GB 23200.69-2016Food safety national standard -- Determination of Dinitroaniline Pesticide Residues in Food by Liquid Chromatography -- Mass Spectrometry / Mass Spectrometry (English PDF)

4.1 Reagents

4.1.1 Methanol (CH3OH). Chromatographic Purification.

4.1.2 acetonitrile (CH3CN). pure chromatography.

4.1.3 n-hexane (C6H14). chromatographic purity.

4.1.4 Formic acid (CH2O2). Chromatographic purity.

4.1.5 Acetone (C3H6O). excellent grade pure.

4.1.6 Sodium chloride (Nacl). Analytical purity.

4.1.7 anhydrous sodium sulfate (Na2SO4). analytical grade, with 650 °C before burning 4 h, set the dryer in the spare.

4.2 Solution preparation

4.2.1 acetonitrile-water solution (1 1, containing 0.05% formic acid). 500 mL of acetonitrile and 0.5 mL of formic acid in 1 L volumetric flask, Set to 1 L, mix well.

4.2.2 n-hexane - acetone solution (2 8). take 20 mL of n-hexane, add 80 mL of acetone, mix.

4.3 Standards

4.3.1 Dinitroaniline Pesticide Reference Material. See Appendix A, Table A.1.

4.4 Standard solution preparation

4.4.1 dinitroaniline pesticide standard stock solution. accurately weighed appropriate amount of dinitroaniline pesticide standard substances, with acetone with A standard stock solution at a concentration of 1000 mg/L was prepared and the standard solution was kept at -18 ° C for a period of 12 months.

4.4.2 dinitroaniline pesticide mixed intermediate standard solution. absorb the appropriate amount of the standard stock solution, diluted with methanol into the concentration of trifluralin for the 5.0mg/L, the other seven kinds of drug concentration of 1.0 mg/L mixed intermediate standard solution, 0 °C ~ 4 °C dark preservation, shelf life of 3 months.

4.4.3 matrix mixed standard working solution. absorb the appropriate amount of mixed intermediate standard solution, with blank sample extract with fen lering concentration 0µg/L, 50µg/L, 100µg/L, 250µg/L, 500µg/L and 1000µg/L, the other seven pesticide concentrations were 0µg/L, 10.0µg /20.0 µg/L, 50.0 µg/L, 100 µg/L and.200 µg/L, respectively. The day of preparation.

4.5 Materials

4.5.1 Graphitized Carbon Black Solid Phase Extraction Column. 3 mL 250 mg, or equivalent. Activate the column with the first 3 mL n-hexane-acetone solution Moist.

4.5.2 Oasis HLB1, 6mL,.200mg, or equivalent. Activate with 5 mL acetonitrile before use to keep the column moist.

6.1 Preparation of the sample

6.1.1 Soybeans, tea and rice

Approximately 500 g of the sample was taken (not with water). After shredding, the sample was processed into a slurry by a crusher and mixed. The samples are divided into Two, into a clean container, sealed, and marked with a mark.

6.1.2 Vegetables, fruits, eggs, pork and chicken liver

Approximately 500 g of the sample was taken (not with water). After shredding, the sample was processed into a slurry by a crusher and mixed. The samples are divided into Two, into a clean container, sealed, and marked with a mark.

Note. The above sample sampling site according to GB 2763 Appendix A implementation.

6.2 Sample storage

During the operation of the sample preparation, the sample should be protected from contamination or changes in the content of the residue. Soybeans, tea and rice samples in the often Save under temperature. Vegetables, fruits, eggs, pork and chicken liver samples are stored at -18 C.

7.1 Extraction

7.1.1 Soybeans, rice, tea, eggs, pork and chicken liver

For tea, soybeans, chestnuts, and corn samples, weigh 2.5 g of the sample (accurate to 0.01 g). For spinach, mushrooms, apples, cow Meat, beef, chicken, fish, milk samples, weighed 5 g sample (accurate to 0.01 g). The weighed sample was placed in a 50 mL centrifuge tube, Add 6 mL of saturated aqueous sodium chloride solution and mix for 30 s on a vortex mixer for 15 min. Add 6 mL of acetone to n-hexane solution Mix the mixers for 2 min. 5 000 r/min centrifugation 1 min, the upper layer of extract in another test tube. And then add 4 mL of acetone - plus the alkane solution was repeatedly extracted twice and the combined extracts were purged with nitrogen at about 45 ° C to about 1 mL to be purified.

