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GB 5009.111-2016

Chinese Standard: 'GB 5009.111-2016'
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GB 5009.111-2016English419 Add to Cart Days<=4 Determination of deoxynivalenol in food -- High performance liquid chromatographic method with immunoaffinity column clean-up Valid GB 5009.111-2016
GB 5009.111-2016Chinese24 Add to Cart <=1-day [PDF from Chinese Authority, or Standard Committee, or Publishing House]

   

BASIC DATA
Standard ID GB 5009.111-2016 (GB5009.111-2016)
Description (Translated English) Determination of deoxynivalenol in food--High performance liquid chromatographic method with immunoaffinity column clean-up
Sector / Industry National Standard
Classification of Chinese Standard X09
Word Count Estimation 26,256
Date of Issue 2016-12-23
Date of Implementation 2017-06-23
Older Standard (superseded by this standard) GB/T 23503-2009; GB/T 5009.111-2003; SN/T 1571-2005
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 5009.111-2016
Determination of deoxynivalenol in food - High performance liquid chromatographic method with immunoaffinity column clean-up
National Standards of People's Republic of China
National Food Safety Standard
Foods deoxynivalenol bacteria enolase
Determination and acetylated derivatives
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 5009.111-2003 "cereal and cereal products deoxynivalenol bacteria enol determination", GB/T 23503-
2009 "Food in deoxynivalenol bacteria enolase measurement immunoaffinity chromatography HPLC purification", SN/T 1571-2005
"Importers and deoxynivalenol in cereals in liquid chromatography test method."
This standard compared with GB/T 5009.111-2003, the main changes are as follows.
--- The name changed to the standard test "national food safety standards in food deoxynivalenol bacteria enolase and acetylated derivatives
set";
--- Increasing the scope of application of the method;
--- Increased DETERMINATION deoxynivalenol bacteria enol acetylated derivatives;
--- Increased isotope dilution liquid chromatography - tandem mass spectrometry;
--- Increasing the solid-phase extraction (SPE) of the pre-treatment;
--- Increased immunoaffinity column clean - high performance liquid chromatography;
--- Increased commercialization immunoaffinity column evaluate technical parameters required.
National Food Safety Standard
Foods deoxynivalenol bacteria enolase
Determination and acetylated derivatives
1 Scope
This standard specifies the method for determination of deoxynivalenol in food fungus alcohol and acetylated derivatives.
The first method is by isotope dilution liquid chromatography - tandem mass spectrometry for cereal and cereal products, wine, soy sauce, vinegar, soy sauce and butter products off
Oxygen snow rot Fusarium enol, 3-acetyl deoxynivalenol and 15-acetyl alcohol bacteria deoxynivalenol fungi alcohol measurement.
The second method is immunoaffinity chromatography and HPLC purification, suitable for grain and its products, wine, soy sauce, vinegar, soy sauce and butter products
Determination of deoxynivalenol bacteria enolase.
The third method of thin layer chromatography assay, a fourth method of ELISA screening method for cereals and products deoxynivalenol bacteria
Determination of alcohol.
The first method isotope dilution liquid chromatography - tandem mass spectrometry
Principle 2
Sample of deoxynivalenol fungi alcohol, 3-acetyl deoxynivalenol and 15-acetyl alcohol bacteria deoxynivalenol fungi with water and alcohol
A mixed solution of acetonitrile extraction, the supernatant after solid phase extraction columns or immuno-affinity column purification, concentration, volume, and after filtration, high pressure liquid chromatography
Spectral separation, tandem mass spectrometry, isotope internal standard.
3 Reagents and materials
Unless otherwise indicated, the methods used were of analytical grade reagents and water as a water GB/T 6682 regulations.
3.1 Reagents
3.1.1 acetonitrile (CH3CN). chromatography.
3.1.2 Methanol (CH3OH). chromatography.
3.1.3 n-hexane (C6H14).
3.1.4 Ammonia (NH3 · H2O).
3.1.5 formic acid (HCOOH).
3.1.6 Nitrogen (N2). purity ≥99.9%.
3.2 reagent preparation
3.2.1 acetonitrile - water (8416). amount of 160mL of water was added to 840mL of acetonitrile and mix.
3.2.2 acetonitrile saturated solution of hexane. amount of n-hexane in 200mL 250mL separatory funnel, adding a small amount of acetonitrile, shake vigorously
A few minutes, standing layer, discarding the lower acetonitrile layer, that is, too.
3.2.3 Methanol - water (595). amount of 5mL of methanol was added to 95mL of water, and mix.
3.2.4 0.01% aqueous ammonia solution. Take 100μL aqueous ammonia was added to 1000mL of water, and mix (for ion source ESI- mode is used).
3.2.5 0.1% formic acid solution. Take 1mL formic acid is added to 1000mL of water, and mix (for ESI ion source mode is used).
3.3 Standard
3.3.1 deoxynivalenol bacteria enolase (DON, C15H20O6, CAS number. 51481-10-8). purity ≥99%, or certified by the state and granted
Standards Standards certificate.
3.3.2 3-acetyl deoxynivalenol bacteria enolase (3-ADON, C17H22O6, CAS number. 50722-38-8). purity ≥99%, or by the State
Certified and standard substance substance certificate.
3.3.3 15- acetyl deoxynivalenol bacteria enolase (15-ADON, C17H22O6, CAS number. 88337-96-6). purity ≥99%, or by State
Hotels certified and reference material reference material certificate.
3.3.4 13C15- deoxynivalenol fungi alcohol standard solution isotopes (13C-DON, 13C15H20O6). 25μg/mL, the purity of ≥99%.
3.3.5 13C17-3- acetyl - deoxynivalenol fungi alcohol standard solution isotopes (13C-3-ADON, 13C17H22O6). 25μg/mL, purity
≥99%.
