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GB 23748-2016: Detection of irradiated foodstuffs -- DNA comet assay -- Screening method
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Detection of irradiated foodstuffs -- DNA comet assay -- Screening method
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Detection of irradiated foodstuffs -- DNA comet assay -- Screening method
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Basic data | Standard ID | GB 23748-2016 (GB23748-2016) | | Description (Translated English) | Detection of irradiated foodstuffs -- DNA comet assay -- Screening method | | Sector / Industry | National Standard | | Classification of Chinese Standard | C53 | | Word Count Estimation | 20,283 | | Date of Issue | 2016-12-23 | | Date of Implementation | 2017-06-23 | | Older Standard (superseded by this standard) | GB/T 23748-2009; NY/T 2214-2012; SN/T 2910.2-2011 | | Regulation (derived from) | National Health and Family Planning Commission Notice No.17 of 2016 | | Issuing agency(ies) | National Health and Family Planning Commission of the People's Republic of China, State Food and Drug Administration | GB 23748-2016: Detection of irradiated foodstuffs -- DNA comet assay -- Screening method---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.
Detection of irradiated foodstuffs - DNA comet assay - Screening method
National Standards of People's Republic of China
National Food Safety Standard
Identification of irradiated food screening
Issued on. 2016-12-23
2017-06-23 implementation
National Health and Family Planning Commission People's Republic of China
China Food and Drug Administration released
Foreword
This standard replaces GB/T 23748-2009 "Identification of irradiated food were screened DNA comet assay method", NY/T 2214-
2012 "Identification of irradiated food OSL Law" and SN/T 2910.2-2011 "Identification of irradiated food outlet - Part 2. Single Cell
Gel electrophoresis. "
This standard compared with GB/T 23748-2009, the main changes are as follows.
--- Standard name was changed to "national food safety standard screening method to identify irradiated foods";
--- Adds two screening methods. optically stimulated luminescence method and microbiological screening method;
--- Increased DNA comet assay method of limiting instructions.
National Food Safety Standard
Identification of irradiated food screening
1 Scope
This standard specifies three rapid screening of food irradiation had accepted identification methods. optically stimulated luminescence method, DNA comet assay method and microorganisms
Learning was screening.
This standard OSL law applies to crustaceans, spices and condiments irradiation identification products; DNA comet assay method is suitable for
Animal products, identification of irradiated grains, nuts, fruits and vegetables; microbiological screening method is suitable for frozen Poultry and Seafood, and other fresh food
Irradiation identification.
The first method OSL method
2 Terms and definitions
2.1 OSL (photostimulatedluminescence, PSL)
After the rich silicate sample capture radiant energy by certain wavelengths of light excitation, in the form of light released from the phenomenon.
2.2 PSL intensity (PSLintensity)
After the amount of light emitted by the sample is detected by optical excitation, the photon count rate indicates.
2.3 Screening PSL (screeningPSL)
PSL initial strength of the sample measurement.
2.4 Calibration PSL (calibratedPSL)
After screening PSL measurement, the same sample with a known dose of radiation after the PSL intensity measurement.
2.5 threshold (thresholds)
In the screening mode, for determining whether or not a sample irradiated PSL strength, comprising a lower threshold (T1) and a high threshold (T2).
2.6 negative PSL (negativePSLresult)
PSL measured intensity below the low threshold value.
2.7 Suspicious PSL (intermediatePSLresult)
PSL measurement of strength between the low threshold and high threshold.
2.8 positive PSL (positivePSLresult)
PSL intensity measured above the high threshold.
2.9 blank (darkcount)
When no light stimulation, empty sample containers obtained photon count rate.
2.10 positive control (lightcount)
The reference light source (such as a scintillator containing 14C or equivalent) placed photon count rate of the sample container obtained.
Principle 3
Rich silicate material samples can be stored ray energy. Can excited by certain wavelengths of light irradiation again, the stored sample
The amount can be released, resulting in the release of an optical signal light. Use light intensity of light release detector test light signal, large signal intensity comparison of different light
Small, it is determined whether the samples irradiated.
4 instruments and equipment
4.1 OSL measurement system. including sample containers, excitation light source, and synchronous pulse stimulator photon counting system.