7.1.2 Vegetables, fruits

Weigh about 5 g (exactly to 0.01 g) of the sample in a 50 mL stoppered centrifuge tube, then add 2 g of sodium chloride, add 15 mL of acetonitrile the homogeneity was extracted for 3 min and centrifuged at 5 000 r/min for 5 min. The acetonitrile layer was transferred to a 25 mL volumetric flask. The residue was repeated with 10 mL of acetonitrile Extract once, combine the extract and freeze to 25 mL with acetonitrile.

7.2 Purification

7.2.1 Soybeans, rice, pork

The whole concentrated extract was aspirated in a Supelclean C18 solid phase extraction column at a flow rate of about 1.5 mL/min.

The column was extracted and washed with 5 mL of acetonitrile and the solid phase extraction was dried. The whole effluent was collected in a 1.5 mL centrifuge tube and treated with nitrogen Air blowing to near dry, the residue with acetonitrile aqueous solution to 1.0 mL. After vortexing, the microporous membrane was passed through a liquid chromatography-mass spectrometer/mass spectrometer Determination.

7.2.2 Vegetables, fruits

Accurate draw 5 mL extract 5.0 mL in 15 mL centrifuge tube, below 40 °C with nitrogen blowing to near dry, with 2.0 mL n-hexane -Acetone solution was dissolved and transferred to an Envi-carb solid phase extraction column. The solution was passed through a solid phase extraction column at a flow rate of about 1.5 mL/min.

Non-commercial statement. Column models are listed here for reference only and are not intended for commercial purposes. Encourage standard users to attempt to use different manufacturers or specifications of the column.

The solid phase extraction column was rinsed with 3 mL of n-hexane-acetone solution and the whole effluent was collected in a 15 mL centrifuge tube.

Air blowing to near dry, the residue with acetonitrile aqueous solution to 1.0 mL. After mixing the vortex, the microporous membrane was measured by liquid chromatography-mass spectrometry/mass spectrometer set.

7.2.3 Tea, eggs and chicken liver

Extraction of tea, eggs and chicken liver concentrated extract in the Oasis HLB solid phase extraction column, the flow rate of about 1.5 mL/min the solid phase extraction column was washed with 5 mL of acetonitrile and the solid phase extraction column was drained. The whole effluent was collected in a 15 mL centrifuge tube at 40 ° C Was purged with nitrogen to near dryness, dissolved in 2.0 mL n-hexane-acetone solution, transferred to Envi-carb solid phase extraction column at about 1.5 mL/min the flow rate was passed through a solid phase extraction column and eluted with 3 mL of n-hexane-acetone solution and the solid phase extraction column was drained.

Liquid in 15 mL centrifuge tube, below 40 °C with nitrogen blowing to near dry, the residue with acetonitrile aqueous solution to 1.0 mL. After mixing the vortex, Microporous membrane for liquid chromatography - mass spectrometry/mass spectrometry.

7.3 Determination

7.3.1 Liquid Chromatographic Reference Conditions

A) Column. C18 column, 50 mm × 2.1 mm (id), 1.7 d., Or equivalent;

B) Column temperature. 40 ° C;

C) Injection volume. 10 µL;

D) The mobile phase, flow rate and gradient elution conditions are shown in Table 1.

Table 1 Mobile phase, flow rate and gradient elution conditions

time

Min

Flow rate

ML/min

0.05% formic acid - 5 mM acetate - aqueous solution Methanol 0 0.30 50 50 5.0 0.30 20 80 8.0 0.30 0 100 9.5 0.30 50 50

7.3.2 Mass spectrometry reference conditions

A) ionization mode. electrospray ion source;

B) Scanning mode. positive ion scanning;

C) Detection method. Multiple reaction monitoring (MRM);

D) Resolution. Unit resolution;

See Appendix B for other reference mass spectrometry conditions.

