GB 23200.102-2016Food safety national standard -- Determination of the content of chlordimeform and its metabolites in royal jelly by gas chromatography-mass spectrometry (English PDF)

4.1 Reagents

4.1.1 Acetonitrile (C2H3N). Chromatographic pure.

4.1.2 Acetone (C3H6O). Chromatographic pure.

4.1.3 n-hexane (C6H14). chromatographic purity.

4.1.4 Trichloroacetic acid (CCl3COOH).

4.1.5 Sodium hydroxide (NaOH).

4.1.6 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 n-Hexane - Acetone (1 1, volume ratio) mixed solvent. take 100 mL of n-hexane, add 100 mL of acetone, shake back.

4.2.2 Trichloroacetic acid solution (5%). Weigh 5 g of trichloroacetic acid, add water and set to 100 ml.

4.2.3 Sodium hydroxide solution (2 mol/L). Weigh 8 g of sodium hydroxide into 100 ml of distilled water.

4.3 Standards

4.3.1 Chlordimeform, C10H13ClN2, CAS No. 6164-98-3, molecular weight 196.68) Reference substance. pure Degree > 98.5%.

4.3.2 4-Chloro-2-methylaniline, C7H8ClN, CAS No. 95-69-2, molecular weight. 141.60) Standard substance. purity > 98.5%.

4.4 Standard solution preparation

4.4.1 Accurately weigh the appropriate amount of chlordimeform and 4-chloro-o-methylaniline standard substance, with a single standard storage solution with a concentration of 100 g/mL Preparation of liquid. According to the need to use acetone diluted mixed to the appropriate concentration of mixed standard working fluid. Store in 4 °C refrigerator.

7.1 Extraction

Weigh 2 g of sample (accurate to 0.01 g) in 50 mL centrifuge tube, add 10 mL of trichloroacetic acid solution vortex mixed for 1 min to 8 000 r/min Centrifuge 3 min. The upper aqueous phase was transferred to another 50 mL centrifuge tube, plus about 4 mL of sodium hydroxide solution adjusted to pH greater than 12. Add 5 g Sodium chloride and 20 mL of n-hexane-acetone (1 1, volume ratio) were mixed and vortexed for 1 min to transfer the upper organic phase into a concentrated flask.

The addition of 20 mL of n-hexane-acetone (1 1, volume ratio) in the aqueous phase was repeated and the extraction was repeated once. The upper organic phase was combined at 45 ° C the bath is concentrated under reduced pressure to near dryness, to be purified.

7.2 Purification

The residue in the concentrated flask was washed twice with 10 mL of acetonitrile and transferred to a 50 mL centrifuge tube. 10 ml of n-hexane was added and the vortex was mixed Min, centrifuged at 3 000 r/min for 3 min, the upper n-hexane phase was transferred to another 50 mL centrifuge tube, 10 mL of n-hexane was added to the acetonitrile phase, the operation was repeated once and the n-hexane phase was combined. Add 10 mL of acetonitrile, vortex mixing 1 min, 3000 r/min centrifugal 3 min, discard the upper layer is Hexane phase. Combined acetonitrile phase, in 45 °C below the water bath under reduced pressure to near dry, dissolved with acetone and set to 1.0 mL, with gas chromatography - quality Spectral determination, 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) Inlet temperature. 250 ° C;

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

E) Injection volume. 2 µL;

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

G) solvent delay time. 10 min;

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

I) Interface temperature. 280 ° C;

J) Ionization energy. 70 eV;

K) ion source temperature. 280 ° C;

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

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

Table 1 Quantitative ion, qualitative ion and relative abundance of chlordimeform and 4-chloro-o-toluidine

Pesticide name Quantitative ion - Qualitative ion 1 Qualitative ion 2 Chlordimeform 196 (100) 181 (85.4) 152 (54.1) 4-Chloro-o-toluidine 141 (100) 77 (25.8) 143 (34.1)

7.3.2 Quantitative determination

According to the sample solution of chlordimeform and 4-chloro o-toluidine content of the case, selected with the sample solution of chlordimeform and 4-chloro o-toluidine concentration similar to the standard Quasi-working solution. The response values of the pesticide and 4-chloro-o-toluidine in the standard working solution and the sample solution should be within the linear range determined by the instrument. Standard work as a solution and sample volume of the same interspersed injection determination. Under the above chromatographic conditions, the retention times of chlordimeform and 4-chloro-o-toluidine were approximately 15.0 min, 12.0 min, the total ion chromatogram of the standard is shown in 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) the presence of chlordimeform and 4-chloro-o-toluidine in the sample. The confirmed samples can be identified as chlordimeform and 4-chloro-o-toluidine positive detection. Chlordimeform and the mass spectrum of the 4-chloro-o-toluidine standard is shown in Appendix A, Figure A.2, Figure A.3.

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 quantitative line of this method for chlordimeform and 4-chloro-o-toluidine is 0.01 mg/kg.

10.2 Recovery rate

The experimental data on the concentration and recovery of the sample are given in Appendix B.

Appendix A

(Informative)

Chromatograms and spectra

Figure A.1 Total ion chromatogram of chlordimeform and 4-chloro-o-toluidine

1. 4-Chloro-o-toluidine 2. Chlordimeform (concentration 0.02 µg/mL)

Figure A.2 EI spectrum of chlordimeform

Figure A.3 EI spectra of 4-chloro-o-toluidine

Appendix B

(Informative)

Sample concentration and recovery of the experimental data

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

Pesticide name Add concentration (mg/kg) Recovery rate range% Precision range% Chlordimeform 0.01 86.7 to 112 10.8 0.02 91.0 to 112 9.5 0.05 81.2 ~ 107 11.6 4-Chloro-o-toluidine 0.01 82.1 to 118 8.9 0.02 85.5 to 107 9.1 0..05 79.4 ~ 102 12.3 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