3.4 Standard Solution
3.4.1 Standard stock solution (100μg/mL). Weigh DON, 3-ADON and 15-ADON1mg (accurate to 0.01mg), points
Do not dissolved in acetonitrile and set the volume to 10mL. The solution was transferred to a vial, sealed and stored at -20 ℃, valid for 1 year.
3.4.2 mixed standard working solution (10μg/mL). Imbibe 100μg/mLDON, 3-ADON and 15-ADON stock standard solution
Each 1.0mL in the same 10mL volumetric flask, add acetonitrile to volume. Sealed and stored at -20 ℃, valid for six months.
3.4.3 Mixed isotope internal standard working solution (1μg/mL). Imbibe 13C15-DON and 13C17-3-ADON isotope internal standard
(25μg/mL) each 1mL to 25mL same flask, add acetonitrile to volume. Sealed and stored at -20 ℃, valid
Six months.
3.4.4 standard series working solution. accurate Pipette the appropriate amount of mixed standard working solution and mixed isotope internal standard working solution with the initial mobile phase
Formulated as 10ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 160ng/mL, 320ng/mL, mixed standard 640ng/mL of
Series, in which the isotope internal standard at a concentration of 100ng/mL. Standard series solution at 4 ℃ preservation, valid 7d.
4 instruments and equipment
4.1 Liquid chromatography - tandem mass spectrometry. a charged ion source.
4.2 Electronic balance. a sense of the amount of 0.01g and 0.00001g.
4.3 high-speed grinder. Speed 10000r/min.
4.4 homogenizer.
4.5 Screen. 0.5mm ~ 1mm aperture.
4.6 Ultrasound/or a vortex shaker.
4.7 nitrogen blowing instrument.
4.8 high-speed centrifuge. speed of not less than 12000r/min.
4.9 Pipette. Range 10μL ~ 100μL and 100μL ~ 1000μL.
4.10 solid phase extraction device.
4.11 Universal SPE. both hydrophilic group (group pyrrolidone) and hydrophobic groups (divinyl benzene) packing solid phase adsorbents
Extraction column, 200mg, 6mL, or equivalent person.
4.12 DONs special solid phase clean-up columns, or equivalent person.
4.13 deoxynivalenol bacteria enol immunoaffinity column. Column capacity ≥1000ng (column size and column recovery verification method, see A.2).
4.14 aqueous phase porous membrane. 0.22μm.
Step 5 Analysis
5.1 Sample Preparation
5.1.1 cereals and cereal products. taking at least 1kg sample was pulverized with a high-speed grinder and sieved to a particle size of less than 0.5mm ~ 1mm
Aperture test sieve, mixed uniformly reduced to 100g, stored in a vial, sealed and stored for detection.
5.1.2 Alcohol. Take bulk wine at least 1L, for bags, bottles and other packaging samples taken at least three packaging (or the same batch number), all the liquid
Body with a sample container after homogenous mixing machine, reduced to 100g (mL) stored in the vial, sealed and stored for detection. Including two
Wine samples of carbon monoxide before you use the refrigerator at 4 ℃ for 30min, filtered before use or ultrasonic degassing.
5.1.3 soy sauce, vinegar, soy sauce and butter products. taking at least 1L samples for bags, bottles and other packaging samples taken at least three packaging (or the same batch
Number), after all the liquid sample in a container with a homogenizer mix, reduced to 100g (mL) stored in the vial, sealed for
Detection.
5.2 Sample Extraction
5.2.1 cereal and its products. Weigh 2g (accurate to 0.01g) sample was 50mL centrifuge tube was added 400μL mixed isotope internal standard
After shaking the working fluid mixed stand 30min. Added 20.0mL of acetonitrile - water (8416), placed in an ultrasonic/or a vortex shaker
Ultrasonic or oscillation 20min. 10000r/min centrifugal 5min, the supernatant was collected A spare in a clean container.
5.2.2 Alcohol. Weigh 5g (accurate to 0.01g) sample was 50mL centrifuge tube was added 200μL mixed isotope internal standard working solution vibrator
After mixing swing standing 30min, with acetonitrile dilute to 10mL, mix, placed in an ultrasonic/Vortex shaker or ultrasonic or oscillation
20min. 10000r/min centrifugal 5min, the supernatant was collected B alternate in a clean container.
5.2.3 soy sauce, vinegar, soy sauce and butter products. Weigh 2g (accurate to 0.01g) sample was 50mL centrifuge tube, mixed with bits added 400μL
Su internal standard working solution After shaking the mixing stand 30min. Added 20.0mL of acetonitrile - water (8416), placed in an ultrasonic/Vortex
Or shaker or ultrasonic oscillations 20min. 10000r/min centrifugal 5min, the supernatant was collected C alternate in a clean container.
5.3 Sample purification
NOTE. The following sample purification process, according to the actual situation, choose one of the methods can be.
5.3.1 Universal solid-phase extraction (SPE)
A take 5mL supernatant or supernatant or supernatant B C a 50mL centrifuge tube, 10mL of acetonitrile was added a saturated solution of n-hexane
Liquid vortex mixing 2min, after 5000r/min centrifugal 2min, discard the layer of n-hexane at 40 ℃ ~ 50 ℃ under dry nitrogen, was added 4mL
The residue was dissolved sufficient water to be purified.
The SPE is connected to the solid phase extraction, washed with methanol and 3mL 3mL water activated balance. The above-mentioned water 4mL reconstituted
The liquid column to control the flow rate drops 1 to 2 drops per second. With 3mL water, 1mL5% methanol - aqueous solution followed by rinsing thoroughly drained after the column. use
4mL methanol, after collecting all the eluent at 40 ℃ ~ 50 ℃ under dry nitrogen. The initial mobile phase was added 1.0mL residue,
Vortex 10s, with 0.22μm filter membrane in an injection vial until injection.