7.3.3 Qualitative determination

Each selected component selected one parent ion, two or more child ions, in the same experimental conditions, the sample retention time of the material to be tested, and the substrate standard solution retention time deviation within ± 2.5%; and the sample of each component of the relative abundance of ions and concentrations close to the matrix Comparing the relative abundance of the corresponding qualitative ions in the standard working solution, the deviation does not exceed the range specified in Table 2, There is a corresponding test substance in the product.

Table 2 shows the maximum allowable error for relative ion abundance when qualitatively by liquid chromatography-tandem mass spectrometry

Relative abundance (base) 50% 20% to 50% 10% to 20% <=10% Allowable relative deviation ± 20% ± 25% ± 30% ± 50%

7.3.4 Quantitative determination

The best working conditions of the instrument, the substrate mixed with the standard working solution into the peak area for the vertical axis, the matrix mixed standard working solution Concentration for the abscissa to draw the standard working curve, with the standard working curve of the sample quantitative. The response value of the analyte in the sample solution should be in the linear range of the assay method, if the linear response range is exceeded, the blank matrix solution is used for proper dilution. In the above liquid chromatography-the retention times of dinitroaniline pesticides were. salbutam (3.21 min), methotrexate (3.41 min) Ammonofural (4.06 min), Ammonolone (5.20 min), Pentylenol (5.36 min), Zhong Dingling (5.70 min), trifluralin (5.70 min) Min and isoproterenol (6.06 min). The chromatogram of mass spectrometry for mass spectrometry/mass spectrometry/mass spectrometry is shown in Figure C.1 in Appendix C.

7.4 Blank experiment

In addition to the sample, according to the above determination steps.

10.1 Quantitation limits

The limit of quantification of dinitroaniline pesticides is 0.01 mg/kg.

10.2 Recovery rate

When adding levels of 0.05 mg/kg, 0.1 mg/kg, 0.2 mg/kg, the recovery of dinitroaniline pesticides is given in Appendix D.

Appendix A

(Informative)

Dinitroaniline pesticide

Table A.1 Related information about dinitroaniline pesticides

Compound name English name Cas No. molecular formula molecular weight molecular structure Trifluralin 1582-09-8 C13H16F3N3O4 335.28 Pendimethalin 40487-42-1 C13H19N3O4 281.31 Zhong Dingling Butralin 33629-47-9 C14H21N3O4 295.33 Isopropalin 33820-53-0 C15H23N3O4 309.36 Amoxicillin Dinitramine 29091-05-2 C11H13F3N4O4 322.24 Nitrone Nitrone Amoxicillin Oryzalin 19044-88-3 C12H18N4O6S 346.36 Ampicillin Prodiamine 29091-21-2 C13H17F3N4O4 350.29 Appendix B (Informative) Mass spectrometry conditions A) electrospray voltage. 3000 V;

B) Auxiliary gas flow rate. 750 L/h;

C) collision gas. argon;

D) curtain air flow rate. 50 L/h;

E) ion source temperature. 105 F) Auxiliary gas temperature. 350 auxiliary;

G) Qualitative ion pair, quantitative ion pair, acquisition time, cone hole voltage and collision energy are shown in Table B.1.

Table B.1 Mass Spectrometric Parameters of Dinitroaniline Pesticide Reference Material

Analyte

Reference retention time Min Mother ion (M/z) Ion (M/z) Acquisition time Cone hole voltage Collision energy EV Trifluralin 5.70 336.2 236 * 0.1 252 0.1 23 Pendimethalin 5.36 282 212 * 0.05 194 0.05 17 Zhong Dingling 5.70 296 240 * 0.05 222 0.05 20 Isopropyl Lose 6.06 310 226 * 0.05 268 0.05 14 Ampicillin 4.06 323 289 * 0.05 247 0.05 16 Methasone Lentin 3.41 346 304 * 0.05 262 0.05 22 Amoxicillin 3.21 347 288 * 0.05 305 0.05 14 Ammonofluramine 5.20 351 267 * 0.05 291 0.05 18 Note. * for the quantitative ions, for different mass spectrometry equipment, instrument parameters may be different, should be measured before the mass spectrometry parameters to optimize the best.

