GB 23200.47-2016Food safety national standard -- Determination of tetramine residues in food by gas chromatography-mass spectrometry (English PDF)

4.1 Reagents

4.1.1 Acetone (CH3COCH3, CAS. 67-64-1). Chromatographic Purification.

4.1.2 Dichloromethane (CH2Cl2, CAS. 75-09-2). Chromatographic pure.

4.1.3 Cyclohexane (C6H12, CAS. 110-82-7). Chromatographic purity.

4.1.4 Ethyl acetate (C4H8O2, CAS. 141-78-6). Chromatographic pure.

4.1.5 n-Hexane (C6H14, CAS. 110-54-3). Chromatographic pure.

4.1.6 anhydrous sodium sulfate (Na2SO4, CAS. 15124-09-1). 650 °C burning 4 h, stored in a sealed container in reserve.

4.2 Solution preparation

4.2.1 sodium sulfate aqueous solution (20 g/L). take 20g anhydrous sodium sulfate, water volume to 1000mL, shake back standby.

4.3 Standards

4.3.1 Tetracycline reference substance. see Appendix A.

4.4 Standard solution preparation

4.4.1 Tetracycine standard stock solution. accurately weighed the appropriate amount of tetramine standard material, with acetone prepared into a concentration of 100 g/mL Standard stock solution, the standard reserve solution away from 0 °C ~ 4 °C preservation, shelf life of 6 months.

4.4.2 Tetracycine standard working solution. According to the testing requirements, respectively, the above standard reserve solution in the volumetric flask, diluted with acetone to the standard preparation of the appropriate concentration of the standard working solution, the standard working solution away from 0 °C ~ 4 °C preservation, shelf life of one month.

4.5 Materials

4.5.1 Florisil Solid phase extraction column. 1.0 g, or equivalent.

6.1 Preparation of the sample

6.1.1 Fruits or vegetables

Take 500 g of fruit or vegetable sample (not water wash), and then cut the sample into a slurry with a food masher.

Mix, are divided into two as a sample, into the clean Sheng Sheng bag, sealed and marked.

6.1.2 Meat and meat products

Remove a representative sample of about 1 kg, crushed by the mashed evenly, are divided into two, respectively, into the clean container as a test kind. Sealed and marked.

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

6.2 Sample storage

The sample was stored at -18 ° C or lower. During sampling and sample preparation, the sample should be protected from contamination or residue the amount of change.

7.1 Extraction

Weigh the sample about 20 g (accurate to 0.1 g) in a 250 mL stoppered flask, add 100 mL of water - acetone (14), shake Extraction for 30 min, the extract was filtered in 250 mL concentrated bottle. Residue and then 50 mL of acetone repeated extraction time, combined filtrate, and concentrated in a water bath at 40 ° C to about 20 mL.

The concentrated extract was transferred to a 250 mL separatory funnel, 150 mL of sodium sulfate aqueous solution and 50 mL of dichloromethane were added, shaking 3 Min, and the layers were separated and the dichloromethane phase was collected. The aqueous phase was extracted twice with 2 x 50 mL of methylene chloride and the dichloromethane phases were combined.

Dehydrated by anhydrous sodium sulfate column, collected in 250 mL concentrated flask, concentrated in a 40 ° C water bath to near dry, add 5 mL of cyclohexane -Ethyl acetate (1 1) The residue was dissolved and filtered with a 0.45 m filter to be purified.

7.2 Purification

7.2.1 Gel Chromatography (GPC)

7.2.1.1 Gel chromatographic conditions

A) Gel purification column. 700 mm × 25 mm, Bio Beads S-X3, or equivalent.

B) Mobile phase. cyclohexane-ethyl acetate (1 1).

C) Flow rate. 5.0 mL/min.

D) Sample dosing ring. 5.0 mL.

E) Pre-elution volume. 50 mL.

F) Elution volume. 150 mL.

G) Collection volume. 95 mL - 135 mL.

7.2.1.2 Gel Chromatographic Purification Procedure

The purified solution of 5 mL was purified under the conditions specified in 6.2.1.1 and the collected fractions in the fraction collector were mixed in 50 mL of concentrated , Concentrated in a 40 ° C water bath to close to dry, add 2 mL of n-hexane - ethyl acetate (4 1) to dissolve the residue, to be purified.

