GB 23200.56-2016Food safety national standard -- Determination of Quinoxaline Residue in Food (English PDF)

4.1 Reagents

4.1.1 N-Hexane (C6H10). Chromatographic pure.

4.1.2 Ethyl acetate (C4H8O2). Chromatographic purity.

4.1.3 Acetonitrile (CH3CN). Chromatographic Purification.

4.1.4 Methanol (CH3OH). Chromatographic Purification.

4.1.5 Cyclohexane (C6H12). Chromatographic pure.

4.1.6 Acetone (C3H6O). Chromatographic pure.

4.1.7 Sodium bicarbonate (NaHCO3).

4.1.8 anhydrous sodium sulfate (Na2SO4). dried at 650 °C for 4 h before use, sealed and stored.

4.2 Solution preparation

4.2.1 Cyclohexane-ethyl acetate solution (1 1, V/V). 50 mL of cyclohexane and ethyl acetate were mixed and mixed.

4.2.2 5% acetone-n-hexane solution. Remove 5 mL of acetone in 95 mL n-hexane and mix well.

4.2.3 50% acetonitrile - water solution. 50 mL of acetonitrile and 50 mL of water were collected and mixed.

4.2.4 saturated sodium bicarbonate solution. Weigh a certain amount of sodium bicarbonate dissolved in aqueous solution to saturation.

4.3 Standards

4.3.1 Quinoxaline standard substance (English name Quinoxyfen, molecular formula C15H8Cl2FNO, CAS No.124495-18-7, molecular weight 308.14). purity >= 98%.

4.4 Standard solution preparation

4.4.1 Quinoxaline standard stock solution (100 mg/L). accurately weighed 0.0100 g quinoxaline standard substance, dissolved in methanol and volume to 100 mL, the standard stock solution at 4 °C can be stored for three months.

4.4.2 quinoxaline standard working solution. according to the need to take appropriate standard stock solution, 50% acetonitrile aqueous solution diluted to the appropriate concentration of standard workers for liquid. The standard stock solution can be stored at 4 ° C for one month.

4.5 Materials

4.5.1 Amino (NH2) solid phase extraction column. 500 mg, 3 mL.

4.5.2 Filtration. 0.45 µm, 0.22 µm, organic.

6.1 Preparation of the sample

6.1.1 Fruits and vegetables

Take a representative sample 500 g, cut it (not water wash), with the tissue crusher to sample processed into a slurry, mix, divided into 2 copies, 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 2.0 mm round hole sieve, mixed, divided into 2 portions, packed in a clean container, 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 °C 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 Juice, wine and so on

All the original samples were poured into a clean mix bucket, stirred thoroughly and mixed, and the mixed samples were dispensed into two portions, each about 500 ML, sealed and identified.

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 °C, vegetables, fruits, meat and meat products stored at -18 °C.

7.1 Extraction

7.1.1 Samples of tea, cereals and nuts

Weigh 5 g (accurate to 0.01 g) sample, placed in a 100 mL stoppered plastic centrifuge tube, add 15 mL of saturated sodium bicarbonate solution Shake for 10 min, add 20 mL of ethyl acetate, vortex 30 s after ultrasonic extraction 20 min, 4 500 r/min centrifugation 3 min, shift the organic phase was added and the residue was further extracted with 20 mL of ethyl acetate. The extracts were combined, dried over anhydrous sodium sulfate and rotated at 50 ° C Evaporated to near dry, add 5 mL n-hexane, vortex 30 s dissolved residue, according to 7.2.2 purification steps.

7.1.2 Vegetables and fruit samples

Weigh 10 g (accurate to 0.01 g) sample, placed in a 100 mL stoppered plastic centrifuge tube, add 30 mL of ethyl acetate, shake all After centrifugation for 20 min, 4 500 r/min centrifugation 3 min, remove the organic phase, the residue and then add 20 mL of ethyl acetate repeat extraction 1 the combined extracts were dried over anhydrous sodium sulfate and evaporated to near dryness at 50 ° C. After addition, 5 mL of n-hexane and vortex Slag, according to 7.2.2 steps purification.

7.1.3 Juice, honey, wine and so on

Weigh 10 g (accurate to 0.01 g) The sample was placed in a 250 mL stoppered flask with 10 mL of saturated sodium bicarbonate solution, 30 mL Ethyl acetate, shake evenly after ultrasonic extraction 20 min, 4 500 r/min centrifugal 5 min, remove the organic phase, the residue and then add 20 mL B Ethyl acetate repeated extraction 1 times, combined extract, dried over anhydrous sodium sulfate after 50 ° C rotary evaporation to near dry, add 5 mL n-hexane, Vortex 30 s dissolved residue, according to 7.2.2 steps purification.

