GB 23200.79-2016Food safety national standard -- Meat and meat products -- Determination of mycophenolate residues -- Gas chromatographic method (English PDF)

4.1 Reagents

4.1.1 Cyclohexane (C6H12). Chromatographic Purification.

4.1.2 Ethyl acetate (C4H8O2). Chromatographic purity.

4.1.3 n-hexane (C6H14). chromatographic purity.

4.1.4 Toluene (C7H8). Chromatographic pure.

4.1.5 anhydrous sodium sulfate (Na2SO4). before burning at 650 °C for 4 h, stored in a dryer, cooling and standby.

4.2 Solution preparation

4.2.1 Cyclohexane-ethyl acetate mixed solvent (1 +1, volume ratio). take 100mL cyclohexane, add 100mL ethyl acetate, shake Evenly used.

4.3 Standards

4.3.1 Pythium Phosphate standard substance (pyrazophos; formula. C14H20N3O5PS; CAS No. 13457-18-6). purity >=97%.

4.4 Standard solution preparation

4.4.1 Pythium phosphate standard stock solution. accurately weighed appropriate amount of Pseudomonas citrate standard substance (accurate to 0.1 mg), with toluene concentration of about A standard stock solution of 1.0 mg/mL.

4.4.2 Pythium standard working solution. According to the testing requirements, respectively, the above standard stock solution diluted with n-hexane diluted to the appropriate concentration of the standard Quasi-working solution.

4.5 Materials

4.5.1 Filtration. 0.45 µm, organic.

4.5.2 anhydrous sodium sulfate column. in the cylindrical funnel (diameter 1.5 cm), containing about 5 cm high anhydrous sodium sulfate.

6.1 Preparation of the sample

Sampling parts according to GB 2763 Appendix A implementation, the sample with meat grinder minced, fully mixed, with a quarter to shrink to less than 500 g, installed Into the clean container, sealed, marked mark.

6.2 Storage of samples

The sample was stored at -18 ° C or lower.

7.1 Extraction

Weigh 10 g of sample (accurate to 0.01 g) in a 100 mL centrifuge tube containing 20 g of anhydrous sodium sulfate, add 30 mL of cyclohexane-acetic acid Ethyl ester mixed solvent, homogenized at 15 000 r/min homogeneous extraction 1.5 min, centrifuged at 3000 r/min for 3 min. Supernatant is filled with an anhydrous sodium sulfate canister was collected in 100 mL pear bottles. The residue was extracted with 30 mL of cyclohexane-ethyl acetate mixed solvent the combined extracts were washed twice and the anhydrous sodium sulphate column was washed with a small amount of cyclohexane-ethyl acetate mixed solvent. The extract was washed with water at 40 ° C Turn to about 2 mL, to be purified.

7.2 Purification

7.2.1 Gel Permeation Chromatography Instrument Conditions

A) Purification column. 400 mm × 25 mm, built with BIO-Beads S-X3 packing or equivalent.

B) Mobile phase. cyclohexane-ethyl acetate (1 + 1, volume ratio).

C) Flow rate. 5 mL/min.

D) Injection volume. 5 mL.

E) Start collection time. 1.200 s.

F) End of collection time. 1 800 s.

7.2.2 Purification steps

The concentrated extract was transferred to a 10 mL graduated centrifuge tube and washed several times with a cyclohexane-ethyl acetate mixed solvent.

And wash the liquid and constant volume to the scale. After centrifugation at 4 000 r/min for 5 min, the supernatant was filtered through a 0.45 µm organic phase filter into a gel permeation chromatograph in the sample bottle. Purified with a gel permeation chromatograph and collected effluent from 1.200 s to 1 800 s. The effluent was dried with a nitrogen dry drier and added 1.0 mL n-hexane dissolved, mixed, gas chromatographic determination.

7.3 Determination

7.3.1 Chromatographic determination of reference conditions

A) Column. HP-5, 30 m x 0.32 mm (id), film thickness 0.25 µm, or equivalent.

B) column temperature. initial temperature 220 °C, keep 10 min; at 10 °C/min rate rose to 250 °C, keep 7 min.

C) Carrier gas (N2). 1.5 mL/min.

D) Hydrogen (H2). 75 mL/min.

E) Air. 100 mL/min.

F) Detector temperature. 280 ° C.

G) Inlet temperature. 250 ° C.

H) Injection volume. 1 µL.

I) Injection method. no split injection, 1.5 min after opening the diverter valve.

7.3.2 Chromatographic determination

According to the content of mycorrhizal phosphorus in the sample, select the appropriate concentration of the standard working solution and the sample to be measured and other volume injection. Standard working fluid and pending the response value of the mycophenolate in the sample solution should be within the linear range of the instrument response. Under the above chromatographic conditions, the retention time of mycorrhizal phosphorus is about 14.9 Min, according to the peak area external standard method. See Appendix A for standard chromatograms.

7.4 Blank test

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

10.1 Quantitation limits

The quantification limit of this method is 0.01 mg/kg.

10.2 Recovery rate

When the levels were 0.01 mg/kg, 0.02 mg/kg, 0.05 mg/kg, the recovery of the mycophenolate was given in Appendix B.

Appendix A

(Informative)

Pseudomonas aeruginosa standard gas chromatogram

Figure A.1 Pyridine phosphorus standard gas chromatogram

Appendix B

(Informative)

Table B.1 Recovery and precision of the three matrix samples

Substrate Name Added Level/(mg/kg) Recovery Range/(%) Precision/RSD (%) pork 0.01 82.5 to 104.2 8.3 0.02 82.1 to 96.7 5.6 0.05 83.5 to 93.1 3.9 chicken 0.01 80.8 to 103.3 8.0 0.02 80.0 to 92.5 5.2 0.05 80.8 to 96.5 5.9 beef 0.01 81.7 to 107.5 8.0 0.02 83.8 to 97.5 5.8 0.05 82.7 to 97.7 5.9 Appendix C (Normative appendix) Laboratory repeatability requirements

Table C.1 Laboratory repeatability requirements

Measured component content Mg/kg RSD <=0.001 36 > 0.01 > 1 14 Appendix D (Normative appendix) Inter-laboratory repeatability requirements

Table D.1 Inter-laboratory repeatability requirements

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