GB 23200.74-2016Food safety national standard -- Determination of Jinggangmycin Residue in Food by Liquid Chromatography -- Mass Spectrometry / Mass Spectrometry (English PDF)

4.1 Reagents

4.1.1 Ammonium acetate (CH3COONH4, CAS number. 631-61-8).

4.1.2 Methanol (CH3OH, CAS No. 67-56-1).

4.1.3 Acetonitrile (CH3CN, CAS No. 75-05-8).

4.1.4 Ethyl acetate (CH3COOCH2CH3, CAS number. 141-78-6).

4.1.5 glacial acetic acid (CH3COOH, CAS number. 64-19-7).

4.2 Solution preparation

4.2.1 methanol solution (9 1). the amount of 90 mL of methanol by adding 10 mL of water, mix.

4.2.2 5 mmol/L ammonium acetate buffer solution. Weigh 0.385 g of ammonium acetate, dissolved in water and fixed to 1 000 mL, adjusted with glacial acetic acid PH to 4.5 ± 0.1.

4.3 Standards

4,3.1 Jinggangmycin standard (Validamycin A, molecular formula. C20H35NO13, CAS. 37248-47-8). purity >= 91.0%.

4.4 Standard solution preparation

4.4.1 Jinggangmycin standard stock solution. Weigh 2 mg (accurate to 0.1 mg) the standard of Jinggangmycin, dissolved in water and set to 25 mL of a brown volumetric flask to prepare a standard stock solution at a concentration of 80 mg/L.

4.4.2 Jinggangmycin matrix standard working solution. according to the need, with blank samples in accordance with the sample treatment steps to get the extract, preparation is not with the concentration of the matrix standard solution, is now available.

4.5 Materials

4.5.1 HLB column. 6 mL /.200 mg or equivalent; activated with 6 mL of methanol and 6 mL of water before use.

4.5.2 Filtration. 0.22 m, biphasic.

6.1 Sample requirements

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

6.2 Tea and cereals

Approximately 500 g of the representative sample, ground and ground through the mill and passed through a 2.0 mm round hole screen, mixed and sealed in a clean container.

Remember, stored at 0 °C ~ 4 °C refrigerated.

6.3 Vegetables and fruits

To replace the sample of about 500 g, chopped, the multi-functional food mixer fully mashed evenly, into a clean container sealed, marked mark, Frozen at -18 °C below the storage.

6.4 Nuts

Approximately 500 g of the sample was taken and ground thoroughly with an attritor and passed through a 20 mm round hole screen, mixed, sealed in a clean container, marked with a mark, Store at 0 °C ~ 4 °C.

6.5 Animal muscle, liver, fish, shrimp

Replace the sample part of about 500 g, chopped, by the multi-functional food mixer fully mashed evenly, into a clean container sealed, marked Note, in -18 °C below the frozen storage.

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

7.1 Extraction

7.1.1 Rice, edible fungus, almonds

Weigh 2.5 g sample (accurate to 0.01 g), placed in 50 mL polytetrafluoroethylene centrifuge tube, add 20 mL of methanol solution, with homogenizer high Rapid homogenate extraction 2 min, 3 000 r/min centrifugation 5 min, collecting supernatant in a scale tube. The residue after centrifugation was dissolved in 5 mL of methanol the supernatant was combined once the above extraction step was repeated and the nitrogen was concentrated to 2.5 mL below the nitrogen bath at 45 ° C to be purified.

7.1.2 cabbage, onions, carrots, tomatoes, cucumber, spinach, pear, lemon, pork, liver, fish, shrimp Weigh 2.5 g sample (accurate to 0.01 g), placed in 50 mL polytetrafluoroethylene centrifuge tube, add 20 mL of methanol, homogenizer high speed uniform Pulp extraction 2 min, 3 000 r/min centrifugation 5 min, collecting supernatant in a scale tube. The residue after centrifugation was diluted with 5 mL of methanol solution Repeat the above extraction step 1, the supernatant, in 45 °C water bath blowing nitrogen concentrated to 2.5 mL below, to be purified.

7.1.3 Tea

Weigh 1 g of sample (accurate to 0.01 g), add 1.5 mL of water, mix, and the rest according to 7.1.1.

7.2 Purification

7.2.1 Solid phase extraction (SPE) purification

The resulting solution from 7.1 was transferred to a pretreated HLB solid phase extraction column and the sample solution was passed through solid phase extraction at a flow rate of about 1 drop/s Column, rinse the column with 2 mL of water, collect all the effluent and eluent to a scale tube, add water to a volume of 5.00 mL, mix, over 0.22 m Filter for determination.

7.2.2 Liquid - distribution purification

The resulting solution in 7.1 was fixed to 5.00 mL with water, mixed into a 50 mL stoppered glass centrifuge tube, 5 mL of ethyl acetate was added, Vortex oscillation 3 min, 3 000 r/min centrifugal 5 min, discard the upper ethyl acetate phase, and then 5 mL ethyl acetate repeated operation once. Too 0.22 m filter for liquid chromatography-tandem mass spectrometry.

7.3 Determination

7.3.1 Liquid Chromatographic Reference Conditions

Column. HILIC (250 mm × 4.6 mm, particle size 5 m), or equivalent.

Mobile phase. acetonitrile 5 mmol/L ammonium acetate buffer solution, gradient elution (see Table 1).

Flow rate. 1 000 L/min.

A) Column temperature. 35 ° C.

B) Injection volume. 10 µL.