7.1.4 Meat and meat products

Weigh 10 g (accurate to 0.01 g) The sample was placed in a 100 mL stoppered plastic centrifuge tube, 10 mL of saturated sodium bicarbonate solution was added, 30 mL of ethyl acetate, homogenized at 10 000 r/min homogenously 30 s, 4 500 r/min centrifugal 5 min, remove the organic phase, residual Slag and then add 20 mL of ethyl acetate repeated extraction 1 times, combined extract, dried over anhydrous sodium sulfate after 50 ° C rotary evaporation to near dry, Add 10 mL of cyclohexane-ethyl acetate, shake the residue for 30 s, centrifuge at 4 500 r/min for 5 min, press 7.2.1 and 7.2.2 Sequence of purification.

7.2 Purification

7.2.1 Gel permeation chromatography

For the extract obtained in 7.1.4, take 5 mL into the gel column through the sample loop and operate according to the following conditions.

A) Purification column. Bio-Beads S-X3 filler, 25 (id) ×.200 mm (id);

B) Mobile phase. cyclohexane-ethyl acetate (1 1, V/V);

C) Flow rate. 4.2 mL/min;

The effluent from 9.5 to 21.5 min was collected and evaporated to near dry at 50 ° C. Add 5 mL of n-hexane to solid phase extraction.

The

Note. If the amount of fat contained in the sample of 5.00 g is greater than 0.5 g, the volume of the sample extract should be adjusted so that it is injected into the 5 mL sample of the gel chromatogram of the amount of fat is not greater than 0.5 g.

7.2.2 Solid phase extraction column purification

Pre-leach the column with 3 mL of methanol and 3 mL of n-hexane before use. The sample extract was washed with 5 mL of n-hexane and discarded the eluent was eluted with 5 mL of 5% acetone-n-hexane to maintain a flow rate of about 1 mL/min. The eluate was collected and blown at 50 ° C to near-with 50% acetonitrile - aqueous solution volume 1 mL, over 0.45 µm filter for high performance liquid chromatography determination; or 50% acetonitrile aqueous solution volume 5 mL, 0.22 µm filter for liquid chromatography-mass spectrometry/mass spectrometry.

Determination of liquid chromatography

7.3.1 Liquid Chromatographic Reference Conditions

A) Column. Waters Symmetry C18 column, 4.6 x 250 mm (id), 5 µm, or equivalent;

B) Mobile phase. acetonitrile - water (75 25, V/V);

C) Flow rate. 1.0 mL/min;

D) Detection wavelength. 235 nm;

E) Injection volume. 40 µL;

7.3.2 Determination by liquid chromatography

The standard working curve was drawn with the standard working solution of quinoxaline standard, and the standard working curve was drawn with the peak area as the ordinate and the working solution concentration as the abscissa.

The working curve is used to quantify the sample. The response values of quinoxaline in standard working fluid and sample solution should be within the linear response range of the instrument. Press 8 Under the provisions of the results of the calculation. Under the present method, the retention time of quinoxaline was about 8.0 min. Refer to Appendix A for standard chromatograms

Figure A.1.

7.4 Determination by liquid chromatography - mass spectrometry/mass spectrometry

7.4.1 Liquid Chromatography - Mass Spectrometry/Mass Spectrometry Reference Conditions

A) Column. Acquity UPLC BEH C18 column, 2.1 x 55 mm (id), 1.7 µm, or equivalent;

B) Column temperature. 30 ° C;

C) mobile phase. acetonitrile - water (70 30, V/V);

D) Flow rate. 300 µL/min;

E) Injection volume. 10 µL.

7.4.2 Mass spectrometry reference conditions

A) ion source. electrospray source (ESI), positive ion mode;

B) scanning mode. multiple reaction monitoring (MRM);

Other reference mass spectrometry conditions are given in Table B.1 in Appendix B.

7.4.3 Determination by liquid chromatography-mass spectrometry/mass spectrometry

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 value of quinoxaline in the solution should be within the linear response range of the instrument. According to the provisions of 8 to calculate the results. Under the conditions of this method, Oxygen retention time of about 1.5 min, the cone voltage of 30 V, the standard secondary mass spectrometry and multi-reaction monitoring (MRM) chromatography See Figure C.1 and Figure C.2 in Appendix C, respectively.

7.4 Qualitative determination

When the sample is measured, if the detected chromatographic peak retention time is consistent with the standard sample, and in the sample spectrum after subtracting the background, the relative abundance of the qualitative ions is close to the standard solution of the standard solution obtained under the same conditions compared to the maximum allowable relative deviation does not exceed the table 1 in the range, you can determine the existence of the corresponding sample 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%

7.5 Blank experiment

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

10.1 Quantitation limits

The limit of quantification for liquid chromatography was 0.01 mg/kg.

The quantitation limit of the liquid chromatography-mass spectrometry/mass spectrometry is 0.001 mg/kg.