4.2 Measurement disc. diameter 5cm.
4.3 sources of ionizing radiation. ionizing radiation sources used and managed according to GB 17568 and GB/T 25306 requirements.
Step 5 Analysis
Note. Place the sample in the dark before measuring. Use disposable measuring disc is measured.
5.1 Preparation of spices and condiments samples
Prepare duplicate samples, spread evenly in the bottom of the measuring plate were detected. If the two test results compared with the determination threshold value is inconsistent, then
After re-detected sample aliquot 4, whichever is the highest value as a result of two detection determination, and so on.
5.2 Preparation of samples crustaceans
The shell or shelled into the test sample dish, help detect the OSL signal when samples with animal intestines. As larger individuals,
The samples can be cut; also desirable intestines (less than 6) in a measuring plate is measured.
5.3 Instrument settings
Screening for irradiated food system set individual measurement parameters.
Check the blank and positive controls.
NOTE. should periodically or when a positive result after the measurement, the containers are empty sample testing, inspection equipment for contamination.
5.4 Determination of the sample
5.4.1 Screening Measurement
Conduct sample testing and the results recorded.
5.4.2 Calibration Measurement
Screening sample measured after capping save to prevent sample loss or contamination. These samples were then irradiated specific dose (spices and shellfish food
Was irradiated 1kGy), room temperature (crustaceans and other perishable samples should be refrigerated after irradiation) from light 12h, calibration measurements.
6 The results presentation
6.1 threshold setting
Spices and condiments threshold value is set. T1 = 700counts/min, T2 = 5000counts/min.
Crustaceans threshold set as. T1 = 1000counts/min, T2 = 4000counts/min.
6.2 Analyzing Results
According to the comparison with the threshold value, the measurement results of the sample can be divided into negative, suspicious, positive three cases were judged.
6.2.1 negative results
6.2.1.1 Screening PSL
PSL screening result was negative, non-irradiated sample can be determined.
6.2.1.2 Calibration PSL
When corrected PSL and PSL with negative screening, the sample can not be determined to be irradiated food. To further confirm whether the sample is then
Received radiation should be used other confirmatory methods of identification of irradiated food.
Calibration PSL negative, but positive screening PSL, indicating that the measuring system error, it should take a fresh sample of the original re-measured.
6.2.2 Suspicious Results
6.2.2.1 Screening PSL
PSL as suspicious at screening results can not be directly determined whether the sample had received radiation.
6.2.2.2 Calibration PSL
When corrected PSL and PSL screening results with the suspect, judging samples received irradiation.
When corrected PSL as suspicious results PSL screening results are negative, indicating that the sample may be hypoallergenic non-irradiated foods. To enter a
Step confirm whether the sample had received radiation, other confirmatory method should be used for identification of irradiated food.
Calibration PSL as suspicious results PSL screening for high value positive results, indicating that the measurement is incorrect.
Calibration PSL as suspicious results, positive results of screening PSL close to T2, indicating that the sample may have received higher doses of radiation correction
Photos. It should be re-measured sample is determined.
6.2.3 positive results
6.2.3.1 Screening PSL
PSL screening when a positive result, the sample can be determined received irradiation.
6.2.3.2 Calibration PSL
When corrected PSL and PSL screening for a positive result at the same level judgment sample received irradiation.
Calibration PSL when a positive result, and the result is far higher than the negative or suspicious screening PSL, indicates that the sample may not irradiated.
Calibration PSL and PSL screening results were positive, but less than the screening correction PSL PSL (a difference of 1-2 orders of magnitude), indicating that the measurement
Error, need to re-measure.
The second method DNA comet assay method
Principle 7
DNA molecules after irradiation fracture occurs. The cells were embedded in agar with lysis reagent dissolve the cell membrane, and then some
Voltage electrophoresis. Damaged DNA fragments move quickly in the direction of the electric field to the anode, forming a tail-like distribution. After staining, DNA that is
Cell loss occurs comet-shaped electrophoresis patterns, non-irradiated cells nearly circular pattern or a slight hangover.
8 kit
Unless otherwise indicated, the methods used were of analytical grade reagents and water for the three water GB/T 6682 regulations.
8.1 dimethyl sulfoxide (DMSO, C2H6OS).