Non-commercial declaration. The reference mass spectrometry conditions listed in B.1 are performed on a Waters Quattro Premier LC/MS, where the test instrument model Only for reference, does not involve commercial purposes, to encourage standard users to try different manufacturers or models of equipment.

Appendix C

(Informative)

Standard Quality Spectrum of 8 Dinitroaniline Pesticides

Figure C.1 Standard spectra of eight dinitroaniline pesticides

Appendix D

(Informative)

The recoveries of dinitroanilines in different matrices

Table D.1 Recovery of dinitroanilines in different matrices

Compounds

name

Add water

Mg/kg

Rice recovery rate

range

Recovery rate of soybeans range Apple recovery rate range Watermelon Recycling range Recovery of melon range Ginger recycling range Spinach recycling range Cabbage recycling range Tea recycling range Egg recycling range Pork recycling range Chicken liver recovery range Trifluralin 50.0 74.7 to 98.8 103 to 116 74.9 to 115 93.3 to 120 79.8 to 98.6 87.3 to 111 72.6 to 82.4 72.5 to 78.4 75.8 to 99.6 100 to 112 71.2 to 75.5 73.1 to 79.1 100 77.1 to 114 89.2 to 119 71.8 to 88.6 79.7 to 102 85.0 to 112 76.3 to 109 71.5 to 92.6 70.5 to 81.5 77.3 to 96.5 99.9 to 111 70.2 to 73.0 70.6 to 77.8 200 76.0 to 115 70.8 to 122 75.1 to 98.9 76.4 to 103 72.4 to 97.4 70.2 to 81.1 70.5 to 86.0 71.5 to 76.8 77.5 to 84.0 74.0 to 85.5 71.5 to 77.3 74.6 to 80.5 Paclitaxel 10.0 88.0 to 118 79.0 to 95.0 95.1 to 121 70.6 to 73.7 74.6 to 118 86.2 to 116 79.0 to 95.0 89.4 to 98.2 71.0 to 79.0 89.0 to 96.0 73.5 to 87.3 85.3 to 96.9 50.0 77.7 to 105 84.6 to 99.6 82.4 to 110 70.5 to 103 83.0 to 114 86.3 to 97.8 84.6 to 95.0 85.9 to 96.8 71.2 to 77.8 81.8 to 91.8 80.2 to 95.9 80.7 to 91.0 200 85.0 to 119 73.5 to 96.0 89.1 to 118 73.8 to 82.0 76.0 to 94.2 83.9 to 101 73.5 to 96.0 91.2 to 96.9 85.5 to 89.0 74.5 ~ 82.5 84.5 ~ 98.5 70.4 ~ 78.8 Ammonolate 10.0 91.0 to 115 80.4 to 96.8 99.1 to 121 72.5 to 115 97.9 to 119 84.7 to 107 83.0 to 101 92.6 to 94.1 80.0 to 99.0 87.6 to 94.2 75.3 to 96.7 83.6 to 98.7 50.0 79.0 to 115 91.0 to 117 91.4 to 108 83.7 to 100 95.1 to 116 79.1 to 104 84.4 to 109 91.7 to 98.4 74.2 to 79.4 78.3 to 83.3 82.9 to 96.4 83.7 to 97.8 100 79.5 to 120 71.0 to 115 90.9 to 121 85.8 to 94.0 88.3 to 108 70.6 to 92.0 84.4 to 109 91.7 to 98.8 72.8 to 88.8 93.0 to 99.0 85.3 to 99.0 91.6 to 95.9 Zhong Dingling 10.0 91.0 to 118 70.7 to 98.0 103 to 113 70.6 to 76.2 95.6 to 120 91.5 to 98.7 75.0 to 98.0 91.9 to 95.7 71.0 to 78.0 81.0 to 94.0 70.6 to 91.5 77.2 to 95.6 20.0 72.5 to 104 74.5 to 94.0 87.5 to 101 