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GB 23200.73-2016: Food safety national standard -- Determination of rotenone and azadirachtin residues in food by liquid chromatography-mass spectrometry / mass spectrometry
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Food safety national standard -- Determination of rotenone and azadirachtin residues in food by liquid chromatography-mass spectrometry / mass spectrometry
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GB 23200.73-2016
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Basic data | Standard ID | GB 23200.73-2016 (GB23200.73-2016) | | Description (Translated English) | Food safety national standard -- Determination of rotenone and azadirachtin residues in food by liquid chromatography-mass spectrometry / mass spectrometry | | Sector / Industry | National Standard | | Classification of Chinese Standard | G25 | | Word Count Estimation | 12,162 | | Date of Issue | 2016-12-18 | | Date of Implementation | 2017-06-18 | | Older Standard (superseded by this standard) | SN/T 3264-2012 | | Regulation (derived from) | State Health Commission, Ministry of Agriculture, Food and Drug Administration Notice No. 16 of 2016 | | Issuing agency(ies) | National Health and Family Planning Commission of the People's Republic of China, State Food and Drug Administration | GB 23200.73-2016: Food safety national standard -- Determination of rotenone and azadirachtin residues in food by liquid chromatography-mass spectrometry / mass spectrometry
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Food safety national standard - Determination of rotenone and azadirachtin residues in food by liquid chromatography - mass spectrometry/mass spectrometry
National Standards of People's Republic of China
GB
Replace SN/T 3264-2012
National standards for food safety
Determination of the residue of rotenone and azadirachtin in food
Liquid chromatography - mass spectrometry/mass spectrometry
National food safety standards-
Determination of rotenone and azadirachtin residues in foods
Liquid chromatography - mass spectrometry
2016-12-18 Release.2017-06-18 Implementation
National Health and Family Planning Commission of the People 's Republic of China
Issued by the Ministry of Agriculture of the People 's Republic of China
State Administration of Food and Drug Administration
Foreword
This standard replaces SN/T 3264-2012 "Detection methods of rotenone and azadirachtin residues in export food by liquid chromatography-mass spectrometry
Spectrum method ".
Compared with SN/T 3264-2012, the main changes are as follows.
- Standard text format is modified to national standard text format for food safety;
- the name and scope of the "export food" to "food";
- increase the "other food reference implementation" in the standard range.
This standard replaced the previous version of the standard release.
-SN/T 3264-2012.
National standards for food safety
Determination of rotenone and azadirachtin residues in food by liquid chromatography - mass spectrometry/mass spectrometry
1 Scope
This standard specifies methods for the determination of rotenone and azadirachtin residues in food by liquid chromatography-mass spectrometry/mass spectrometry.
This standard applies to rice, cauliflower, apple, fungus, tea, honey, liver, fish, shrimp, chicken, milk rotenone
And azadirachtin residues in the determination and confirmation, other food can refer to the implementation.
2 normative reference documents
The following documents are indispensable for the application of this document. For dated references, only the dated edition applies to this article
Pieces. For undated references, the latest edition (including all modifications) applies to this document.
GB 2763 National Standard for Food Safety - Maximum Residue Limit of Pesticides in Foodstuffs
GB/T 6682 Analytical laboratory water specifications and test methods
3 principle
The residual rotenone and azadirachtin in the sample were extracted with acetonitrile, and the extract was salted out by sodium chloride and degreased with n-hexane. The polystyrene-
Dimethylbenzene-pyrrolidone polymer filler solid phase extraction column purification, liquid chromatography - mass spectrometry/mass spectrometry detection and confirmation, external standard statutory
the amount.
4 reagents and materials
Unless otherwise specified, all reagents are of analytical grade and water is in accordance with the primary water specified in GB/T 6682.
4.1 Reagents
4.1.1 Acetonitrile (CH3CN). Chromatographically pure.
4.1.2 n-hexane (C6H10). pure chromatography.