8.2 Sodium chloride (NaCl).
8.3 Potassium chloride (KCl).
8.4 Disodium hydrogen phosphate (Na2HPO4 · 12H2O).
8.5 potassium dihydrogen phosphate (KH2PO4).
8.6 disodium edetate (Na2EDTA, C10H14N2O8Na2 · 2H2O).
8.7 Sodium hydroxide (NaOH).
8.8 Tris (Tris, C4H11NO3).
8.9 boric acid (H3BO3).
8.10 sodium dodecyl sulfate (SDS, C12H25O4NaS).
8.11 Acridine Orange (C17H19N3 · HCl · ZnCl2).
8.12 ethidium bromide (C21H20BrN3).
8.13 trichloroacetic acid (TCA, C2HCl3O2).
8.14 zinc sulfate (ZnSO4).
8.15 glycerol (C3H8O3).
8.16 sodium carbonate (Na2CO3).
8.17 ammonium nitrate (NH4NO3).
8.18 silver nitrate (AgNO3).
8.19 tungstosilicic acid (H4 [Si (W3O10) 4] · xH2O).
8.20 Formaldehyde (CH2O).
8.21 Glacial acetic acid (CH3COOH).
8.22 sulfonated pyridine (C10H12O4N2S).
8.23 agarose.
8.24 low melting point agarose.
Reagent preparation 9
Test used in the reagent preparation see Appendix A.
10 instruments and equipment
10.1 horizontal electrophoresis tank.
10.2 Electronic balance. a sense of the amount of 0.1g.
10.3 Electric heating magnetic stirrer.
10.4 microwave.
10.5 porous membrane. pore size 100μm, 200μm, 500μm.
10.6 microscope slides. 76mm × 26mm, with frosted edges.
10.7 fluorescence microscope. 100 to 400-fold; blue excitation wavelength of 460nm ~ 485nm, for acridine orange staining; green excitation wave
Length 515nm ~ 560nm, for sulfonated pyridine or ethidium bromide staining.
11 analysis steps
11.1 Sample Preparation
11.1.1 animal tissues
11.1.1.1 marrow sample preparation
Cut bone marrow weigh about 50mg a test tube, add 3mL ice-cold PBS buffer. Stirring with a glass rod, the fine
Hanging from the cell, the cell suspension was filtered with a microporous membrane 100μm, and the filtrate was collected and the filtrate was placed on ice boxes, alternate (cell suspension can be placed in ice
Placed, no more than 10min; after adding 5% to 10% DMSO as antifreeze, the cells can be placed at -20 ℃ 26h).
11.1.1.2 muscle tissue sample preparation
Muscle tissue with a scalpel to cut into thin slices (not visible fat). Weigh 1g muscle placed in a small beaker, add ice-cold 5mL
PBS buffer. The beaker was placed in an ice box, a glass rod stirring 5min, followed by 500μm and 200μm microporous membrane filtration,
The filtrate was collected and placed 5min, on standby in the ice box.
11.1.2 Plant Tissue
11.1.2.1 raw grain, nuts and other food seasonings
Sample Weigh 0.25g, ground with a mortar, placed in a small beaker, add 3mL ice-cold PBS buffer. The beaker was placed in an ice
Box with a glass rod stirring for 5min, followed by 500μm and 200μm microporous membrane filtration, the filtrate was collected and placed in an ice box
15min ~ 60min, standby.
11.1.2.2 strawberries and other berries
Weigh 0.25g strawberry berry, ground with a mortar, placed in a small beaker, add 3mL ice-cold PBS buffer. The beaker is set
Ice box with a glass rod stirring for 5min, followed by 500μm and 200μm microporous membrane filtration, the filtrate was collected and placed in an ice box
15min ~ 60min, standby.
11.1.2.3 vegetables (not including edible fungi)
The edible part of vegetables sliced with a scalpel, take 4g, was added 5mL ice-cold PBS buffer, ground with a mortar and placed
Small beaker, add 3mL ice-cold PBS. The small beaker placed in an ice box, a glass rod stirring 5min, followed by 500μm and
200μm microporous membrane filtration, the filtrate was collected and placed 15min ~ 60min, standby on an ice box.
