GB 23200.94-2016Food safety national standard -- Determination of Trichlorfon, Dichlorvos and Phytophthora Phosphorus Residues in Animal Foods by Liquid Chromatography-Mass Spectrometry / Mass Spectrometry (English PDF)

4.1 Reagents

4.1.2 acetonitrile (CH3CN). high performance liquid chromatography grade.

4.1.3 Methanol (CH3OH). High performance liquid chromatography grade.

4.1.4 Ethyl acetate (C4H8O2).

4.1.5 Bichloromethane (CH2Cl2).

4.1.6 Cyclohexane (C6H12).

4.1.7 sodium sulfate (Na2SO4). 650 °C burning 4 h, in the dryer to cool to room temperature, stored in a sealed bottle in reserve.

4.1.8 ammonium acetate (CH3COONH4). excellent grade pure or equivalent.

4.2 Solution preparation

4.2.1 50 mmol/L ammonium acetate solution. Weigh 0.385 g of ammonium acetate in 1 000 mL of water

4.3 Standards

4.3.1 Trichlorfon (CAS. 52-68-6, Molecular formula. C4H8Cl3O4P) Standard. purity >= 98.0%.

4.3.2 dichlorvos (Dichlorvos, CAS. 62-73-7, molecular formula. C4H7Cl2O4P) standard. purity >= 97.6%.

4.3.3 Flamingophos (Coumaphos, CAS. 56-72-4, Molecular formula. C14H16ClO5PS) Standard. Purity >=99.9%.

4.4 Standard solution preparation

4.4.1 standard stock solution. respectively, accurately weighed appropriate trichlorfon, dichlorvos, flies phosphorus standard, with methanol prepared into a concentration of 100 µg/mL Concentration of mixed standard stock solution. The solution in 0 °C ~ 4 °C refrigerator in the dark, can be used for 3 months.

4.4.2 blank sample extract. with no trichlorfon, dichlorvos, flies phosphorus residue samples, according to 7.1 prepared blank sample extract.

4.4.3 Standard working solution. Standard stock solution is diluted to 50 ng/mL, 100 ng/mL,.200

Ng/mL, 500 ng/mL mixed standard working solution. Placed in 0 °C ~ 4 °C refrigerator in the dark, can be used for 3 days.

6.1 Preparation of the sample

6.1.1 Split meat

Take a typical sample of about 500 g, broken with a tissue crusher, into a clean container as a sample, sealed and labeled.

6.1.2 Casing

Take a representative sample of about 100 g, cut with scissors to less than 2 mm, into a clean container as a sample, sealed and labeled.

6.1.3 Honey

Take a representative sample of about 500 g, stir evenly into the clean container sealed and do a good job.

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

6.2 Sample storage

Split meat, casings, honey and other samples in -18 °C below the frozen storage.

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 and purification

7.1.1 Split meat

A) accurately weighed 5 g homogeneous sample (accurate to 0.01 g) in 50 mL stopper tube, add 5 g of anhydrous sodium sulfate to mix, add Into 15 mL of dichloromethane, homogenizer (10000 r/min) homogeneous 2 min, 4 000 r/min centrifugal 3 min, the organic phase to Transferred to 100 mL pear-shaped distillation bottle, the residue and then 2 × 10 mL dichloromethane homogeneous extraction twice.

B) The organic phases were combined by centrifugation and evaporated to 2 mL at 40 ° C. The sample was transferred to a 5 mL graduated tube and washed with a small amount of dichloromethane Wash the pear-shaped distillation bottle, combine the washing liquid to the scale tube, room temperature nitrogen concentrated to dry. Quantitative addition of 1.0 mL of acetonitrile dissolved After adding 2 mL of cyclohexane vortex for 2 min, centrifuge at 2 500 r/min for 3 min and add the lower acetonitrile to 0.45 µm Microporous membrane, for liquid chromatography - mass spectrometry/mass spectrometry.

7.1.2 Casing

Accurately weighed 5 g sample (accurate to 0.01 g) in 50 mL stoppered centrifuge bottle, add 2 g of anhydrous sodium sulfate mix, then add 15 mL Dichloromethane, covered with a lid and placed in an ultrasonic cleaner for 30 min. After cooling, the organic phase was transferred to 100 mL of pear-shaped distillation Bottle, the residue and then 2 × 10 mL with dichloromethane mixed ultrasonic extraction twice, centrifugal organic phase. Next, follow the "Centrifugal" in 7.1.1 Merge organic phase "after the steps.