5.3.2 DONs special solid phase purification column purification
A glass tube take 8mL supernatant or supernatant or supernatant B C DONs dedicated to solid phase clean-up columns will fill the purification column
Feeding tube inserted into a glass tube and slowly push the filler tube to purify liquid precipitation, pipette 5mL decontamination liquid at 40 ℃ ~ 50 ℃ under dry nitrogen. plus
The initial mobile phase into 1.0mL residue was vortexed 10s, with 0.22μm filter membrane in an injection vial, until injection.
Note. The operational aspects of the use of different vendors DONs special type of solid-phase purification column, in the sample, purification may be slightly different, can be carried out in accordance with the specification requirements
operating.
5.3.3 immunoaffinity column purification
In advance of recovering cryopreserved immunoaffinity column to room temperature.
Pipette accurately 5mL supernatant A or B supernatant or supernatant C, at 40 ℃ ~ 50 ℃ under dry nitrogen, was added 2mL water sufficient
The residue was dissolved, to be immunoaffinity column within the original liquid flow after doing the above sample solution was transferred to a glass syringe barrel. The air pressure injection pump and glass
Emitter connected to adjust the dripping speed, control sample solution with 1 drop per second flow rate through the immunoaffinity column, until the air into the affinity column. use
5mLPBS buffered saline solution and 5mL water leaching has immunoaffinity column at a flow rate of approximately 1 drop to 2 drops per second, until the air and into the pro-Note
The discarded all effluent, drained cartridge.
Accurate added 2mL methanol affinity column, under the control of one drop per second, the speed drops to collect all the eluent to a test tube, at 50 ℃
Firmly eluent blown with nitrogen to nearly dry, add 1.0mL initial phase flow, vortex 30s dissolve the residue, 0.22μm filter membrane,
The filtrate was collected in an injection vial to prepare injections.
Note. Use different vendors immunoaffinity column, the sample in the sample, operational leaching and elution may be slightly different, should be carried out in accordance with specification requirements
operating.
5.4 Liquid chromatography - tandem mass spectrometry reference conditions (refer to one of these methods can be based on the actual situation)
5.4.1 Ion Source mode. ESI
Liquid chromatography - mass spectrometry reference conditions are listed below.
a) LC Column. C18 column (column length 100mm, column diameter 2.1mm; particle size 1.7μm), or equivalent person;
b) Mobile phase. A phase. 0.1% formic acid; B phase. 0.1% formic acid - acetonitrile;
c) gradient. 2% B (0min ~ 0.8min), 24% B (3.0min ~ 4.0min), 100% B (6.0min ~
6.9min), 2% B (6.9min ~ 7.0min);
d) flow rate. 0.35mL/min;
e) Column temperature. 40 ℃;
f) Injection volume. 10μL;
g) Capillary voltage. 3.5kV; cone voltage. 30V; desolvation temperature. 350 ℃; desolvation gas flow rate. 900L/h;
h) deoxynivalenol bacteria enolase and acetylated derivatives thereof MS conditions refer to Table 1.
Table 1 deoxynivalenol bacteria enolase and Acetyl Derivatives Mass reference conditions (ESI)
Compound
name
Parent ion
(M/z)
Cone voltage
eV
Quantitative ion
(M/z)
Collision voltage
eV
Qualifier ion
(M/z)
Collision voltage
eV
DON 297 20 249 10 203 16
3-ADON 339 17 231 13 203 13
15-ADON 339 18 137 9 321 7
13C-DON 312 20 263 10 245 16
13C-3-ADON 356 17 245 13 - -
Note. 3-ADON and 15-ADON calculated as isomers, 15-ADON optional 13C-3-ADON isotope internal standard as appropriate quantification.
5.4.2 Ion Source mode. ESI-
Liquid chromatography - mass spectrometry reference conditions are listed below.
a) LC Column. C18 column (column length 100mm, column diameter 2.1mm; particle size 1.7μm), or equivalent person;
b) Mobile phase. A phase. 0.01% aqueous ammonia solution; phase B. acetonitrile;
c) gradient. 2% B (0min ~ 0.8min), 24% B (3.0min ~ 4.0min), 100% B (6.0min ~ 6.9min),
2% B (6.9min ~ 7.0min);
d) flow rate. 0.35mL/min;
e) Column temperature. 40 ℃;
f) Injection volume. 10μL;
g) capillary voltage. 2.5kV; cone voltage. 45V; desolvation temperature. 500 ℃; desolvation gas flow rate. 900L/h;
h) deoxynivalenol bacteria enolase and acetylated derivatives thereof MS conditions refer to Table 2.
Table 2 deoxynivalenol bacteria enolase and Acetyl Derivatives Mass reference conditions (ESI-)
Compound
name
Parent ion
(M/z)
Cone voltage
eV
Quantitative ion
(M/z)
Collision voltage
eV
Qualifier ion
(M/z)
Collision voltage
eV
DON 295 14 265 12 138 18
3-ADON 337 12 307 10 173 12
15-ADON 337 12 150 12 219 12
13C-DON 310 16 279 12 145 14
13C-3-ADON 354 18 323 14 230 18
Note. 3-ADON and 15-ADON calculated as isomers, 15-ADON optional 13C-3-ADON isotope internal standard as appropriate quantification.
Ion scan of each compound, multiple reaction monitoring (MRM) ion channel FIG see Figure B.1 ~ Figure B.8.
5.5 Qualitative Determination
The target compound in the sample peak retention time compared with the corresponding standard chromatographic retention time, the range of variation should be ± 2.5%
within.
MS qualifier ion for each compound should appear, including at least a parent ion and two ions, and detect the same batch of
The same compound, the relative abundance of the two sub-target in the sample ion compound concentration equivalent ratio compared to the standard solution, which is not allowable deviation
Table 3 exceeds the range specified.
The maximum permissible relative ion abundances Table 3 Qualitative deviations
Relative ion abundances> 50% 20% ~ 50% 10% ~ 20% ≤10%
Permissible relative deviation of ± 20% ± 25% ± 30% ± 50%
5.6 standard curve
Under 5.4 liquid chromatography tandem mass spectrometry analysis conditions, the standard solution series from low to high concentration injection testing to DON, 3-
Peak area ratio ADON and 15-ADON peaks corresponding to each of the internal standard peak - concentration plotted standard curve regression equation, which
Linear correlation coefficient should be greater than 0.99.