11.2 precoated slides
Prior to coating, the slides should be soaked with methanol to remove surface oil. After air drying, a drop (approximately 50μL) agarose solution in the carrier coating
On the slide, immediately take another cover slides on agarose, agarose solution so spread out, remove the upper layer slides, air-dried 30min, and set aside.
11.3 embedding gel
Take 100μL cell suspension was mixed with 1mL embedded gel solution, and then take the mixture with a micropipette 100μL pipette tip light
Light coated on a pre-coated glass slides, coverslipped immediately spread the gel evenly, careful not to create a bubble. The slides were placed on ice box
5min, then peel off the cover slip from the agarose using a dissecting tip.
11.4 cells were lysed
The slides were immersed in 11.3 fully prepared lysis buffer (animal cell ≥5min, plant cells ≥15min), the cells were dissolved
Solution, do not touch agarose dissolution process.
11.5 rewet
The slides were immersed in the running buffer, time 5min.
11.6 electrophoresis
The slides were placed side by side horizontal electrophoresis tank, scrub edge toward the cathode. Electrophoresis tank electrophoresis buffer was added not too slides 2mm ~
4mm. Electrophoresis at room temperature for 20min, voltage 2V/cm. After turning off the power, the slides into the water 5min, air-dried at room temperature for 1h.
11.7 dyeing
11.7.1 acridine orange, sulfonated pyridine or ethidium bromide staining
The slides were immersed in staining solution (acridine orange staining solution, sulfonated pyridine or ethidium bromide staining solution staining solution) 3min ~ 5min, and then immersed
Water cleaning 0.5min ~ 1min, wipe excess water slides under fluorescence microscope.
11.7.2 silver staining
The slides were placed in a mixture of A10min, washing 1min, 40 ℃ ~ 50 ℃ constant temperature drying oven to dry at room temperature or 1h. then
The slides were immersed in a mixture of D10min ~ 20min, repeat the steps 1 to 2 times, dyeing time 5min ~ 10min, until slides
Rendering brown. Washing 1min, with a stop solution soak 5min E staining reaction is stopped, and then washed with water 1min. Finally, the slides were placed at room temperature,
Dry naturally. Stained slides will not fade, can be stored for a long observation.
Note. The fluorescent dye easily quenched rapidly to staining; staining solution toxic, after the end of the test, to deal with the waste disposal purification treatment again.
Observation 11.8 Identification
11.8.1 fluorescence observation
Acridine orange stained slides using fluorescence microscopy (460nm ~ 485nm, blue light excitation) observed, staining DNA emits green
Fluorescence.
Sulfonated pyridine or stained with ethidium bromide fluorescence microscope slides (515nm ~ 560nm, green excitation) with 590nm filter
Rays observed red fluorescence staining DNA.
11.8.2 White Observation
Silver nitrate stained slides can be observed using an ordinary microscope.
12 analysis results presentation
12.1 Expression of results
Through the microscope, the irradiated sample almost no intact cells, all comet image (see Figure B.1 and B.2); undamaged
Cells do not have light or tailing tailing (see Figure B.3 and B.4).
12.2 determination result
When the image appears comet, results are reported as positive, the sample may be irradiated.
When the comet image does not appear, the result is reported as negative, non-irradiated samples.
13 limiting illustration
In fact the comet samples to produce different shapes will be significantly different, but other processes may also affect DNA form, which gives the convergence
According to the determination of food difficult. Therefore, DNA comet assay as a screening method can only approach irradiated foods, and ultimately determine whether to accept food
Irradiation, irradiated foods need to meet the other confirmatory methods for testing.
The third method microbiology screening
14 Terms and Definitions
Endotoxin (endotoxin)
General toxic substances in the presence of gram-negative bacteria cell is endemic on the bacterial cell wall, and its toxic components primarily for the class
Lipid A. Only after lysis of bacteria, endotoxins will be released, the host can cause fever and endotoxin shock and other symptoms.
Principle 15
Under suitable conditions, bacterial endotoxin activates the pro-clotting enzyme in LAL, LAL make produce agglutination reaction to form a gel. Endotoxin
Gram-negative bacteria was positively correlated with the content, so the content can be determined according to the content of Gram-negative bacteria endotoxin.
