GB 23200.96-2016Food safety national standard -- Determination of the content of chlordimeform and its metabolites in honey by liquid chromatography-mass spectrometry / mass spectrometry (English PDF)

4.1 Reagents

4.1.1 acetonitrile (CH3CN), high performance liquid chromatography pure.

4.1.2 methanol (CH3OH), high performance liquid chromatography pure.

4.1.3 Sodium hydroxide (NaOH).

4.1.4 sulfuric acid (H2SO4).

4.1.5 Formic acid (HCOOH), purity >=99%.

4.2 Solution preparation

4.2.1 0.02 mol/L sodium hydroxide solution. dissolved 800 mg sodium hydroxide in 1 L water, the use of a period of 1 month.

4.2.2 1 mol/L sulfuric acid solution. in the amount of water slowly into the 54.3 mL concentrated sulfuric acid, while stirring and stirring, diluted with water to 1 L, so that with a period of 3 months.

4.2.3 10 mmol/L sulfuric acid solution. in the amount of water slowly into 10 mL 1 mol/L sulfuric acid, while stirring and stirring, diluted with water to 1 L, Use for 1 month.

4.2.4 acetonitrile aqueous solution. acetonitrile. water (3 7, V/V).

4.2.5 0.1% formic acid solution. 1 mL formic acid dissolved in water and fixed to 1 L.

4.3 Standards

4.3.1 Pesticide standard (chlordimeform, C10H13ClN2, CAS. 6164-98-3. purity >=99%) and 4-chloro-o-toluidine standard (4-chloro-o-toluidine, C7H8ClN, CAS. 95-69-2. purity >=99%).

4.4 Standard solution preparation

4.4.1 Preparation of standard stock solution. accurately weighed the appropriate amount of chlordimeform and 4-chloro o-toluidine standard, with acetonitrile prepared into a concentration of 1.0 Mg/mL standard stock solution. The solution was stored in a refrigerator at -18 ° C. Valid for 12 months.

4.4.2 Preparation of the standard intermediate solution. diluted the standard stock solution with acetonitrile to a final concentration of about 1.0 µg/mL, below 4 ° C.

Save, valid for 6 months.

4.4.3 matrix standard working solution preparation. according to need, when used to absorb a certain amount of standard intermediate solution, with the matrix blank solution with Mix the standard working solution with the appropriate concentration. Less than 4 °C dark storage, is now available with.

4.5 Materials

4.5.1 HLB solid phase extraction column (hydrophilic lipophilic balance column). 60 mg (filler. polystyrene-divinylbenzene-pyrrolidone), 3 mL or equivalent. Activate with 3 mL of methanol and 3 mL of water before use.

4.5.2 Microporous membrane. 0.22 µm, organic system.

6.1 Preparation of the sample

Take about 500 g of the representative honey sample, the sampling site according to GB 2763 Appendix A implementation, the crystallization of honey samples will be evenly stirred.

For samples of crystalline honey, in a sealed case, the vials are placed in a water bath of not more than 60 ° C and warmed and shaken until all the samples Melt and stir well, quickly cooled to room temperature, in the melting must pay attention to prevent moisture evaporation. Load a clean container, seal, mark the mark.

6.2 Sample storage

The sample is stored at room temperature.

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

7.1 Extraction

Weigh the sample 1 g (accurate to 0.01 g) in a 15 mL centrifuge tube, add 10 mL of 0.02 mol/L sodium hydroxide solution at 2000 r/min Under the vortex shock, mix.

7.2 Purification

Transfer the extract to the HLB solid phase extraction column and add 3 mL of water to the centrifuge tube. Use 3 mL of water and 1 mL 10 mmol/L sulfuric acid solution leaching column, discard the leaching solution, dry. Eluted with 2 mL of acetonitrile and collected in 10 mL of the test tube at room temperature, nitrogen blowing near dry, accurately add 1.0 mL of acetonitrile. water (3 7, V/V), the oscillation dissolved, over 0.22 µm filter for determination.

7.3 Determination

7.3.1 Liquid Chromatography-Mass Spectrometry Reference Conditions.

A) Column. C18 column length 50 mm, inner diameter 4.6 mm, particle size 1.8 µm, or equivalent;

B) mobile phase. acetonitrile - 0.1% formic acid solution, gradient elution program in Table 1;

Table 1 Mobile phase gradient elution program

Time (min) 0.1% Formic acid solution (%) Acetonitrile (%) 0.0 70 30 2.0 70 30 4.0 5 95 7.0 5 95 7.1 70 30 13.0 70 30 C) Flow rate. 0.50 mL/min;

D) Column temperature. 30 ° C;

E) Injection volume. 10 µL;

F) ion source. electrospray ion source (ESI);

G) Scanning mode. positive ions;

H) Monitoring methods. Multiple reaction monitoring (MRM);

I) Mass spectrum conditions are given in Appendix A.