7.1.3 Honey

Weigh 5 g of sample (accurate to 0.01 g) in 50 mL stoppered centrifuge tube, add 10 mL of water, add 25 mL of ethyl acetate, mix in vortex and the mixture was centrifuged at 3 000 r/min for 5 min. The upper layer of ethyl acetate extract was collected in a concentrated flask and the residue was further added to 20 mL Ethyl acetate, the above procedure was repeated and the ethyl acetate extraction solution was combined. Concentrated to about 3 mL under reduced pressure at 50 ° C and transferred to 10 mL centrifugation Tube, with 5 mL of ethyl acetate in two batches of concentrated bottles, combined washing solution in a centrifuge tube, dry with nitrogen. Add 1.0 mL of acetonitrile to dissolve residue Slag and pass through a 0.45 µm microporous membrane for liquid chromatography-mass spectrometry/mass spectrometry.

7.2 Determination

7.2.1 Liquid Chromatography - Mass Spectrometry/Mass Spectrometry Reference Conditions

A) Column. Column packed with octadecyl silane bonded phase column, 4.6 x 150 mm, 5 µm or equivalent;

B) Mobile phase. methanol. 50 mmol/L ammonium acetate, gradient elution procedure See Appendix A;

C) Flow rate. 0.50 mL/min or optimized according to instrument conditions;

D) Column temperature. 40 ° C;

E) Injection volume. 10 µL;

F) ion source. electrospray ion source;

G) Scanning mode. positive ions;

H) Detection methods. multiple reaction monitoring (MRM);

I) Mass Spectrometry/Mass Spectrometry Refer to Appendix B for reference conditions

7.2.2 Chromatographic determination and confirmation

The sample solution and standard working solution were determined according to 7.2.1 liquid chromatography-mass spectrometry/mass spectrometry conditions. The external standard method was used to determine the enemy Insects, dichlorvos, flies phosphorus residues. Residue of the sample to be measured in the sample should be within the standard curve, if the residue exceeds the standard curve and the blank sample extract is used for proper dilution. In the above chromatographic conditions, trichlorfon, dichlorvos, flies phosphorus quality peak protection the time is about 13.2 min, 14.1 min, 15.9 min. The multi-reaction monitoring chromatogram of the standard is shown in Figure C.1 in Appendix C.

Under the same experimental conditions, the relative retention time of the sample and the mass chromatographic peak of the test substance in the standard working fluid was within 2.5%, and in the sample quality chromatogram after subtracting the background, the selected pair of ions appears, and the relative abundance of the standard is not allowed to exceed

Table 1 specified range, you can determine the existence of the corresponding sample in the sample.

Table 1 Maximum Adsorption Error for Relative Ion Abundance Using Qualitative Liquid Chromatography-Mass Spectrometry/Mass Spectrometry

Relative ion abundance > 50% > 20% to 50% > 10% to 20% <=10% Maximum permissible deviation ± 20% ± 25% ± 30% ± 50%

7.3 Blank experiment

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

10.1 Quantitation limits

The limits of determination of trichlorfon, dichlorvos and flies were 0.010 mg/kg.

10.2 Recovery rate

10.2.1 Concentration and recovery of trichlorfon in casings Data.

When the concentration was 0.010 mg/kg, the recoveries ranged from 73.0% to 94.0% when the concentration was 0.020 mg/kg, the recoveries ranged from 78.0% to 95.0% when the concentration was 0.040 mg/kg, the recovery was between 78.0% and 95.0%.

10.2.2 Addiction concentration and recovery rate of dichlorvos in casings Data.

When the concentration was 0.010 mg/kg, the recoveries ranged from 76.0% to 98.0% when the concentration was 0.020 mg/kg, the recoveries ranged from 78.0% to 98.0% when the concentration was 0.040 mg/kg, the recoveries ranged from 86.0% to 95.0%.

10.2.3 Concentration and recovery of poisonous phosphorus in casings.

When the concentration was 0.010 mg/kg, the recoveries ranged from 75.0% to 95.0% when the concentration was 0.020 mg/kg, the recoveries ranged from 80.0% to 96.0% when the concentration was 0.040 mg/kg, the recoveries ranged from 77.0% to 92.0%.