7.2.2 Solid Phase Extraction Purification (SPE)

The Florisil solid phase extraction column was pre-eluted with 5 mL of n-hexane and the sample was poured into the column and then treated with 8 mL N-hexane-ethyl acetate (4 1). The whole eluate was collected in a 50 mL concentration flask and concentrated in a 40 ° C water bath dry. Dissolved in acetone and set to 2 mL, gas chromatography - mass spectrometry and confirmed.

7.3 Determination

7.3.1 Gas Chromatography - Mass Spectrometry Reference Conditions

A) Column. DB-5 MS quartz capillary column, 30 m × 0.25 mm (id), film thickness 0.25 m, or equivalent.

B) Column temperature. 50 ° C (for 2 min) 30 ° C/min 180 ° C (1 min) 10 ° C/min 270 ° C 10 min);

C) Inlet temperature. 280 ° C.

D) chromatographic - mass spectrometer interface temperature. 270 ° C.

E) Carrier gas. helium, purity >=99.999%, flow rate 1.2 mL/min.

F) Injection volume. 1 µL.

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

H) ionization mode. EI.

I) Ionization energy. 70 eV.

J) Measurement method. Select the ion monitoring mode.

K) Monitoring ions (m/z). See Appendix A.

L) solvent delay. 5 min.

7.3.2 Determination and confirmation of chromatography

According to the content of tetracycine in the sample solution, the standard working solution with similar peak area is selected, and the standard working solution and the sample solution the response value of tetramidazine in standard working solution and sample solution should be within the linear range of instrument detection.

Under the same experimental conditions, the mass retention time of the material to be tested in the sample solution was the same as that of the standard working solution and the background the selected ions in the sample mass chromatogram appear after comparison with the abundance of the selected ions and the abundance of the corresponding ions Within the allowable range (see Table 1 for the permissible range), it is determined that there is a corresponding analyte in the sample. Under the above chromatographic conditions, tetracycline (M/z) abundance ratio is shown in Appendix A. Determination of tetramine standard by gas chromatography - mass spectrometry ion chromatography and the spectra are given in Appendix B, B.1 and Appendix C, C.1.

Table 1 Maximum allowable error for relative ion abundance using gas chromatography-mass spectrometry

Relative abundance (base) > 50% > 20% to 50% > 10% to 20% <=10% Allowable relative deviation ± 10% ± 15% ± 20% ± 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 tetracycline is 0.010 mg/kg.

10.2 Recovery rate

When the addition level was 0.010mg/kg, 0.050mg/kg, 0.200mg/kg, the addition of tetrathione in different substrates was See Appendix D.

Appendix A

(Informative)

The name of the tetramidazine, the retention time and the monitoring of the ion abundance ratio

Table A.1 CAS Number, retention time and monitoring ion abundance ratio of tetracycline

No. Chinese name English name CAS number Molecular formula Molecular weight retention time Min Monitor the ion abundance ratio 1 tetramidazine Clofentezine 74115-24-5 C14H8Cl2N4 303.15 16.10 102 (82), 138 (100) 304 * (98), 306 (60) Note. "*" mark ions as quantitative ions Appendix B (Informative) Gas Chromatography - Mass Spectrometry Ion Chromatography

Figure B.1 Chromosomal Chromatography-Mass Spectrometry Ion Chromatogram of Tetracycline Reference Substance

Min

Appendix C

(Informative)

Gas Chromatography - Mass Spectrometry of Tetrahamidazine Standard

Figure C.1 Gas Chromatography-Mass Spectrometry for Tetramine Standards

Appendix D

(Informative)

The recovery of tetracycine in different matrices

Table D.1 Recovery of tetramidazine in different matrices

NO pesticide name

Sample matrix

Citrus apple spinach broccoli liver chicken kidney 1 tetramidazine 83.8-91.2% 87.0-91.6% 80.8-94.8% 90.5-91.2% 83.8 ~ 95.0 87.5-97.2% 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