7.3.2 Determination and confirmation of chromatography

According to the content of the test compound in the sample solution, the standard working solution with similar peak area is selected, and the standard working solution and sample solution the reaction values of the test compounds in the standard working solution and the sample solution shall be within the linear range of the instrument detection, and the standard working curve According to external standard method. The reference retention times of chlordimeform and 4-chloro-o-toluidine were 1.3 and 4.4 min, respectively, under the above chromatographic conditions, and 4-Chloro-o-toluidine standard Multi-reaction detection (MRM) chromatograms are given in Appendix B, Figure B.1.

The sample and standard working solution were determined according to the liquid chromatography-mass spectrometry conditions. The mass retention time of the sample was corresponding to that of the standard the retention time deviation is within ± 2.5%; and the relative abundance of the qualitative ions of each component in the sample is corresponding to the standard working solution in close proximity the relative abundance of qualitative ions is compared, the deviation does not exceed the range specified in Table 2, it can be determined that there is a corresponding analyte in the sample.

Table 2 The maximum allowable deviation of relative ion abundance when qualitative confirmation

Relative ion abundance > 50% > 20% ~ 50% > 10% ~ 20% <=10% Allowable relative deviation ± 20% ± 25% ± 30% ± 50%

7.4 Blank experiment

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

10.1 Quantitation limits

The quantification limit of this method is 5 µg/kg.

10.2 Recovery rate

The addition rates of chlordimeform and 4-chloro-o-toluidine in different matrices are shown in Appendix C when the level is 5,10,20 µg/kg.

Appendix A

(Informative)

Electrospray ionization source Reference conditions.

A) ionization source mode. electrospray ionization;

B) ionization source polarity. positive mode;

C) Detection method. multiple reaction monitoring;

D) atomization gas. nitrogen;

E) Atomizing gas pressure. 0.207 MPa (30 psi);

F) Capillary voltage. 4000 V;

G) Dry gas temperature. 350 ° C;

H) Dry gas flow rate. 9 L/min;

I) Resolution. Unit resolution;

(E), qualitative ion pairs (m/z), and quantitative ion pairs (m/z) are shown in Table A.1;

Table A.1 Qualitative ion pairs of chlordimeform and 4-chloro-o-toluidine, quantitative ion pair, fragmentation voltage, collision energy

name

Qualitative ion pair (M/z) Quantitative ion pair (M/z) Fragmentation voltage (V) Collision energy (EV) Chlordimeform 197.1/117.1 197.1/117.1 130 197.1/125 33 4-Chloro-o-toluidine 142/106.7 21 Non-commercial declaration. The reference mass spectrometry conditions listed in Appendix A are performed on an Agilent 6410B LC/MS/MS LC/MS, where the test instrument the model is for reference only and does not involve commercial purposes and encourages standard users to try different manufacturers or models of instruments.

Appendix B

(Informative)

Chlordime and 4-chloro-o-toluidine standard multi-reaction detection (MRM) chromatograms

Figure B.1 Chlordimeform and 4-Chloro-o-toluidine Standard Multi-Reaction Monitoring (MRM) Mass Chromatogram (5 ng/mL)

Appendix C

(Informative)

The recovery of chlordimeform and 4-chloro-o-toluidine in different matrices

Table C.1 Recovery range of the method (n = 10)

sample name

Add concentration

(Mg/kg)

4 - Chloro - o - toluidine Recovery rate range (%) Coefficient of variation% Recovery rate range (%) Coefficient of variation% Acacia honey 5 86.4 ~ 102.0 7.5 86.2 ~ 105.3 8.6 10 88.3 ~ 110.3 8.3 87.1 ~ 111.4 7.0 20 86.9 to 111.2 7.6 88.8 to 111.4 8.3 Vitex 5 86.7 ~ 109.6 8.6 86.2 ~ 109.3 8.7 10 76.2 ~ 100.5 7.5 92.3 ~ 108.8 9.8 20 81.5 to 110.2 10.2 88.0 to 114.3 9.0 Honeycomb honey 5 86.3 ~ 109.8 9.0 85.6 ~ 112.2 9.9 10 75.8 ~ 101.0 10.2 90.8 ~ 110.8 10.0 20 82.6 ~ 109.8 9.7 90.7 ~ 105.0 6.7 Mango 5 86.9 to 99.7 4.8 87.4 to 107.6 6.2 10 82.6 ~ 101.5 7.9 87.9 ~ 112.5 9.7 20 83.0 to 112.2 9.7 89.5 to 106.9 6.4 Wild honey 5 86.1 to 113.8 9.2 86.2 to 112.2 9.8 10 81.4 ~ 101.2 8.1 93.4 ~ 110.5 8.6 20 83.1 to 101.1 7.4 87.8 to 110.6 8.0 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