10.2 Recovery rate

When the levels were 0.01 mg/kg, 0.02 mg/kg, 0.04 mg/kg, the recovery of quinoxaline was measured by liquid chromatography.

Appendix D (Table D.1).

When the levels were 0.001 mg/kg, 0.005 mg/kg, 0.01 mg/kg, the concentration of quinoxaline was determined by liquid chromatography-mass spectrometry the recovery is shown in Appendix D (Table D.2).

Appendix A

(Informative)

The chromatogram of quinoxaline

Figure A.1 Liquid chromatogram of quinoxaline

Appendix B (1)

(Informative)

Reference to mass spectrometry conditions Reference mass spectrometry conditions.

Capillary voltage. 0.5 kV;

Source temperature. 120 ° C;

Solvent temperature. 450 ° C;

Taper hole air (nitrogen). 45 L/Hr;

Solvent gas stream (nitrogen). 900 L/Hr;

Collision air pressure (argon). 2.20 × 10-6 Pa;

Duration. 0.20 s

Other mass spectral parameters are shown in Table C.1

Table B.1 Basic reference mass spectra of quinoxaline

(M/z) ion (m/z) dwell time (s) taper hole voltage (V) collision energy (eV) Quinoxaline 308.2 272.1 197.1 * 0.2 0.2 Note. the table with * ions for the quantitative ions; for different mass spectrometry equipment, instrument parameters may be different, should be measured before the mass spectrometry the number of optimization to the best.

(1) Non-commercial declaration. The reference to the mass spectrometric conditions listed in Appendix C is performed on a Waters UPLC/Priemer LC/MS.

The list of test instruments listed below is for reference only and does not involve commercial purposes and encourages standard users to try different manufacturers or models of instruments.

Appendix C

(Informative)

The secondary spectrum of quinoxaline and the multi - reaction monitoring (MRM) ion chromatogram

Figure C.1 The quaternary spectrum of quinoxaline

Figure C.2 Quinoxyline Multiple Reaction Monitoring (MRM) Ion Chromatography

Appendix D

(Informative)

Sample concentration and recovery of the experimental data

Table D.1 Data on the recovery of quinoxaline residues by liquid chromatography

Sample Name Add Concentration (mg/kg) Recovery (%) Soybeans 0.010 75.2 ~ 104.5 0.020 76.2 to 92.9 0.040 81.8 ~ 93.0 cauliflower 0.010 76.6 ~ 106.5 0.020 90.1 to 102.4 0.040 94.9 to 100.3 Cherry 0.010 81.4 ~ 102.8 0.020 83.2 ~ 98.0 0.040 86.6 ~ 98.4 Fungus 0.010 89.6 ~ 108.2 0.020 84.2 to 104.4 0.040 88.9 to 104.8 wine 0.010 77.2 to 102.8 0.020 79.2 ~ 98.2 0.040 82.8 to 97.2 tea 0.010 74.6 ~ 107.6 0.020 76.2 ~ 100.3 0.040 78.9 to 94.8 honey 0.010 79.4 ~ 102.8 0.020 91.9 to 99.7 0.040 84.8 ~ 95.6 Liver 0.010 74.6 ~ 106.5 0.020 76.3 to 101.5 0.040 80.7 to 96.9 eel 0.010 74.6 to 100.3 0.020 76.2 to 97.7 0.040 80.9 to 98.3 chicken 0.010 74.6 ~ 104.5 0.020 78.3 ~ 93.8 0.040 81.6 to 94.1

Table D.2 Liquid Chromatography Quirgine Ling Drug Residue Recovery Recovery Data

Sample Name Add Concentration (mg/kg) Recovery (%) Soybeans 0.001 83.5 to 102.0 0.005 84.6 ~ 101.8 0.010 79.3 ~ 97.8 cauliflower 0.001 81.0 to 103.0 0.005 79.4 ~ 96.8 0.010 78.7 to 97.7 Cherry 0.001 81.0 to 99.0 0.005 79.6 ~ 98.4 0.010 78.4 to 100.3 Fungus 0.001 81.0 to 96.0 0.005 79.0 to 99.8 0.010 81.4 ~ 104.5 wine 0.001 81.0 to 97.0 0.005 80.6 ~ 100.8 0.010 81.3 to 96.3 tea 0.001 78.0 to 106.0 0.005 78.8 ~ 103.8 0.010 77.4 to 99.7 honey 0.001 84.0 to 101.0 0.005 80.2 ~ 97.6 0.010 77.1 to 99.7 Liver 0.001 75.0 to 98.0 0.005 78.4 to 103.2 0.010 76.8 to 100.1 eel 0.001 79.0 to 96.0 0.005 79.6 ~ 101.2 0.010 76.2 to 103.0 chicken 0.001 84.0 to 97.0 0.005 77.8 to 96.6 0.010 81.2 to 96.9 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