GB 23200.89-2016Food safety national standard -- Determination of ethoxyquinoline residues in animal-derived foods by liquid chromatography (English PDF)

4.1 Reagents

4.1.1 Acetone (C3H6O, 67-64-1). Chromatographic pure.

4.1.2 n-hexane (C6H14,110-54-3). chromatographic purity.

4.1.3 acetonitrile (C2H3N, 75-05-8). pure chromatography.

4.1.4 Sodium carbonate (Na2CO3, 497-19-8).

4.1.5 Ammonium Acetate (C2H7NO2,631-61-8). Chromatographic Purification.

4.2 Solution preparation

4.2.1 sodium carbonate solution (10%). accurately weighed 100 g of sodium carbonate in the beaker by adding the appropriate amount of water dissolved, transferred to 1L volumetric flask, constant volume to the scale, shake.

4.2.2 ammonium acetate solution (20mmol/L). accurately weighed 1.54 g ammonium acetate, dissolved in 1L water.

4.3 Standards

4.3.1 ethoxyquinoline standard substance (C14H19NO, 91-53-2). purity >= 98.0%.

4.4 Standard solution preparation

4.4.1 ethoxyquinoline standard stock solution. accurately weighed the amount of standard, diluted with acetonitrile prepared 100 µg/mL standard stock solution, in 0 °C ~ 4 °C dark preservation.

4.3.2 ethoxyquinoline standard working solution. according to the need to remove a certain volume of standard intermediate solution with acetonitrile gradually diluted to the appropriate concentration of the standard working solution. Standard working solution is ready for use.

6.1 Preparation of the sample

6.1.1 Meat and internal organs

Take a representative sample 500 g, cut it, and then use the tissue crusher to process the sample into a slurry, mix it, and mix it into a clean sample Inside, sealed and marked.

6.1.2 Honey and milk

Take a representative sample of 500 g, pour it into a clean enamel mix bucket, stir well and mix it into a clean container.

And mark the mark.

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

6.2 Sample storage

Milk, honey samples stored at 0 °C ~ 4 °C; meat and visceral products such as samples at -18 °C frozen storage.

During the operation of the sample preparation, the sample should be protected from contamination or changes in the content of the residue.

7.1 Extraction

Weigh 2 g homogeneous sample (accurate to 0.01 g) in a 50 mL stoppered centrifuge tube and add 5 mL of sodium carbonate solution to the vortex Mix for 5 minutes, add 5 mL of acetone to the vortex mixer for 2 min, add 15 mL of n-hexane, in the vortex mixer Extraction of 2 min, 6000 r/min centrifugation 3min, take the upper layer of n-hexane in.200 mL chicken heart bottle, with 15 mL n-hexane and then repeat Take the n-hexane layer in the heart of the heart, and extract the extract to a concentration of about 1 mL in a 30 ° C water bath and blow it with a gentle nitrogen stream Close to dry, dissolved in acetonitrile and set to 1.0 mL, filter membrane, for high performance liquid chromatography and high performance liquid chromatography - tandem mass spectrometry Confirmation.

7.2 Determination

7.2.1 Reference conditions for high performance liquid chromatography

A) Column. C18, 150 mm x 4.6 mm (inner diameter), 5 µm, or equivalent;

B) Column temperature. 25 ° C;

C) mobile phase. ammonium acetate aqueous solution (20 mmol/L) acetonitrile = (7 3, V/V);

D) Fluorescence detector. excitation wavelength 360nm, emission wavelength 435nm;

E) Flow rate. 0.50 mL/min;

F) Injection volume. 10 µL.

7.2.2 High Performance Liquid Chromatography - Tandem Mass Spectrometer Determination of Reference Conditions

7.2.2.1 High Performance Liquid Chromatography Reference Conditions

A) Column. C18, 150 mm × 4.6 mm (inner diameter), 5 µm or equivalent.

B) Column temperature. 25 ° C;

C) Flow rate. 0.50 mL/min;

D) gradient elution procedure. see Table 1;

E) Injection volume. 5 µL.

Table 1 High Performance Liquid Chromatography - Tandem Mass Spectrometer Gradient Elution Procedure

Time/min flow rate/(mLmin) 5 mmol/L ammonium acetate aqueous solution /% acetonitrile /% 0 0.5 60 40 3 0.5 40 60 6 0.5 10 90 9 0.5 10 90 11 0.5 60 40 14 0.5 40 60

7.2.2.2 Mass spectrometry reference conditions

See Appendix A for reference conditions.

7.2.3 Determination by high performance liquid chromatography

According to the content of ethoxyquinoline in the sample solution, the standard working solution of ethoxyquinoline with similar concentration was selected. Standard working solution and sample solution the response value of ethoxyquinoline should be within the linear range of the instrument. On the standard working fluid and sample volume of alternating injection to determine the volume Time to qualitative, measured sample solution with the standard working fluid peak area compared to quantitative. In the above high performance liquid chromatography conditions, ethoxyquinoline standards See Figure B.1 for chromatograms.