4.1.3 Methanol (CH3OH).
4.1.4 Formic acid (HCOOH). Chromatographic pure.
4.1.5 Sodium chloride (NaCl).
4.1.6 Ammonium acetate (CH3COONH4).
4.1.7 Sodium bicarbonate (NaHCO3).
4.2 solution preparation
4.2.1 Saturated sodium bicarbonate solution. Weigh a certain amount of sodium bicarbonate dissolved in water to saturation.
4.2.2 5mmol/L ammonium acetate buffer. Weigh 0.38 g of ammonium acetate dissolved in 800 mL of water, add 2 mL of formic acid, the water volume to 1000
ML.
4.3 standards
4.3.1 Reference substance (rotenone. English name Rotenone, molecular formula C23H22O6, CAS No. 83-79-4, molecular weight 394.42;
Azadirachtin. English name Azadirachtin, molecular formula C35H44O16, CAS No.11141-17-6, molecular weight 720.71). purity ≥98%.
4.4 standard solution preparation
4.4.1 rotenone and azadirachtin standard stock solution (100 mg/L). accurately weighed 0.0100 g of rotenone and azadirachtin standard substance, with a
The alcohol was dissolved and set to 100 mL, and the standard stock was kept at 4 ° C for no more than one month.
4.4.2 rotenone and azadirachtin standard working solution. according to the need to take appropriate standard stock solution, 20% acetonitrile aqueous solution diluted to the appropriate concentration
Of the standard working fluid, temporary with the distribution.
4.5 Materials
4.5.1 Solid phase extraction column of polystyrene-divinylbenzene-pyrrolidone polymer filler. 60 mg, 3 mL. Use 3 mL before use
Methanol, 3 mL water pretreatment.
4.5.2 Filtration. 0.22 μm, organic.
5 instruments and equipment
5.1 Liquid Chromatography-Mass Spectrometry/Mass Spectrometer, Distribution Spray (ESI) Source.
5.2 Analysis of balance. 0.01 g and 0.0001 g.
5.3 Centrifuge. 4 500 r/min with 50 mL stoppered plastic centrifuge tube.
5.4 pulverizer.
5.5 Tissue crusher.
5.6 Scroll Mixer.
5.7 Ultrasonic Cleaner.
5.8 solid phase extraction device.
5.9 nitrogen blowing instrument.
6 Preparation and storage of samples
6.1 Preparation of the sample
6.1.1 Fruits and vegetables
Take a representative sample 500 g, chopped (not washed), with the tissue crusher to sample processed into a slurry, mix, divided into 2,
Into a clean container, sealed and identified.
6.1.2 Tea, grain and nuts
Take a representative sample of 500 g, crushed by a pulverizer and passed through a sieve having a diameter of 2.0 mm, mixed, divided into 2 portions, packed in a clean container
Inside, sealed and identified.
6.1.3 Meat and meat products
Take a representative sample 500 g, chopped after the use of tissue crusher sample processed into a slurry, mix, into a clean container, divided into 2
Part, sealed and identified.
6.1.4 honey
Take a representative sample of 500 g, and stir the honey sample without crystallization. For crystalline samples, in confined cases,
Placed in a water bath of not more than 60 ℃ in the warm, shaking, until the sample all melt and stir well, quickly cooled to room temperature, divided into 2, into the clean
Net vial, sealed and identified.
6.1.5 milk
Take a representative sample of 500 g, mix thoroughly, divide it into 2, into a clean vial, seal and mark it.
In the sample preparation process, should prevent the sample contamination or the occurrence of residue content changes.
Note. The above sample sampling site according to GB 2763 Appendix A implementation.
6.2 Sample storage
Tea, wine, honey, milk, grain stored at 0 ~ 4 ℃, vegetables, fruits, meat and meat products stored at -18 ℃.