5.7 Determination of the sample solution
Take the test solution injection treatment was 5.2, 5.3, calculate the mass concentration of the target substance in the test solution of the internal standard method, the sample is calculated by 6
Content of the analyte. Analyte response test solution should be within the linear range of the standard curve over the linear range should be appropriate to reduce the amount of sampling
After the re-determination.
5.8 blank test
Except that no sample addition, according to the steps 5.3 and 5.4 of the blank experiment. Confirm contain interfering substances should not be tested components.
6 expression analysis
Sample DON, 3-ADON or 15-ADON content according to formula (1).
X = ρ
× V1 × V3 × 1000
V2 × m × 1000
(1)
Where.
X --- sample DON, 3-ADON or 15-ADON content in micrograms per kilogram (μg/kg);
ρ --- sample DON, 3-ADON or 15-ADON internal standard method in accordance with the corresponding concentration of the standard curve, the unit is
Nanograms per milliliter (ng/mL);
Vl --- sample extract volume in milliliters (mL of);
V3 --- the final volume of the sample, in milliliters (mL);
1000 --- conversion factor;
--- For purifying V2 of the dispensing volume, in milliliters (mL of);
Sample weight m --- sample in grams (g).
The results to three significant figures.
7 precision
Two independent determination results under the absolute difference in repeatability condition must not exceed 23% of the arithmetic mean.
8 Other
When weighed and cereal products, wine, soy sauce, vinegar, soy sauce and butter products sample 2g, methods deoxynivalenol bacteria enolase, 3-B
Acyl deoxynivalenol fungi alcohol, 15-acetyl deoxynivalenol bacteria enolase detection limit of 10μg/kg, the limit of quantification of 20μg/kg.
When the sample liquor weighed 5g, methods deoxynivalenol fungi alcohol, 3-acetyl deoxynivalenol fungi alcohol, 15-acetyl-deoxy
Snow rot Fusarium enol detection limit of 5μg/kg, the limit of quantification of 10μg/kg.
The second method of immune affinity chromatography and HPLC purification
Principle 9
Sample of deoxynivalenol fungi alcohol extracted with water, after immunoaffinity column purified by high performance liquid chromatography - ultraviolet detector measurement,
External standard.
10 Reagents and materials
Unless otherwise indicated, the methods used were of analytical grade reagents and 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 polyethylene glycol [molecular weight of 8000, HO (CH2CH2O) nH].
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 hydrochloric acid (HCl).
10.2 reagent preparation
10.2.1 phosphate buffer solution (hereinafter referred to as PBS). Weigh 8.00g of sodium chloride, 1.20 g of disodium hydrogenphosphate, potassium dihydrogenphosphate 0.20 g,
0.20g potassium chloride, dissolved with 900mL of water, adjusted to pH 7.0 with hydrochloric acid, water volume to 1000mL.
10.2.2 methanol - water (2080). amount of 200mL of methanol was added to 800mL of water, and mix.
10.2.3 acetonitrile - water (1090). Measure 100mL acetonitrile was added to 900mL of water, and mix.
10.3 Standards
Deoxynivalenol bacteria enolase (C15H20O6, CAS number. 51481-10-8). purity ≥99%, or granted by the National Certification and Standards
Standard Substance certificate.
10.4 Standard Solution
10.4.1 Standard stock solution (100μg/mL). Weigh deoxynivalenol bacteria enol 1mg (accurate to 0.01mg), dissolved in acetonitrile and
Volume to 10mL. The solution was transferred to a vial, sealed and stored at -20 ℃, valid for 1 year.
10.4.2 series of standard working solution. accurate Pipette the appropriate amount deoxynivalenol fungi alcohol standard stock solution was diluted with an initial phase flow, preparation
To 100ng/mL, 200ng/mL, 500ng/mL, 1000ng/mL, 2000ng/mL, 5000ng/mL working solution of the standard series,
4 ℃ preservation, valid 7d.
11 instruments and equipment
11.1 High performance liquid chromatograph. equipped with an ultraviolet detector or diode array detector.
11.2 Electronic balance. a sense of the amount of 0.01g and 0.00001g.
11.3 High-speed grinder. Speed 10000r/min.
11.4 Screen. 1mm ~ 2mm aperture.
11.5 Ultrasound/or a vortex shaker.
11.6 nitrogen blowing instrument.
11.7 high-speed centrifuge. speed ≥12000r/min.
11.8 pipettes. Range 10μL ~ 100μL and 100μL ~ 1000μL.
11.9 deoxynivalenol bacteria enol immunoaffinity column. Column capacity ≥1000ng. (Column size and column recovery verification method, see A.2).
Note. For different batches affinity column in quality verification before use.
11.10 Glass fiber filter. diameter 11cm, pore size 1.5μm.
11.11 aqueous phase microporous membrane. 0.45μm.
11.12 Polypropylene graduated centrifuge tube. a plug, 50mL.
11.13 glass syringes. 10mL.
11.14 air pressure pump.
12 analysis steps
Use different vendors immunoaffinity column, the sample in the sample, operational leaching and elution may be slightly different, should be in accordance with the instructions
Requested operation.
12.1 Sample Preparation
With 5.1.
12.2 Sample Extraction
12.2.1 cereal and its products. Weigh 25g (accurate to 0.1g) ground sample in 100mL flask was added 5g stoppered polyethylene
Alcohol, add water 100mL, mix, placed in an ultrasonic/Vortex shaker or ultrasonic or oscillation 20min. Glass fiber filter paper to
The filtrate was clarified (or 6000r/min centrifugation 10min), A filtrate collected in a clean container. 10000r/min centrifugal 5min.