Table 1 Gradient elution conditions for liquid chromatography

Time/min 5 mmol/L ammonium acetate buffer solution /% acetonitrile /% 0.0 20 80 4.0 45 55 10.0 45 55 10.1 20 80 16.0 20 80

7.3.2 Mass spectrometry reference conditions

See Appendix A for the mass spectrometric conditions.

7.3.3 Determination and confirmation of chromatography

Under the optimum working condition of the instrument, according to the content of the compound in the sample solution, the substrate standard working solution with similar peak height is selected, Quality standard working solution and sample solution volume measurement. With the peak area as the ordinate, the concentration of abscissa to draw the standard working curve, with the standard working curve is used to quantify the sample, and the response values of the test compound to be tested in the working solution and the sample solution should be measured linearly Within range. In the above instruments, the reference retention time of Jinggangmycin is about 8.10 min; the calibration curve for the calibration product of Jinggangmycin is given in Appendix B Figure B.1.

Table 2 in the same experimental conditions, the sample solution to be tested material retention time, and the matrix standard working solution retention time deviation of ± 2.5%

And the relative abundance of the qualitative ion pairs in the sample solution is compared with the matrix standard working solution close to the concentration. The deviation does not exceed the standard it is determined that there is a corresponding analyte in the sample.

Table 2 Maximum allowable deviation of relative ion abundance using liquid chromatography-mass spectrometry/mass spectrometry

Relative ion abundance 50% 20% to 50% 10% to 20% <=10% Permissible relative deviation +/-20% +/-25% +/- 30% +/- 50%

7.4 Blank test

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

10.1 Quantitation limits

The limit of quantification of this method was 10 µg/kg.

10.2 Recovery rate

When adding levels of 10 µg/kg, 25 µg/kg, 50 µg/kg, the recovery rates of Jinggangmycin in different substrates are given in Appendix C.

Appendix A

(Informative)

Reference to mass spectrometry conditions Reference mass spectrometry conditions.

A) Ionization mode. Atmospheric pressure Chemical ionization positive ion mode (APCI).

B) Mass Spectrometry. Multiple Reaction Monitoring (MRM).

C) Resolution. Unit resolution.

D) air curtain gas pressure (CUR). 207 KPa (nitrogen).

E) Corona discharge current (NC). 5.00 µA.

F) Atomization temperature (TEM). 600 ° C.

G) Atomizing gas pressure. 138 KPa (nitrogen).

H) Impact air pressure (CAD). 34.5 KPa (nitrogen).

I) Other mass spectral parameters are shown in Table A.1.

Table A.1 Main reference mass spectrometry parameters

Compounds

Ion pair

Dwell time

To cluster voltage

(DP)/V

Inlet voltage

(EP)/V

Collision energy

(CE)/eV

Collision cell outlet voltage (CXP)/V Jinggangmycin 498.3/336.3 100 75 3 31.74 12.08 498.3/178.1a 100 75 3 38.57 12.08 498.3/142.2 100 75 3 48.77 12.08 Note. For different mass spectrometry instruments, the instrument parameters may be different, before the determination of the mass spectrometry parameters should be optimized to the best.

A ion pair is a quantitative ion pair Appendix B (Informative) Standard multi-reaction monitoring (MRM) chromatograms

Figure B.1 Multi-reaction monitoring (MRM) chromatogram of Jinggangmycin standard solution

Ion pair 498.3/336.3 Ion pair 498.3/178.1 Ion pair 498.3/142.2 Appendix C (Informative) Addition Recovery of Jinggangmycin in Different Substrates

Table C.1 Recovery of Jinggangmycin in Different Substrates

Sample Concentration/µg/kg Recovery Range /% Sample Concentration/µg/kg Recovery Range /% Rice 10 87.2 ~ 101.8 10 86.6 ~ 111.0 25 92.8 ~ 101.6 25 87.2 ~ 108.8 50 89.2 ~ 100.8 50 87.2 ~ 113.6 cabbage 10 96.8 to 109.4 lemon 10 86.2 to 111.6 25 94.4 ~ 108.0 25 89.6 ~ 102.4 50 96.0 ~ 108.0 50 87.6 ~ 106.8 10 94.6 ~ 108.4 almond 10 90.6 ~ 105.4 25 93.6 ~ 109.6 25 87.2 ~ 111.2 50 93.6 ~ 108.0 50 99.6 ~ 113.2 carrot 10 91.6 ~ 109.2 tea 10 86.0 to 107.8 25 91.2 ~ 105.6 25 87.2 ~ 106.4 50 91.2 ~ 108.4 50 86.8 ~ 104.0 tomato 10 91.6 ~ 107.2 pork 10 89.4 ~ 105.6 25 91.2 ~ 111.2 25 87.2 ~ 108.0 50 94.8 ~ 111.6 50 88.0 ~ 101.6 cucumber 10 98.6 ~ 111.4 Liver 10 90.0 to 106.4 25 89.6 ~ 108.8 25 92.0 ~ 106.4 50 95.2 ~ 111.6 50 87.2 ~ 106.4 spinach 10 87.0 ~ 104.2 Tilapia 10 88.8 ~ 107.2 25 92.8 ~ 108.8 25 96.8 ~ 112.8 50 89.6 to 108.0 50 88.4 to 113.2 Fungus 10 92.8 to 107.2 10 93.4 ~ 106.2 25 92.8 ~ 109.6 25 93.6 ~ 113.6 50 89.2 ~ 108.8 50 88.0 ~ 112.0 IX 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