Food irradiation after a dose of food in Gram-negative bacteria are killed substantially, but not because of residual endotoxin exposure
Treatment disappears. Therefore, the concentration of endotoxin can be measured to calculate the total number of irradiated foods dead and alive Gram-negative bacteria, while
Surviving on food Gram-negative bacteria were cultured counted by the difference between the two, it can determine whether irradiated food. If the two numbers
The obvious difference value, indicating that the sample may have received radiation.
16 Reagents and materials
16.1 Reagents
16.1.1 LAL. Sensitivity 0.5EU/mL.
16.1.2 non-pyrogenic saline injection.
16.2 Materials
16.2.1 peptone solution (see C.1).
16.2.2 nutrient agar (see C.2).
16.2.3 milk agar (see C.3.1).
16.2.4 crystal violet solution (see C.3.2).
16.2.5 penicillin solution (see C.3.3).
16.2.6 Gram-negative bacteria selective medium (see C.3.4).
16.3 Standards
Endotoxin standard sample (CAS No. 1235503). 50EU/tube, purity ≥99%.
16.4 Standard Solution
Endotoxin standard solution. take endotoxin standard sample, no heat is added 1mL saline dissolution of 50EU/mL of the standard solution.
17 instruments and equipment
17.1 thermostat water bath.
17.2 Autoclave.
17.3 Electronic balance. a sense of the amount of 0.1g.
17.4 oven thermostat.
17.5 Vortex.
17.6 Heated Incubators.
17.7 homogenizer. 8000r/min ~ 10000r/min.
18 analysis steps
Note. Samples should be stored at 4 ℃ within 24h test. If you can not verify immediately, it should be placed at -20 ℃, the longest storage time is not more than 30d.
18.1 sample preparation
Under sterile conditions, in different parts of the sample of the sample, for a total 10g. The sample was placed in a sterile plastic container using a disposable syringe added
90mL nonpyrogenic saline. 8000r/min homogeneous 1min ~ 2min, to a concentration of the sample solution 10-1, 4 ℃ stored for use,
Maximum storage time is not more than 24h.
18.2 Gram-negative bacterial culture counts
18.2.1 media preparation
Preparation of peptone solution, nutrient agar, Gram-negative bacteria selective medium (see Appendix C), pour the melted front plate
Training based on 45 ℃ ± 1 ℃ water bath insulation.
18.2.2 dilution and culture
18.2.2.1 The sample contamination of the sample solution (10-1) further diluted with peptone solution for sample dilution in accordance with
9mL 1mL volume ratio than 10-fold dilution, the sample diluent to give a concentration of 10-2 to 10-5.
18.2.2.2 The coated sample diluent 0.1mL different dilutions in nutrient agar plates at room temperature, allowed to stand for 90min up front.
Each dilution do two plates.
18.2.2.3 Each plate covering 10mL Gram-negative bacteria selective medium. Horizontally, so that solidification of the agar. Invert the plate on
21 ℃ ± 1 ℃ incubator for 24h ± 1h.
18.2.2.4 selected colonies of two consecutive dilutions plate count of 15 to 300 (see Table 1 Example D.1). Per gram of sample Gram
Negative bacteria according to equation (1).
N = Σ
V × n1 0.1 × n2 () [] × d
(1)
Where.
N --- gram negative bacteria per gram of the sample, in units of colony forming units per gram (CFU/g);
Σc --- long or 15 to 300 colonies of two successive dilutions and plate the colonies;
The V --- volume of broth per plate inoculated in milliliters (mL of);
Count the number of flat n1 --- first dilution;
Count the number n2 --- flat second dilution;
d --- plate count valid first dilution.
If only a dilution plate length 15 to 300 colonies, then n2 counted as 0 (see Table D.1 Example 2).
If the plate count all dilutions are less than 15, or the highest number of colonies colonies flat estimates as N values (see
Table D.1 Example 3).
If all dilutions of colony no longer bacteria, results were expressed as gram-negative bacteria is not detected, N value as 0 (see Table D.1
Example 4).
Within 18.3 endotoxin detection
18.3.1 LAL detection
18.3.1.1 LAL dissolved and dispensing
According to the labeled amount of reagent was added pyrogen saline solution, and mix with each 0.1mL bottle packed in amperes.