12.2.2 alcohol. from wine samples 20g (accurate to 0.1g), 1g of polyethylene glycol, water volume to 25.0mL, mix, placed in an ultrasonic /
Vortex shaker or ultrasonic or oscillation 20min. Filtered through a glass fiber filter paper filtrate to clarify (or 6000r/min centrifugation
10min), the filtrate was collected in a clean container B.
12.2.3 soy sauce, vinegar, soy sauce and butter products. Weigh sample 25g (accurate to 0.1g), 5g of polyethylene glycol, water volume to 100mL,
Mix, put the ultrasonic/Vortex shaker or ultrasonic or oscillation 20min. Glass fiber filter paper to clarify the filtrate (or
6000r/min under centrifugal 10min), C and the filtrate was collected in a clean container.
12.3 Purification
In advance of recovering cryopreserved immunoaffinity column to room temperature. Immunoaffinity column to be within the original liquid flow after doing the above sample was transferred
Glass syringe barrel, pipette accurately above filtrate A or B or filtrate filtrate C2.0mL, put in a glass syringe. The air pressure pump
Connected with the glass syringe, dripping adjust speed, control sample solution with 1 drop per second flow rate through the immunoaffinity column, until the air enters affinity
Column. With 5mLPBS buffered saline solution and 5mL water leaching has immunoaffinity column at a flow rate of approximately 1 drop to 2 drops per second, until the air
Into the affinity column, discard all effluent, drained cartridge.
12.4 elution
Accurate added 2mL methanol affinity column, under the control of one drop per second, the speed drops to collect all the eluent to a test tube, at 50 ℃
Firmly eluent blown with nitrogen to nearly dry, add 1.0mL initial phase flow, vortex 30s dissolve the residue, 0.45μm filter membrane,
The filtrate was collected in an injection vial to prepare injections.
12.5 Reference conditions Liquid Chromatography
HPLC reference conditions are listed below.
a) LC Column. C18 column (column length 150mm, column diameter 4.6mm; particle size 5μm), or equivalent person;
b) Mobile phase. methanol-water (2080);
c) flow rate; 0.8mL/min;
d) Column temperature. 35 ℃;
e) Injection volume. 50μL;
f) detection wavelength. 218nm.
Quantitative Determination 12.6
12.6.1 standard curve
In deoxynivalenol standard working fungi alcohol concentration as abscissa to peak area values ordinate the standard solution series from low to
Followed by injection of high concentration detector, standard curve regression equation.
12.6.2 Determination of the sample solution
Response analyte in the sample solution should be within the linear range of the standard curve over the linear range should be appropriate to reduce the sample weight, re-press
Injection analysis 12.2, 12.3 and 12.4 are processed before.
12.7 blank test
In addition to not weighed samples were used, 12.2, 12.3 and 12.4 blank test. Claim does not contain interfering substances tested components.
13 analysis results presentation
Content of the sample deoxynivalenol fungi alcohol according to formula (2).
X =
(Ρ1-ρ0) × V × f × 1000
m × 1000
(2)
Where.
Content X --- sample deoxynivalenol fungi alcohol, in units of micrograms per kilogram (μg/kg);
Concentration ρ1 --- sample deoxynivalenol fungi alcohol, unit nanograms per milliliter (ng/mL);
Concentration ρ0 --- blank sample deoxynivalenol fungi alcohol, unit nanograms per milliliter (ng/mL);
V --- sample of the final volume of the eluent, the unit milliliter (mL);
f --- sample dilution factor;
1000 --- conversion factor;
Sample weight m --- sample, in grams (g).
The results to three significant figures.
14 Precision
Two independent determination results under the absolute difference in repeatability condition must not exceed 23% of the arithmetic mean.
Other 15
When referring to the time taken and cereal products, soy sauce, vinegar, soy sauce and butter products sample 25g, deoxynivalenol bacteria detection limit of alcohol
100μg/kg, the limit of quantification of 200μg/kg; when 20g of alcohol when weighed sample deoxynivalenol bacteria alcohol detection limit of 50μg/kg,
Limit of quantification is 100μg/kg.
Thin Layer Chromatography Method III
Principle 16
Sample of deoxynivalenol fungi alcohol by extraction, purification, concentration and silica gel G was launched, after heating a thin layer to expand, thin heating
Laminates. Since the preparation of thin plate when added aluminum trichloride, make deoxynivalenol bacteria alcohol at 365nm was blue fluorescence under ultraviolet light
Light, compared with the standard.
17 Reagents and materials
Unless otherwise indicated, the methods used were of analytical grade reagents.
17.1 Reagents
17.1.1 chloroform (CHCl3).
17.1.2 absolute ethanol (CH3CH2OH).
17.1.3 methanol (CH3OH).
17.1.4 petroleum ether (CnH2n 2).
17.1.5 ethyl acetate (CH3COOCH2CH3).
17.1.6 acetonitrile (CH3CN).
17.1.7 acetone (CH3COCH3).
17.1.8 isopropanol (CH3CH2OHCH3).
17.1.9 ether (CH3CH2OCH2CH3).
17.1.10 aluminum chloride (AlCl3 · 6H2O). chemically pure.
17.1.11 neutral alumina. activation at 300 ℃ 4h, the dryer use.
17.1.12 Activated Carbon. 20g activated carbon, hydrochloric acid solution with 3mol L/soak overnight, after filtration, washed with hot distilled water until no chlorine ions in
120 ℃ drying spare.
17.1.13 silica gel G. thin-layer chromatography.
17.2 Standards
Deoxynivalenol bacteria enolase (C15H20O6, CAS number. 51481-10-8). purity ≥99%.
17.3 Standard Solution
Standard stock solution (25μg/mL). Weigh deoxynivalenol fungi alcohol 5.0mg (accurate to 0.1mg), add ethyl acetate - methanol
(191) dissolved into 10mL volumetric flask, and dilute to 10mL draw solution 0.5mL, ethyl acetate - methanol (191) dilute
Diluted to 10mL.