7.2.3.1 High performance liquid chromatography - tandem mass spectrometry

If the retention time of the detected peaks is consistent with the standard sample, the mass of the sample is determined by the mass spectrometry.

certificate. When the sample was confirmed by high performance liquid chromatography-tandem mass spectrometry, the relative abundance of each qualitative ion and the concentration of close to the same conditions obtained under the standard solution spectrum comparison. Under the same experimental conditions, the mass of the test substance in the sample solution the peak retention time is consistent with that in the standard solution, and the selected qualitative ions are present, and the relative abundance of the selected qualitative ions is the same as that of the standard sample of the relative abundance of uniform (allowable deviation range in Table 2), you can determine the existence of the corresponding sample in the sample. If you can not confirm, you should re-enter , With a scanning method (with sufficient sensitivity) or by adding other confirmed ions or with other higher sensitivity analytical instruments certificate. In the above-mentioned high performance liquid chromatography-tandem mass spectrometry conditions, the selective ion monitoring chromatogram of the ethoxyquinoline standard is shown in Figure C.1.

Table 2 The 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%

7.3 Blank test

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

10.1 Quantitation limits

The method of quantification of ethoxyquinoline is 0.01 mg/kg.

10.2 Recovery rate

When the levels were 0.01 mg/kg, 0.05 mg/kg, 0.1 mg/kg, the addition of ethoxyquinoline in different substrates was See Appendix D.

Appendix A

(Informative)

Mass spectrometry conditions A.1 Mass spectrometry conditions are given in Table A.1

Table A.1 Mass spectrometry conditions

Ion source electrospray ionization source (ESI), positive ion monitoring Scan mode selection Ion monitoring (SRM) Air Curtain (CUR) 68.95 kPa (10 psi) Atomization gas (GS1) 275.8 kPa (40 psi) Auxiliary heating gas (GS2) 344.75 kPa (50 psi) Collision gas (CAD) 48.27 kPa (7 psi) Electromagnetic spray voltage (IS) 5 000 V Ion source temperature (TEM) 450 ° C A.2 Select the ion monitoring conditions shown in Table A.2

Table A.2 Select the ion monitoring conditions

To be measured

To cluster voltage

DP/V

Collision voltage

CE/V

Collision chamber voltage EP/V the chamber voltage of the collision chamber CXP/V Ethoxyquinoline 218.1 148.1a 74 31 10 12 190.4 74 30 10 6 202.1 74 28 10 9 Note. "a" is a quantitative ion pair

1) Non-commercial notices. The parameters listed in Appendix A are done on the API4000 mass spectrometer. The test instrument model listed here is for reference only and does not Involving commercial purposes, to encourage standard users to try to use different manufacturers or models of equipment.

Appendix B

(Informative)

Ethoxyquinoline standard liquid chromatography B.1 Ethoxyquinoline standard liquid chromatogram shown in Figure B.1 Min0 2.5 5 7.5 10 12.5 LU Gt;

Figure B.1 Ethoxyquinoline standard (0.2 mg/kg) Liquid chromatogram

Appendix C

(Informative)

Ethoxyquinoline standard selection ion monitoring (SRM) chromatogram C.1 Ethoxyquinoline Standard Selective Ion Monitoring (SRM) Chromatogram See Figure C.1

Figure C.1 Ethoxyquinoline standard (0.02 mg/kg) Select ion monitoring (SRM) chromatogram

1 2 3 4 5 6 7 8 9 10 11 12 13 0.0 2.0e4 4.0e4 4.8e4 10.28 1 2 3 4 5 6 7 8 9 10 11 12 13 0.0 2.0e4 4.0e4 4.8e4 10.28 1 2 3 4 5 6 7 8 9 10 11 12 13 T/min 0.0 1.0e4 2.0e4 3.0e4 3.7e4 10.28 1 2 3 4 5 6 7 8 9 10 11 12 13 0.0 1.0e4 2.0e4 2.8e4 10.28 TIC 218.1 > 148.1 218.1 > 190.41 218.1 > 202.1 T/min T/min Appendix D (Informative) the recovery of ethoxyquinoline in different sample substrates D.1 Recovery of ethoxyquinoline in different sample substrates is shown in Table D.1

Table D.1 Recovery of ethoxyquinoline in different sample substrates

Sample Substrate Add Concentration/(mg/kg) Recovery /% Sample Substrate Add Concentration/(mg/kg) Recovery /% pork 0.01 89.5 108.0 honey 0.01 89.2 100.3 0.05 84.2 102.2 0.05 88.8 101.2 0.10 86.898.2 0.10 84.190.5 Liver 0.01 92.7 109.0 egg 0.01 90.3 101.4 0.05 93.2 109.2 0.05 93.7 107.5 0.1 90.6 98.4 0.10 96.6 101.6 Pig kidney 0.01 85.8 102.0 Fish 0.01 94.7 102.1 0.05 91.3 105.6 0.05 87.7 108.0 0.10 92.0105.6 0.10 92.097.7 chicken 0.01 84.8 101.1 milk 0.01 91.41010.1 0.05 85.8 102.6 0.05 83.9 95.2 0.10 86.199.5 0.10 87.489.8 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