18.3.1.2 sample dilution
Use 96-diluted sample. Each well was added a 650μL sample was diluted in pyrogen-free saline. Take 300μL sample solution
A diluted sample is added to the first wells, mixed uniformly, a sample diluent to obtain 10-0.5. The 300μL10-0.5 sample diluent was added to the first
Samples were diluted two wells, sample diluent to give 10-1. Then 300μL10-1 sample diluent was added to the third sample dilution holes,
Get 10-1.5 sample dilution. Continue diluted the sample until a final dilution of the toxin test is negative (see Figure D.3). finally
The dilution depends on sample availability.
Diluted toxin standard solution within 18.3.1.3
Formulated within good endotoxin standard solution in accordance with 18.3.2.2 sample dilution dilution different multiples.
18.3.1.4 LAL detection
The reaction carried out in 96-well plates. Within endotoxin standard solution was used as a positive control, non-pyrogenic saline as a negative control. Each
Wells diluted sample was added 0.1mL and 0.1mL LAL, and mix. Cover with a lid 96, placed 37 ℃ ± 0.5 ℃ water bath
The reaction 1h.
18.3.2 Results count
When the reagent occurs completely aggregated as "" part when aggregated as "/ -" when no agglutination as "-."
Endotoxin within 18.3.3
LAL detection sensitivity is determined based on the detection of the situation within the different dilutions of endotoxin standard solution. In the last solidified
Samples used to calculate the titer of the diluted solution, if completely solidified, is used to calculate the actual titers (see Table D.2 in Example 1), if the Department of
Solidification points "/ -" Later, the places of the index minus a multiple of 0.25 as a value used to calculate the titer (see Table D.2 in Example 2). Per gram of sample
Endotoxin content according to formula (2).
LAL =
c × 10t × 10
10T
(2)
Where.
Within LAL --- endotoxin content per gram of sample units of endotoxin units per gram (EU/g);
C --- inside the standard endotoxin units of endotoxin units per milliliter (EU/mL);
--- t titer of the sample;
T --- standard dilution.
19 analysis results presentation
19.1 Analysis
19.1.1 Calculation lgLAL, lgN and lgLAL-lgN value.
19.1.2 lgLAL-lgN > 0, indicates that the sample tested contains a high content of endotoxin, but the number of Gram-negative bacteria than the cultured
There are not detected or less can be cultured Gram-negative bacteria.
19.1.3 lgLAL-lgN value ≤0, indicates that the sample tested contains a high content of endotoxin, and the number of gram-negative bacteria cultured
More.
19.1.4 lgLAL < 2 indicates a lower content of endotoxin in the sample.
19.1.5 lgN < 2 indicates a smaller number of Gram-negative bacteria culture.
19.2 determination result
19.2.1 lgLAL-lgN > 0, it is determined that the sample may after irradiation.
19.2.2 lgLAL-lgN value ≤0, determined that the sample has not been irradiated.
19.2.3 lgLAL < 2 and lgN < 2, NA 19.2.1 and 19.2.2 determination condition undecidable whether the sample after irradiation.
Appendix A
The method of reagent preparation
A.1 hydrochloric acid (1mol/L)
Measure 90mL of hydrochloric acid, diluted with an appropriate amount of water to 1000mL.
A.2 PBS buffer (pH 7.4)
Weigh 8.0g sodium chloride, 0.2g KCl, 2.94g disodium hydrogen phosphate dodecahydrate, 0.24g potassium dihydrogen phosphate dissolved in 900mL water
Mix well, adjust the pH with hydrochloric acid (A.1) = 7.4, then dilute to 1000mL, after autoclaving spare.
A.3 coating agarose solution (0.5%)
10mL of distilled water was added 50mg of agarose, boil or microwave heating, 45 ℃ water bath until needed.
A.4 embedded gel solution (0.8%)
10mLPBS (A.2) was added 80mg low melting point agarose, boil or microwave heating, 45 ℃ water bath until needed.
A.5 sodium hydroxide solution (40%)
Weigh 40g of sodium hydroxide dissolved in 60mL water.
A.6 EDTA stock solution (0.5mL/L)
Weigh 93.05g Disodium edetate was dissolved in 300mL of water, mixed with sodium hydr...
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