18 instruments and equipment
18.1 Small grinder.
18.2 electric oscillator.
18.3 glass evaporating dish. 75mL.
18.4 Column. inner diameter of 2cm, length 10cm, does not have pistons.
18.5 concentrate bottle stopper. 10mL, 0.2mL bottom of the scale with the tailpipe.
18.6 the glass. 5cm × 20cm.
18.7 thin applicator. 0.3mm.
18.8 Expand tank. inner length 26cm, width 6cm, height 4cm.
18.9 UV light. 365nm.
18.10 micro syringe. 10μL, 50μL.
18.11 flat tube. 50mL.
18.12 pairs wavelength TLC scanner. with a data processor.
19 analysis steps
19.1 Sample Extraction
Weigh 20g pulverized sample is placed in 200mL stoppered Erlenmeyer flask, add 8mL and 100mL chloroform - absolute ethanol (82), dense
Plug. Cork coating on the water, reclosable leak, shaking 1h, by folding the rapid qualitative filter paper, take 25mL to 75mL glass filtrate evaporated
Hair dish, ventilation evaporated to dryness on a water bath set to 90 ℃.
19.2 Purification
19.2.1 liquid - liquid partition
19.2.1.1 cereals. petroleum ether several times with 50mL dissolve residue evaporating dish, wash 100mL separating funnel, and then 20mL (Jade
Rice sample was 30mL) methanol - water (41) was washed several times evaporation pan, into the same separatory funnel.
19.2.1.2 cereal products (cakes, cookies, bread, etc.). petroleum ether several times with 100mL dissolve residue evaporating dish, wash 250mL points
Funnel, and then 30mL of methanol - water (41) was washed several times evaporation pan, into the same separatory funnel. Oscillation separating funnel 1.5min,
Stand for about 15min so stratified, the lower methanol - water extract was purified by column, not the white floc at the junction of the two phases into the column.
19.2.2 purification column
19.2.2.1 wheat and its products. cotton plugs about 0.1g column in the lower end of the small tube at the link, try plugged, first into neutral 0.5g
Alumina, knocked flat surface, together with 0.4g of activated carbon, knocking tight. The lower end of the column tube is inserted into a small rubber plug, plug in the suction bottle suction bottle
Put in a flat tube column was received, the suction bottle connected to the pump or vacuum pump, the pump is turned slightly, so that the activated carbon pressing the separatory funnel methyl
Alcohol - water extract carefully to each column along the wall, to control the flow rate of 18 drops per 15 seconds to 20 drops (3mL/min), methanol - water extract over
When the column is completed quickly, was added 10mL of methanol - water (41) column leaching, the leaching no more liquid flows out until the column. By column speed control
2mL/min ~ 3mL/min, too fast purification effect is not good, too slow too time-consuming.
19.2.2.2 Corn. with 20.2.2.1, except that the amount of activated carbon to 0.3g.
19.3 preparative thin layer chromatography with sample solution
The eluate was passed through the column was poured into 75mL glass evaporating dish, with a small amount of methanol - water (41) was washed with flat tubes. The evaporating dish set boiling
Concentrated to dryness on a water bath.
a) Wheat. hot add 3mL of ethyl acetate, heated to boiling in a water bath and gently rotated repeatedly evaporating dish several times to full boil
Proton The residue was dissolved in DON, and ethyl acetate was evaporated to dryness, then add 3mL of ethyl acetate treated in the same time, the solution
Agent evaporated to dryness, and finally add 3mL of ethyl acetate, heated to boiling, let cool to room temperature and concentrated into the bottle, then 3 parts 1.5mL B
Washed with ethyl evaporation pan, into the concentrate bottle.
b) wheat and corn products. hot add 3mL of ethyl acetate, heated to boiling in a water bath and gently rotated repeatedly evaporating dish number
Times to full boil, and the residue was dissolved in DON, let cool to room temperature and concentrated into the bottle. Plus about 0.5mL methanol - acetone
(12) in the evaporating dish with a glass stirring to dissolve the residue, evaporated on a water bath pot dish solvent was evaporated to dryness, added 3mL acetic acid
Ethyl, heated to a boil, turn the evaporating dish to make full boil, let cool to room temperature and concentrated into the same bottle, and then 0.5mL A
Alcohol - acetone (12) and 3mL of ethyl acetate treated in the same time, the ethyl acetate extract was concentrated incorporated into the bottle.
The concentrate bottle set on about 95 ℃ water bath, heated with steam blowing nitrogen and concentrated to dryness, let cool to room temperature after adding 0.2mL chloroform -
Acetonitrile (41) The residue was dissolved reserved by thin layer chromatography.
19.4 Determination
19.4.1 Preparation of TLC plates. 4g silica gel G plus about 9mL15% aluminum chloride (AlCl3 · 6H2O) solution, was sticking to grind about 2min
The thick, thin plate three paved 5cm × 20cm, the post-dried at room temperature at 105 ℃ activation 1h, storage desiccator.
19.4.2 Spotting. spotted on each plate with the lower end of 2.5cm from the baseline.
The first plate. the first point for each sample first piece of thin plate, away from the left edge of the plate at the point 1.8cm 25μL sample solution, away from the upper plate
1.5cm at the point on the line and the baseline sample point corresponding to the position 2μLDON standard solution (50ng).
Second board. At a board need not explicitly fluorescent samples from the left edge of 0.8cm ~ 1cm at second thin plate-like dropping
Dots (according to the situation estimate dropping amount, diluted or quantitative), away from the plate at the left edge of 2cm and 1.2cm away from the right edge of the dropping respectively
Standard two points, the amount of DON may be 50ng, 75ng, 100ng. Again on the line from the plate at the upper end of 1.5cm DON standard three point
Points (each 50ng), make three points on the baseline, respectively.
19.4.3 Expand.
Horizontal Exhibition solvent. diethyl ether, ethyl ether - acetone (955) or anhydrous ether. Optionally one, departing from the origin point of the sample DON 0.7cm ~
1cm, just separated from impurities fluorescence.