7 Analysis steps
7.1 Extraction
7.1.1 samples of tea, cereals and nuts
Weigh 1 g (accurate to 0.01 g) sample, placed in a 50 mL stoppered plastic centrifuge tube, add 5 mL of saturated sodium bicarbonate solution
Shake and soak for 10 min, add 15 mL of acetonitrile, vortex 30 s after ultrasonic extraction 10 min, 4 500 r/min centrifugal 3 min, shift
The organic phase, the residue and then add 10 mL of acetonitrile repeated extraction 1 times, combined extract, add about 3 g sodium chloride salting out, 4 500 r/min
Centrifuge 3 min centrifugation, take the supernatant, add 2 mL by acetonitrile saturated n-hexane, shaking 1 min, 4 500 r/min centrifugal 3 min
Centrifuged, discarded n-hexane layer, acetonitrile layer at 45 ℃ under reduced pressure evaporation to near dry, with 5 mL of 20% methanol water to dissolve the residue, according to 7.2 steps
Purification.
7.1.2 Vegetables and fruit samples
Weigh 2 g (accurate to 0.01 g) sample, placed in 50 mL stoppered plastic centrifuge tube, add about 2 g sodium bicarbonate and 15 mL
Acetonitrile, shake evenly after ultrasonic extraction 10 min, 4 500 r/min centrifugal 3 min, remove the organic phase, the residue and then add 10 mL of acetonitrile weight
Extraction of the extraction 1, combined extract, add about 3 g sodium chloride salting out, 4 500 r/min centrifugal 3 min, the supernatant, at 45 ℃ by
Pressure rotation to near dry, with 5 mL of 20% methanol water to dissolve the residue, according to 7.2 steps purification.
7.1.3 honey
Weigh 2 g (accurate to 0.01 g) The sample was placed in a 50 mL stoppered plastic centrifuge tube, 5 mL of saturated sodium bicarbonate solution was shaken,
Add 15 mL of acetonitrile, vortex 30 s after ultrasonic extraction 10 min, 4 500 r/min centrifugal 3 min, remove the organic phase, the residue and then add
10 mL of acetonitrile was repeated 1 times, the combined extract, add about 3 g sodium chloride salting out, 4 500 r/min centrifugation 3 min, take the supernatant,
At 45 ° C under reduced pressure rotary evaporation to near dry, with 5 mL of 20% methanol water to dissolve the residue, according to 7.2 steps purification.
7.1.4 milk, fruit juice and wine
Weigh 2 g (accurate to 0.01 g) The sample was placed in a 50 mL stoppered plastic centrifuge tube, about 2 g of sodium bicarbonate and 15 mL of acetonitrile was added,
After centrifugation for 30 s, the mixture was centrifuged at 4 500 r/min for 3 min, and the organic phase was removed. The residue was added with 10 mL of acetonitrile.
Take 1, the combined extract, add about 3 g sodium chloride salting out, 4 500 r/min centrifugal 3 min, take the supernatant, at 45 ℃ under pressure
Evaporated to near dry, with 5 mL of 20% methanol water to dissolve the residue, according to 7.2 steps purification.
7.1.5 Fish, meat and meat products
Weigh 2 g (accurate to 0.01 g) The sample was placed in a 50 mL stoppered plastic centrifuge tube, 5 mL of saturated sodium bicarbonate solution was shaken,
Add 15 mL of acetonitrile, vortex 30 s after ultrasonic extraction 10 min, 4 500 r/min centrifugal 3 min, remove the organic phase, the residue and then add
10 mL of acetonitrile was repeated 1 times, the combined extract, add about 3 g sodium chloride salting out, 4 500 r/min centrifugation 3 min, take the supernatant,
Add 5 mL n-hexane, shaking 1 min, 4 500 r/min centrifugal 3 min, discard the n-hexane layer, acetonitrile layer at 45 ℃ under reduced pressure steam
Sent to near dry, with 5 mL of 20% methanol water to dissolve the residue, according to 7.2 steps purification.
