1.3., after drying and heating, spot 2LDCN standard (5g) on the baseline 1cm away from the starting edge of the layer plate. Second board: The fluorescent sample on the first fast tax needs to be 6 meters away from the left side of the second reflection layer board.Add 25g of 1 standard solution at three locations (2m away), add 1 standard solution at 25g) at a distance of 2m to the right, and fill the sample with 2! Weigh the standard solution. Take a quantitative measurement of these samples: add a drop of a machine point at a distance of 08c~1c from the edge of the thin plate (the machine point can be used for the measurement of the sample, or the method of measuring the sample by the user), fill two standard points at 1.2cm from the edge of the plate: 50g for small plates, 75ng for small plates, and 10"g for small plates, and then add a DC standard point (about 5R) on the horizontal line 1.5cm from the top of the plate, which corresponds to the three points on the baseline. 5.4.3 Spreading agent: acetaldehyde, acetaldehyde-1 (S5+5) or water-soluble acetaldehyde. Choose one of them, make the test point 1D0> deviate from the origin by 0.7ctl--1cm·cut and impurity fluorescence separation,
cut dimethyl ether-propanol (8+1+1>trifluoromethane-propanol-isopropanol-water (7.:+1+1.i-0.1). 5.4.3.1 Full total I take the inner guard 10 mL horizontal agent. The thin plate with good opening point is not the long side of the side of the fence. The exhibition is requested to take out the wind and beat the product for a small safety product. mL is not aldehyde 3~l description exhibition-happy, 1min ventilation selection min
5.4.3.2 effect exhibition: the room slot is 10m1. The weave is reported, the horizontal expansion rate of the thin layer of the board is afraid of the internal fee 159, take out the ventilation and stir 1! . Because the effect of the soft method is greatly affected by the humidity of the air, when the board separation result is not the first fast thin layer of the test point, there is no interference, the following development methods can be changed according to the polarity. s) trichloromethane propanol [8+1+1) 1) trifluoromethane-two ketone two alcohol (1+1) and in the stock five increase the surface of the disk waterproof saturated paper c) berry Huan-reduced ketone isoalcohol-water 7.5+1 2. = +(.1). ）-methane isoflash alcohol water (7,5111.5. Apply water and release the fiber inside the skirt. Change the longitudinal expansion direction, and open two thin layer boards. If the lower opening of the tree version is too large, it will flash quickly. 5.4.4 Display: First look at the heated thin layer board and you can see that it is blue-purple. At this time, TX does not show light. After the hot thin layer is reported, the mixed origin is still hot. It is close to the D fluorescent point, but not XN. Then put this thin morning version at 139C in the hot m--0 crazy out on the cold surface-after m Rong external prohibition 5.4,5 observation and evaluation, the thin layer recovers through the horizontal embedding material, and the board L moves with the IXN of the model (.7m~1). It is reported that the ON point of the sample used in the proposed exhibition is compensated for the affinity of the fluorescent material. Light resistance. The three FOY standard points on the upper and lower layers of the plate can be used as the horizontal positioning points of the sample KN point and a standard X point respectively. The N points are arranged and compared with the N standard points after the K: standard agreement. In this way, the horizontal and horizontal positioning of the sample 1XN points are determined by the two-dimensional diagram to determine the quality of the sample. A standard point is selected at random.
If the sample solution on the first thin layer plate shows light loss, and the sample solution on the first thin layer plate is full, the laser strength can be equal to 25% of the standard or less than .05g. When the non-strategic quantitative measurement is performed, there is no effect on the accuracy of the sample. Two standard points are selected. The yellow light intensity can be compared with the sample II point. GR/T5009.111-2003 5.4.6 Photometric determination: The excitation light is 340nm, the emission wavelength is 400nm, and the standard 10 small fluorescent points on the thin layer plate are at least 120g200g.490g. The obtained value should be in a positive relationship with the amount of D0V. For samples with a DC content of more than 1.3mg/kg, the light intensity is determined. When the DN content is 0.mg/kg, the sample wave is added so that the measured DON amount falls between 130-200g. When using a thin layer photometric density meter to determine, drop two standard points on each plate. The amount of the standard point is 00:200n. g or 20Crg-150ng, take the measured single-surface value as the vertical coordinate, 1)CN as the horizontal coordinate, draw the standard line 5.4.7 Calculation of results
Calibration formula (1) Calculation:
Where:
The content of D(>N in the test group, the unit is grams per gram (m/kg); A
The amount of V measured on the thin-point plate, the unit is microgram (μg): The volume of the melt or flow of the added methyl cyanide-acetyl cyanide mixture, the unit is milliliter (L); The volume of the droplet, the unit is milliliter (ml). The total volume of the sample!
The mass of the residual sample dissolved in the tri-anion-acetyl cyanide mixture, the unit is milliliter (g). --( 1 )
5.4. Daily standard pit width: When the amount of DON added to the indoor wheat is n.3, 0.5, 1ug/kg, and each level is n=4, the daily visual comparison determination is c.2378±G, 323k.0.5=0.681g, 0.(97gU.112g: the full layer light density [calculated by the station results are 0.290g13.05.0.450.0771=0.15g respectively. The 0N recovery of the small product is 80~10056, and the amount of DON added to the blank rice is 0. 3.0, 1mg/%, each half water = 4 hours, visual comparison test results are C.234 + 5.05g, 0.415.0, G.OO = n, the above data is X-SD,
Second method immunoassay (ELISA)
6 Principle
Adsorb the known antigen on the surface of the solid phase, wash off the adsorbed antigen: add a certain amount of antibody and the test solution (containing the antigen) to compete with the reaction mixture to form an antibody-antibody complex on the surface of the solid phase carrier. Remove the remaining antibody components, then add the enzyme-labeled anti-immune protein complex to combine with the antigen-antibody complex adsorbed on the surface of the solid phase carrier, and then the substrate is degraded under the action of chemistry to produce negative products. The degradation amount of the test substance is measured by the aldehyde detector, and the amount of antigen in the test substance is calculated according to the standard.