Longitudinal exhibition solvent. chloroform - acetone - isopropanol (811);
Chloroform - acetone - isopropanol - water (7.5 1 1.5 0.1).
19.4.3.1 horizontal Exhibition. In the expanded cross-slot poured into 10mL exhibition agent. The good kind of lamina point by point the long side of the ramp-like liquid immersion solvent, to show
Board side 1min ~ 2min, remove the ventilation evaporated 3min. Wheat products must then 10mL petroleum ether (30 ℃ ~ 60 ℃) show a cross,
Exhibition panels to side over 1min, remove the ventilation evaporated 5min.
19.4.3.2 Longitudinal exhibition. the expansion tank into the 10mL vertical development agent. The exhibition was evaporated to dryness after a cross TLC plate was expanded longitudinal slot exhibition 15cm, remove
Ventilation evaporated 10min, due to the effects of DON and separation of impurities influenced by air humidity, when the board is not very good separation effect that is first
When impurities fluorescence interference near the sample point block lamina, according to the size of the polarity switch to successively expand the following ways.
a) Chloroform - acetone - isopropanol (811).
b) chloroform - acetone - isopropanol (811) and the inner surface of the expansion slot cover labeled water-saturated filter paper.
c) chloroform - acetone - isopropanol - water (7.5 1 1.5 0.1).
d) chloroform - acetone - isopropanol - water (7.5 1 1.5 0.1) and expand the tank surface labeled water-saturated filter paper. Change vertical Exhibition
Way to start the second block TLC plate. As polar expansion tank is too big, DON point becomes biased.
19.4.4 significant fluorescence. the first observation of the unheated lamina visible blue-violet fluorescence interference significant point, then DON no significant fluorescence, heating lamina
After point impurities remained fluorescence, which fluorescence near DON point, but do not interfere with DON. This thin layer plate was then heated in an oven at 130 ℃
7min ~ 10min, take out place on a cold surface 1min ~ 5min after 365nm UV lamp observation.
19.4.5 Observation and evaluation. the TLC plate after a cross exhibition, DON spotting board dots moving 0.7cm ~ 1.0cm, it is in accordance with this
The samples show the posterior longitudinal DON point to get rid of the interference of impurities fluorescence. DON standard three point TLC plate without longitudinal upper exhibition respectively as
Exhibition posterior longitudinal lateral positioning point and two-point sample DON DON standard points. DON DON sample point but also with standard vertical point after the show than
Rf value than the qualitative. DON position thus determined sample from both horizontal and vertical, to achieve a qualitative purpose. At a thin layer
DON board as the sample points have a very shallow oblique by fluorescence, this is no time to master too good column rate, clean enough, it could be air
Humidity changes, the impact of separation, but no impurity fluorescence interference positions of two points on the standard DON.
As the first piece of thin plate-like fluorescent spots was not significant, while in the second plate with the sample solution 25μL was added to the standard 25ng fluorescence intensity
25ng of standard equal, the amount of negative samples DON or 50μg/kg or less. Positive sample outline quantitation, although TLC
DON board three points are a slight movement in the horizontal exhibition, but had no effect on the fluorescence intensity of each point, two points can be used for standard and sample DON
Point comparison of fluorescence intensity.
19.4.6 thin optical density was measured by. an excitation wavelength of 340nm, emission wavelength 400nm. TLC plate standard DON phosphor dots
At least 100ng, 200ng, when 400ng, the amount of the measured response of DON was linear. DON content of 300μg/kg
The above sample was measured with a densitometer. When the DON content at 300μg/kg, spotting solution 20μL make the measured amount of DON falls
100ng ~ between 200ng. When measured by TLC scanner, each plate with two standard DON dropped points for the amount of DON
100ng, 200ng or 200ng, 400ng. At an excitation wavelength of 340nm, 400nm wavelength emission conditions were measured to the measured peak
Area value of the vertical coordinate, DON amount abscissa, the standard curve.
20 analysis results presentation
Content of the sample deoxynivalenol fungi alcohol according to formula (3) Calculated.
X =
C × V1 × f × 1000
V2 × m × 1000
(3)
Wherein
X --- sample DON content in micrograms per kilogram (μg/kg);
C --- amount TLC plate measured on a sample point DON, measured in nanograms (ng);
V1 --- chloroform was added - a mixture of the residue was dissolved in acetonitrile volume in milliliters (mL);
Dilution factor f --- sample solution;
1000 --- conversion factor;
--- Dropwise V2 of the volume of the sample solution, in milliliters (mL of);
m --- chloroform - acetonitrile mixture of the residue was dissolved quite sample in grams (g).
Results are expressed to the nearest whole number of measured values.
DON GB sample content exceeds the limit value of 2761 the first law required further confirmation.
21 Precision
Weigh each sample was measured in duplicate parallel arithmetic average thereof as the analysis result.
The relative difference between the results of its analysis should not exceed 60%.
Other 22
The minimum detectable amount TLC plate deoxynivalenol bacteria enolase is 100ng, a detection limit of 300μg/kg.
The fourth method ELISA screening method
Principle 23
Sample of deoxynivalenol bacteria alcohol extracted with water, homogenization, vortex, centrifugation (or filtration) and other pre-treatment to obtain a supernatant. Microtiter
Remember deoxynivalenol enolase bacteria even conjugate, and the sample supernatant or standard of deoxynivalenol micro fungi enol competitive binding
The wells are pre-coated with specific antibodies of. After washing by adding the appropriate chromogenic reagent, the reaction was terminated by an inorganic acid, at 450nm or 630nm
Wavelength detection. Sample of deoxynivalenol fungi alcohol and absorbance is inversely proportional to a certain concentration range.
24 Reagents
Solution preparation necessary reagents were of analytical grade, water GB/T 6682 provisions of two water.
According to the selected kit instructions described in the preparation of the required solution.