10.2.4 Concentration and recovery of trichlorfon in divided meat Data.

When the concentration was 0.010 mg/kg, the recoveries ranged from 92.0% to 115% when the concentration was 0.020 mg/kg, the recoveries ranged from 86.5% to 113% when the concentration was 0.040 mg/kg, the recovery was between 80.8% and 101%.

10.2.5 Add the concentration of dichlorvos in the divided meat and its recovery rate Data.

When the concentration was 0.010 mg/kg, the recoveries ranged from 93.0% to 110% when the concentration was 0.020 mg/kg, the recoveries ranged from 88.5% to 108% when the concentration was 0.040 mg/kg, the recovery was between 84.3% and 110%.

10.2.6 Concentration and recovery of poisonous phosphorus in divided meat.

When the concentration was 0.010 mg/kg, the recoveries ranged from 91.0% to 112% when the concentration was 0.020 mg/kg, the recoveries ranged from 79.0% to 102% when the concentration was 0.040 mg/kg, the recoveries ranged from 73.9% to 95.3%.

10.2.7 Concentration and recovery of trichlorfon in honey Data.

When the concentration was 0.010 mg/kg, the recoveries ranged from 70.0% to 110% when the concentration was 0.020 mg/kg, the recoveries ranged from 79.0% to 104% when the concentration was 0.040 mg/kg, the recovery was between 80.0% and 107%.

10.2.8 Additive concentration of honey dichlorvos and its recovery rate Data.

When the concentration was 0.010 mg/kg, the recoveries ranged from 70.0% to 92.0% when the concentration was 0.020 mg/kg, the recoveries ranged from 71.0% to 91.0% when the concentration was 0.040 mg/kg, the recoveries ranged from 70.0% to 96.0%.

10.2.9 Concentration and recovery of poisonous phosphorus in honey.

When the concentration was 0.010 mg/kg, the recoveries ranged from 79.0% to 110% when the concentration was 0.020 mg/kg, the recoveries ranged from 74.0% to 105% when the concentration was 0.040 mg/kg, the recovery was between 71.0% and 104%.

Appendix A

(Informative)

Gradient elution reference conditions

Table A.1 Mobile phase gradient elution program

Time/min methanol/(%) 50 mmol/L ammonium acetate/(%) 0 10.0 90.0 5.00 10.0 90.0 10.00 95.0 5.0 18.00 95.0 5.0 18.01 10.0 90.0 25.00 10.0 90.0 Appendix A (Informative) API 4000 LC-MS/MS system for detection of trichlorfon, dichlorvos, flies phosphorus reference conditions A.1 API 4000 LC-MS/MS System Electrospray Ion Source Reference Conditions Monitoring of ion pairs and voltage parameters.

A) Curtain gas (CUR). 15.00 Pa;

B) Atomizing gas (GS1). 40.00 Pa;

C) Auxiliary heated gas (GS2). 45.00 L/min;

D) collision gas (CAD). 7.00 Pa;

E) Ion source nebulizer voltage (IS). 5000.00 V;

F) spray needle temperature. (TEM). 550.00 ° C;

G) Qualitative ion pair, quantitative ion pair, de-cluster voltage, collision energy, collision chamber outlet voltage See Table B.1.

Table B.1 Qualitative ion pairs, Quantum ion pairs, Cladding voltage, Collision energy, and collision chamber outlet voltage

To be measured

To cluster voltage

Collision energy

Collision chamber outlet voltage Trichlorfon Trichlorfon 259.0a 109.2 63.0 26.5 19.0 256.9 221.2 63.0 16.0 13.0 Dichlorvos Dichlorvos 221.3 a 109.2 63.0 25.5 19.0 223.3 109.0 63.0 25.0 18.0 Flies phosphorus Coumafos 363.3 a 307.0 96.0 25.0 20.0 363.3 227.0 96.0 36.0 13.0 A is a quantitative ion pair.

Appendix B

(Informative)

B.1 Standard multi-reaction monitoring chromatogram

Figure C..1 Trichlorfon, dichlorvos, flies toxic phosphorus standard solution multi-reaction monitoring chromatogram

* Non-commercial statement. The equipment and models used in this standard do not involve commercial purposes, encourage standard messengers to try different manufacturers or models of equipment.

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