7.2 Purification
The sample extract was rinsed with 5 mL of water and the whole eluent was discarded, dried, eluted with 5 mL of acetonitrile to maintain a flow rate of about 1
ML/min, the eluent was collected and blown to near dry at 45 ° C. The mixture was allowed to settle 1 mL in 20% aqueous acetonitrile and tested for 0.22 μm.
7.3 Determination
7.3.1 Liquid Chromatographic Reference Conditions
A) Column. Phenomenex Luna C18 column, 150 mm x 2.0 mm (id), 3 [mu] m, or equivalent;
B) Column temperature. 35 ° C;
C) mobile phase. acetonitrile - 5 mmol/L ammonium acetate buffer (35 65, V/V);
D) Flow rate. 400 μL/min;
E) Injection volume. 10 μL.
7.3.2 Mass spectrometry reference conditions
A) ion source. electrospray source (ESI), positive ion mode;
B) scanning mode. multiple reaction monitoring (MRM);
Refer to Table A.1 in Appendix A for other reference mass spectrometry conditions.
7.3.3 Determination and confirmation of chromatography
According to the sample content of the sample, select the appropriate response value of the standard working solution for chromatographic analysis. Standard working fluid and sample to be tested
The response values of rotenone and azadirachtin in the solution should be within the linear response range of the instrument. According to the formula 8 under the results of the calculation. In this method
The retention time of rotenone and azadirachtin was about 5.6 min and 4.2 min, and the multi-reaction monitoring (MRM) of rotenone and azadirachtin standards,
See Appendix B for chromatograms.
Qualitative criteria For sample determination, if the detected chromatographic peak retention time is consistent with the standard sample, and after deducting the background
In the spectrum, the relative abundance of each qualitative ion is close to the standard solution spectrum obtained under the same conditions, and the maximum allowable relative deviation
The difference does not exceed the range specified in Table 1, it can be judged that there is a corresponding analyte in the sample.
Table 1 Maximum allowable deviation of relative ion abundance when qualitative confirmation
Relative abundance (base) 50% 20% to 50% 10% to 20% ≤10%
Allowable relative deviation ± 20% ± 25% ± 30% ± 50%
7.4 blank experiment
In addition to the sample, according to the above steps.
8 results are calculated and expressed
Use the data processing software or according to formula (1) to calculate the samples of rotenone and azadirachtin drug residues, the results need to deduct the blank value.
X = C ×
(1)
Where.
Residue of rotenone or azadirachtin in the sample, in micrograms per kilogram, μg/kg;
The concentration of rotenone and azadirachtin solution from the standard working curve, in micrograms per liter, μg/L;
The final volume of the sample solution, in milliliters, mL;
The mass of the sample represented by the final sample, in grams, g.
Note. The result of the calculation shall be deducted from the blank value. The result of the measurement shall be expressed as the arithmetic mean of the parallel measurement, and two valid digits shall be retained.
9 precision
9.1 The ratio of the absolute difference between the two independent determinations obtained under reproducibility and its arithmetic mean (percentage) shall be in accordance with
Appendix D requirements.
9.2 The ratio of the absolute difference between the two independent determinations obtained under reproducibility and its arithmetic mean (percentage) shall be in accordance with
Appendix E requirements.
10% limit and recovery rate
10.1 Quantitation limits
The limits of quantification of rotenone and azadirachtin were 0.0005 mg/kg and 0.002 mg/kg, respectively.
10.2 Recovery rate
The added recoveries of rotenone and azadirachtin when added at levels of 0.0005 mg/kg, 0.001 mg/kg, 0.005 mg/kg
Record C.
Appendix A (1)
(Informative)
Reference to mass spectrometry conditions
Reference mass spectrometry conditions.
Capillary voltage. 4 kV;
Shielding gas temperature. 320 ℃;
Shielding air flow. 10 L/min;
Dry air flow. 3 L/min;
Collision pressure. 50 psi;
Other mass spectral parameters are shown in Table B.1
Table A.1 The main reference mass spectra of rotenone and azadirachtin
Compound Monitoring Ion Dwell Time (ms) Voltage (V) Collision Energy (eV)
Rotenone 395 > 213 50 160 24
395 > 192 25
Azadirachtin 743 > 725 50 140 28
743 > 625 36
Note. The figures shown in the boldface in the table are quantitative ions. For different mass spectrometry instruments, the instrument parameters may be different and the mass spectrometry parameters should be optimized before the measurement
To the best.