25 instruments and equipment
25.1 microtiter plate spectrophotometer. with 450nm and 630nm (optional) filter.
25.2 mill.
25.3 oscillator.
25.4 Electronic balance. a sense of the amount of 0.01g.
25.5 Centrifuge. Speed ≥6000r/min.
25.6 rapid quantitative filter paper. aperture 11μm.
25.7 Screen. 1mm ~ 2mm test sieve apertures.
Other instruments 25.8 kit required.
26 analysis steps
26.1 Extraction
Weigh at least 100g sample was pulverized with a mill, pulverized sample over 1mm ~ 2mm test sieves. 5.0g sample was taken
50mL centrifuge tube, was added the required kit extract were determined according to the test method described in the specification cassette.
26.2 ELISA Detection
ELISA kits according to the procedure treated test sample (liquid) is detected quantitatively.
27 analysis results presentation
According to the calculation method or computer software kit instructions provided, the standard according to the standard concentration and absorbance change the relationship between
Working curve.
27.1 analyte concentration calculated
According to the calculation methods and computer software kit instructions provided by the absorbance of the test solution was drawn substituting standard working song
Line, calculated test concentration (ρx).
27.2 Calculation Results
In Food deoxynivalenol fungi alcohol according to formula (4) Calculated.
X = ρx
× V × f
(4)
Where.
X --- content in foods deoxynivalenol fungi alcohol, in units of micrograms per kilogram (μg/kg);
ρx --- test solution deoxynivalenol fungi alcohol concentration, in micrograms per liter (μg/L);
V --- extract volume in liters (L);
f --- pretreatment process of dilution;
m --- sample sampling amount, in kilograms (kg).
The results reservations to one decimal place.
DON GB sample content exceeds the limit value of 2761 the first law required further confirmation.
28 Precision
Two independent determination results under the absolute difference in repeatability condition must not exceed 25% of the arithmetic mean.
Other 29
When weighed sample of grain and its products 5g, the method detection limit of 200μg/kg, the limit of quantification of 250μg/kg.
Appendix A
A.1 Standard Basic Information
Standard basic information Table A.1.
Table A.1 Standard Basic Information
Chinese name English name abbreviation CAS Number Molecular Formula Molecular Weight
Deoxynivalenol bacteria enolase Deoxynivalenol DON 51481-10-8 C15H20O6 296
3-acetyl deoxynivalenol bacteria enol 3-acetyl-deoxynivalenol 3-ADON 50722-38-8 C17H22O6 338
15- acetyl deoxynivalenol bacteria enol 15-acetyl-deoxynivalenol 15-ADON 88337-96-6 C17H22O6 338
A.2 immunoaffinity column capacity quality authentication method column
A.2.1 column capacity verification. in 5mL water was added 6000ngDON standard stock solution and mix thoroughly. The same batch were taken
3 immunoaffinity columns, each column of the sample volume was 1mL. After loading, washing, elution, eluate dried with nitrogen to 1mL, with early
Beginning with mobile phase to 1mL, separation and determination of DON was determined by liquid chromatography.
Results found. Results DON≥1000ng (recovery ≥80%, RSD% ≥15%), can be used as a commodity.
A.2.2 column recoveries authentication methods. in 5mL water was added 6000ngDON standard stock solution and mix thoroughly. Take the same respectively
A batch of three immunoaffinity columns, each column of the sample volume was 1mL. After loading, washing, elution, eluate dried with nitrogen to
1mL, using an initial mobile phase volume to 1mL, separation and determination of DON was determined by liquid chromatography.
Results found. Results column recovery ≥80% (RSD% ≥15%), can be used as a commodity.
Appendix B
Standard Substance chromatography - mass spectrum
B.1 deoxynivalenol bacteria enolase (ESI-) ion scan is shown in Figure B.1.
Figure B.1 deoxynivalenol bacteria enolase (ESI-) ion scan
B.2 3- acetyl deoxynivalenol bacteria enolase (ESI-) ion scan is shown in Figure B.2.
Figure B.2 3- acetyl deoxynivalenol bacteria enolase (ESI-) ion scan
B.3 15- acetyl deoxynivalenol bacteria enolase (ESI-) ion scan is shown in Figure B.3.
Figure B.3 15- acetyl deoxynivalenol bacteria enolase (ESI-) ion scan
B.4 deoxynivalenol bacteria enolase and Acetyl Derivatives multiple reaction monitoring (MRM-ESI-) chromatogram shown in Figure B.4.
Figure B.4 deoxynivalenol bacteria enolase and acetylated derivatives
(DON/3-ADON/15-ADON) and multiple reaction monitoring (MRM-ESI-) chromatogram
B.5 deoxynivalenol bacteria enolase (ESI) ion scan is shown in Figure B.5.
Figure B.5 deoxynivalenol bacteria enolase (ESI) ion scan
B.6 3- acetyl deoxynivalenol bacteria enolase (ESI) ion scan is shown in Figure B.6.
Figure B.6 3- acetyl deoxynivalenol bacteria enolase (ESI) ion scan
B.7 15- acetyl deoxynivalenol bacteria enolase (ESI) ion scan is shown in Figure B.7.
Figure B.7 15- acetyl deoxynivalenol bacteria enolase (ESI) ion scan
B.8 deoxynivalenol bacteria enolase and Acetyl Derivatives multiple reaction monitoring (MRM-ESI) chromatogram shown in Figure B.8.
Figure B.8 deoxynivalenol bacteria enolase and Acetyl Derivatives multiple reaction monitoring (MRM-ESI) chromatogram
B.9 deoxynivalenol bacteria enolase HPLC standard solution is shown in Figure B.9.
twenty one
Figure B.9 deoxynivalenol fungi alcohol standard solution HPLC diagram
Related standard:   GB 5009.118-2016  GB 5009.91-2017
Related PDF sample:   GB 5009.118-2016  GB/T 5009.116-2003
   
 
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