7 reagents
7.1 anti-mixing, hybrid tobacco cell line 3? Specific monoclonal antibodies against adrenocortical agents were generated 7.2 Conjugate of antigen binding protein and carrier protein-bovine serum albumin (BSA) (LKOV-BSA) 7.3 Bovine serum albumin (RSA), tetramethylrhodamine: conjugate of rabbit anti-rat immunoglobulin and horseradish peroxide 7.4 Temperature 20, 3% hydrogen peroxide, methanol, petroleum aldehyde, anhydrous, 1% ethanol. All other chemicals were analytical reagents and were required to be water or equivalent. 7.5 The washing liquid was 1H 2 SO 4 carbonate standard dilution. The preparation method was to weigh 1.50 g NuO 2, 2.5 g carbon monoxide and add 1 ml HO. 7.6 The washing liquid was 15% Na 2 O in H 2 SO 4 solution. 7.4 Carbonate solution (abbreviated as PHST) was used. The preparation method is:
GB5009.111-2003
Weigh 2.0 mL of phosphate chloride (KHF()>0.2), 2.0 mL of sodium phosphate (NaHPO4,12HO), 3.0 mL of sodium phosphate (NaC1), 0.25 mL of potassium phosphate (KCl), add water to [FC.,7.7] and dilute to pH 5.0. The preparation method is 3.2 mol/L citric acid (CH30,HO), which is called citric acid. 1 mol/L hydrogen phosphate is called NgTIT(). Take 284 g of phosphate distillate and add water to 1300 to make ethyl. Take 24.3 mL of liquid A, 25.7 mL of liquid B and cut into 100 mL of water. 7. Use the standard drop solution of emetic toxin in the same area before the test, %/m. emetic toxin case warning, "-80 crystal sugar paste. On the day of the test, take the end of the liquid, use 2% alcohol PRS (preparation method 1S-1 this essence 2C that is) to bail or make the required electroporation device. The old blood sample was washed with tap water and rinsed with steamed water. 8.1 Alcohol standard test instrument: 6.2 Enzyme label knock-out plate (4 holes or starting hole) 3 Electric pump 8.4 Electric heating constant run water bath ladder: 8, E tool 0. 2 ml. 1 room tube ml-small Zhang Zhizhi h25tm1. Divide into buckets.
9 Analysis step
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20 broken wells and 20 days of good quality samples, write 20 people three single burn, add 8 water and 100 one level of anhydrous ethanol (4+1). Apply a layer of water on the bottle stopper to prevent full, after 1h, pass through the ball qualitative filter paper, collect 25m1. The filtrate is evaporated in the evaporation door, 90 water is ventilated. 6 yuan petroleum ether is dissolved in several times or made, and poured into a 250 decanter funnel, and then washed with 20ml, methyl alcohol-water (-1) in several times and transferred to the same decanter funnel, and sent to 1,5min, collect the lower layer of methanol water extraction and purify it through the chromatography column. Add about 1g of cotton wool at the lower end of the chromatography column, plug it as tightly as possible, first load 0.5g of neutral oxidizing agent-original surface, then add 3,4 active ingredients, knock it). The eluted solution after long injection is then poured into the warm water, put the hair into the water 5% concentrated water, add 3M acetic acid, heat to 10, and then repeat the last 10mL acetaldehyde acetate, and then transfer it to a 10mL bottle at room temperature. Wash the evaporated blood with a drop of acetaldehyde and put it into the concentrated auxiliary bottle, heat the reduced bottle in a 95% water bath with steam, evaporate it, and then use 0.2M pH 5.0 containing 20% acetaldehyde to discard it for EI.ISA detection. 9.2 ELISA test
9.2.1 Label the microplate with DON-BSA (20/ml) and 10L of aldehyde, incubate overnight. 9.2.2 Wash the labeled plate with P1ST 3 times each time. min later, add the standard ON with different concentration (as standard sample extraction) (as test sample, the sample contains heavy > antibody (11), the mixture should be prepared on the day before, overnight, and set aside at 37℃:1. 9.2.3 Wash the plate 3 times, each time with a small amount, add the secondary antibody, 13μ1 per well, for 41.5h. 9.2.4 After washing as above, add substrate solution L, i.e. 51.TM10mgTMB in 1ml. Add 10ml dimethyl fluoride solution. Add 103% oxygen to the substrate, 10Vr per well, 39min. 9.2.5T2m01.1 Please adjust the acid mixture to 450℃. The temperature should be 50℃. 57
GR/T 5009.111—2003
10 Calculation of results
Calculate according to formula (2,
Where:
K——the content of vomiting agent per gram, the unit is nanogram per gram (m/s); the content of vomiting agent measured by the micropore test of the enzyme standard (obtained according to the standard), the unit is nanogram (m); the volume collected by the formula is milliliters (L); V——the volume of the added sample, the unit is liters (mL); D——the total dilution multiple of the sample solution!
The mass of the sample, the unit is gram (g)
11 The absolute difference between two independent determination results obtained under the conditions of precision and reproducibility shall not exceed 15% of the arithmetic mean.
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