(1) Non-commercial declaration. The reference mass spectrometry conditions listed in Appendix C are performed on an Agilent 6460 LC/MS, where the test instrument model is only listed
For reference, does not involve commercial purposes, encourage standard users to try different manufacturers or models of equipment.
Appendix B
(Informative)
Multi - reaction monitoring chromatogram of rotenone and azadirachtin standard solution
Figure B.1 Multi-reaction monitoring chromatograms of rotenone and azadirachtin standard solutions
Appendix C
(Informative)
The recovery of rotenone and azadirachtin in different matrices
Table C.1 Data on the recovery of rotenone and azadirachtin residues
Sample name rotenone azadirachtin
Add concentration (μg/kg) Recovery rate range (%) Add concentration (μg/kg) Recovery rate range (%)
Rice
0.5 83.5 to 102.0 2 75.2 to 104.5
1 84.6 ~ 101.8 4 79.6 ~ 105.2
5 79.3 ~ 97.8 20 80.5 ~ 101.3
cauliflower
0.5 81.0 to 103.0 2 84.8 to 106.5
1 79.4 to 96.8 4 80.1 to 102.4
5 80.8 ~ 108.7 20 78.9 ~ 100.3
apple
0.5 81.0 to 96.0 2 81.4 to 102.8
1 79.6 ~ 98.4 4 86.6 ~ 106.5
5 78.4 ~ 100.3 20 90.1 ~ 102.4
Fungus
0.5 81.0 to 99.0 2 82.1 to 108.2
1 79.0 ~ 99.8 4 85.6 ~ 104.4
5 81.4 ~ 104.5 20 81.4 ~ 104.8
tea
0.5 78.0 to 103.0 2 74.6 to 107.6
1 78.8 ~ 102.2 4 76.2 ~ 100.3
5 77.4 ~ 99.7 20 78.9 ~ 104.8
honey
0.5 84.0 to 101.0 2 79.4 to 102.8
1 80.2 ~ 97.6 4 80.5 ~ 103.2
5 80.5 to 99.7 20 80.5 to 100.5
milk
0.5 76.2 ~ 104.3 2 78.6 ~ 109.6
1 76.4 ~ 106.6 4 76.2 ~ 95.5
5 76.2 ~ 102.8 20 81.9 ~ 106.8
Liver
0.5 75.0 to 98.0 2 77.2 to 106.5
1 78.4 ~ 103.2 4 76.3 ~ 101.5
5 76.8 ~ 100.1 20 81.8 ~ 100.7
Fish
0.5 79.0 to 105.0 2 77.5 to 100.3
1 79.6 ~ 101.2 4 76.2 ~ 101.7
5 80.7 to 103.0 20 80.9 to 98.4
Shrimp
0.5 81.0 to 97.0 2 77.2 to 102.8
1 80.6 ~ 101.8 4 79.2 ~ 105.3
5 80.3 ~ 96.3 20 82.7 ~ 101.4
chicken
0.5 83.0 to 97.0 2 78.6 to 104.5
1 80.4 ~ 104.2 4 79.3 ~ 93.8
5 81.2 ~ 96.9 20 81.9 ~ 106.8
Appendix D
(Normative appendix)
Laboratory repeatability requirements
Table D.1 Laboratory repeatability requirements
Measured component content
Mg/kg
Precision
0.001 36
> 0.01
> 1 14
Appendix E
(Normative appendix)
Inter-laboratory reproducibility requirements
Table E.1 Inter-laboratory reproducibility requirements
Measured component content
Mg/kg
Precision
0.001 54
> 0.01
> 1 19
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