70.6 to 87.8 91.7 to 106 86.5 to 96.8 74.5 to 94.0 91.8 to 95.5 92.0 to 105 75.5 to 83.6 83.9 to 98.5 79.9 to 97.9 50.0 81.4 to 119 74.8 to 89.8 85.8 to 116 70.8 to 72.8 87.6 to 99.8 79.6 to 88.3 74.8 to 89.8 91.8 to 94.1 98.0 to 101 84.0 to 95.0 86.9 to 97.9 87.5 to 98.4 Isoprofen 10.0 86.0 to 119 76.0 to 83.0 96.6 to 122 70.0 to 75.1 96.1 to 122 71.7 to 81.2 76.0 to 83.0 91.5 to 98.5 70.0 to 81.0 81.5 to 94.0 70.6 to 90.2 76.2 to 93.2 20.0 78.5 to 107 77.5 to 89.5 85.6 to 97.7 72.2 to 89.8 85.3 to 106 75.7 to 83.0 79.0 to 94.0 90.9 to 98.8 87.5 to 100 74.2 to 78.0 81.4 to 89.3 78.1 to 91.4 50.0 70.8 to 120 73.0 to 89.0 76.8 to 104 70.9 to 79.9 71.3 to 95.1 70.5 to 75.0 71.0 to 89.0 91.2 to 95.7 86.1 to 95.7 74.2 to 78.0 82.6 to 90.5 83.1 to 91.6 Ammonofen 10.0 88.0 to 120 71.0 to 89.0 102 to 116 72.1 to 82.0 96.4 to 115 78.4 to 95.6 79.0 to 91.5 89.1 to 97.4 70.0 to 81.0 94.0 to 102 72.1 to 98.9 71.8 to 92.3 20.0 76.5 to 119 82.1 to 104 90.5 to 111 70.5 to 91.8 97.0 to 118 78.4 to 90.8 79.0 to 91.4 91.3 to 99.0 87.5 to 100 83.5 to 92.5 89.9 to 98.8 84.8 to 96.7 50.0 83.8 to 119 79.0 to 91.5 93.4 to 110 71.3 to 86.8 86.7 to 107 78.3 to 92.5 70.0 to 95.4 90.8 to 96.1 78.8 to 88.6 80.2 to 109 92.0 to 98.8 90.8 to 98.7 Methrone 10.0 91.0 to 112 70.0 to 95.4 95.3 to 120 76.9 to 118 98.1 to 125 88.9 to 111 74.4 to 94.0 91.9 to 97.2 86.0 to 99.0 100 to 109 74.2 to 92.5 77.8 to 97.7 20.0 70.7 to 105 74.0 to 94.0 92.7 to 104 91.1 to 98.2 96.5 to 118 84.9 to 103 84.0 to 96.8 90.7 to 97.4 91.0 to 110 83.0 to 103 91.3 to 98.6 91.7 to 95.1 50.0 83.2 to 115 84.0 to 97.5 94.2 to 112 83.4 to 96.6 97.7 to 118 81.4 to 92.4 83.0 to 96.8 91.2 to 93.6 82.5 to 91.5 88.0 to 95.0 92.1 to 97.3 91.7 to 97.6 Amaranth 10.0 77.5 to 101 79.5 to 112 93.5 to 123 71.1 to 91.5 77.9 to 119 88.2 to 107 72.0 to 89.0 91.9 to 92.9 70.0 to 80.0 81.5 to 97.0 91.9 to 96.2 85.1 to 99.4 20.0 75.5 to 114 71.0 to 99.5 82.2 to 106 71.9 to 84.7 95.5 to 106 87.9 to 106 71.0 to 99.5 90.8 to 92.6 91.5 to 96.5 81.2 to 83.6 82.9 to 96.4 80.1 to 98.1 50.0 74.8 to 113 80.4 to 100 83.1 to 108 71.9 to 81.4 85.3 to 105 80.0 to 87.2 80.4 to 100 88.3 to 96.3 94.5 to 105 80.2 to 101 87.3 to 96.6 76.1 to 87.1 Appendix E (Normative appendix) Laboratory repeatability requirements

Table E.1 Laboratory repeatability requirements

Measured component content Mg/kg Precision <=0.001 36 > 0.01 > 1 14 Appendix F (Normative appendix) Inter-laboratory reproducibility requirements

Table F.1 Inter-laboratory reproducibility requirements

Measured component content Mg/kg Precision <=0.001 54 > 0.01 > 1 19