GB 23200.103-2016Food safety national standard -- Determination of Residual Amidamidine and Its Metabolites in Royal Jelly by Gas Chromatography -- Mass Spectrometry (English PDF)

4.1 Reagents

4.1.1 n-hexane (C6H14). chromatographic purity.

4.1.2 ether (C4H10O). pure chromatography.

4.1.3 Acetone (C3H6O). Chromatographic pure.

4.1.4 sodium chloride (NaCl). 650 °C burning 4 h, in the dryer to cool to room temperature, stored in a sealed bottle in reserve.

4.2 Solution preparation

4.2.1 hydrochloric acid solution (2.0 mol/L). with a pipette to remove 90 mL of hydrochloric acid, diluted with water to 500mL.

4.2.2 sodium hydroxide solution (10 mol/L). Weigh 40 g of sodium hydroxide, dissolved in water to 100mL.

4.3 Standards

4.3.1 Amitraz (amitraz, C19H23N3, CAS No. 33089-61-1, molecular weight 293) Standard substance. purity > 99%.

4.3.2 2,4-Dimethyl aniline (C8H11N, CAS No. 95-68-1, molecular weight. 121) Reference substance.

Purity > 99%.

4.4 Standard solution preparation

4.4.1 Amitrazine standard solution. accurately weighed the appropriate amount of formamidine standard material, with acetone dubbed the concentration of 100 g/mL standard stock solution.

According to the need to use acetone diluted to the appropriate concentration of standard working fluid. Store in 4 °C refrigerator.

4.4.2 2,4-dimethylaniline standard solution. accurately weighed the appropriate amount of 2,4-dimethyl aniline standard material, with acetone dubbed the concentration of 100 g/mL standard stock solution. According to the need to use acetone diluted to the appropriate concentration of standard working fluid. Store in 4 °C refrigerator.

7.1 Hydrolysis

Weigh 4 g of sample (accurate to 0.01 g) in a 100 mL flask and add 15 mL of hydrochloric acid solution (4.5) to mix on a vortex mixer.

Install the reflux condenser, heat reflux at 120 °C for 2 h, stop heating, continue to cool water at room temperature for 15min, move to the ice bath to cool 15min.

7.2 Extraction and purification

Transfer the hydrolyzate to a 50 mL stoppered centrifuge tube with 10 mL of n-hexane-diethyl ether (2 1, by volume) mixed solvent plus 4 ML of sodium hydroxide solution, vortex and mix well, cooled in ice bath for 5 min, centrifuged at 4000 r/min for 3 min. Transfer the upper organic phase to another A 50 mL stopper with a centrifuge tube. The aqueous phase was extracted twice with a mixed solvent of 2 x 10 mL n-hexane-diethyl ether (2 1, v/v). Merge the upper layer the organic phase. With 2 × 5 mL hydrochloric acid solution extraction 2 times, combined with hydrochloric acid extract and constant volume to 10mL, take 5mL hydrochloric acid extract in 10 mL with a plug in the centrifuge tube. Add 2 mL of n-hexane - acetone (2 1, volume ratio) mixed solvent, 2 mL of sodium hydroxide solution extraction 1, centrifugal Layer, upper organic phase for gas chromatography - mass spectrometry, external standard method.

7.3 Determination

7.3.1 Gas Chromatography - Mass Spectrometry Reference Conditions

A) Column. DB-5ms quartz capillary column, 30 m x 0.25 mm (id) x 0.25 µm, or equivalent;

B) Column temperature. 50 °C for 2 min, at 10 °C/min heating rate rose to 160 °C, keep 1 min to 30 °C/min of the heating rate rose to 320 °C, keep 5min;

C) Carrier gas. helium, purity greater than or equal 99.999%, flow rate. 1.0 mL/min;

D) Injection volume. 2 µL;

E) Injection method. pulse splitless injection, pulse pressure 25 psi ,, 0.75 min after the opening valve;

F) solvent delay time. 8 min;

G) Inlet temperature. 250 ° C;

H) Interface temperature. 280 ° C;

L) solvent delay time. 8 min;

J) ion source. electron bombardment source (EI);

K) ionization energy. 70 eV;

L) Determination of the way. select the ion monitoring mode;

M) Select the ion and relative abundance. see Table 1.

Table 1 Selective ion and relative abundance of 2,4-dimethylaniline

Select ion (m/z) 121 (quantitative) 120 77 Relative abundance (%) 100 85.2 21.3

7.3.2 Quantitative determination

According to the sample solution 2,4-dimethyl aniline content of the situation, selected with the sample of 2,4-dimethyl aniline concentration similar to the standard working solution liquid. The reaction value of the 2,4-dimethylaniline in the standard working solution and the sample solution should be within the linear range of the instrument. Standard working solution and sample solution Equal volume interspersed injection assay. Under the above chromatographic conditions, the retention time of 2,4-dimethylaniline is about 10.0 mim, and the total Subflow diagram See Appendix A, Figure A.1.

7.3.3 Qualitative determination

The standard solution and sample solution are determined according to the conditions specified in 7.3.1, if the sample solution and the standard solution in the same retention time peak Now, it is confirmed by mass spectrometry, after deducting the background of the sample spectrum, the selected ions all appear, while the selected ion from the the abundance ratio is consistent with the relative abundance of the ions associated with the standard, and the fluctuation range is within the maximum allowable deviation of Table 2 (see Table 2) There was 2,4-dimethylaniline in the sample. The confirmed sample can be judged as 2,4-dimethylaniline positive detection. 2,4-dimethylaniline the spectrum of the standard is shown in Figure A.2 in Appendix A.

Table 2 Mass Spectrometry Relative Ion Abundance Maximum Allowable Deviation

Relative ion abundance (% base) > 50% > 20% to 50% > 10% to 20% <=10% Allowable relative error ± 10% ± 15% ± 20% ± 50%

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

The recovery of this method is given in Appendix B.

Appendix A

(Informative)

Chromatograms and spectra

Figure A.1 Total ion chromatogram of the 2,4-dimethylaniline standard (concentration 0.01 j/mL)

Figure A.2 Mass spectrum of 2,4-dimethylaniline standard substance

Appendix B

(Informative)

Sample concentration and recovery of the experimental data

Table B.1 Experimental data on the concentration and recovery of the sample

Compound added concentration/(mg/kg) Recovery /% Precision /% Formamidine 0.01 76.7% to 88.2 9.8 0.02 76.9 to 88.0 10.2 0.05 78.4 to 90.4 9.0 Appendix C (Normative appendix) Laboratory repeatability requirements

Table C.1 Laboratory repeatability requirements

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

Table D.1 Inter-laboratory reproducibility requirements

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