GB 23200.104-2016Food safety national standard -- Meat and meat products -- Determination of 2-methyl-4-chloro and 2-methyl-4-chlorobutyric acid residues by liquid chromatography-mass spectrometry (English PDF)

4.1 Reagents

4.1.1 dichloromethane (CH2Cl2). chromatographic purity.

4.1.2 Methanol (CH3OH). Chromatographic Purification.

4.1.3 Formic acid (HCOOH). Chromatographic pure.

4.1.4 concentrated sulfuric acid (H2SO4). excellent grade pure.

4.1.5 sodium chloride. excellent grade pure.

4.2 Solution preparation

4.2.1 Sulfuric acid - water (19, V/V). take 100 mL concentrated sulfuric acid, add 900mL of water, shake back.

4.2.2 0.1% formic acid aqueous solution. take 1 mL of formic acid, the volume of water to 1000 mL.

4.3 Standards

2 a 4 chlorobutyric acid standard. purity >= 99%, CAS. 94-82-6.

4.4 Standard solution preparation

4.4.1 0.1 mg/mL standard stock solution. accurately weighed the appropriate amount of 2 a 4 chlorine, 2 a 4 chlorobutyric acid standard, with methanol prepared into the concentration Prepare the solution for 0.1 mg/mL standard. Store in 4 °C refrigerator.

4.4.2 mixed standard working fluid. the use of the corresponding matrix solution prepared with the appropriate concentration of the standard working solution, stored in 4 °C refrigerator.

6.1 Preparation of the sample

Take a representative sample 500g, sample sampling site according to GB 2763 Appendix A implementation, crushing machine, and then ground into fine powder Shaped, packed into a clean glass container, sealed and marked.

6.2 Sample storage

The samples were cryopreserved at -18 ° C.

7.1 Extraction and purification

Weigh the sample 4 g (accurate to 0.01 g) in a 50 mL centrifuge tube, add 3 mL of methanol, 1 mL of sulfuric acid-water, 2 g of sodium chloride and 20 mL Dichloromethane. In the high-speed homogenizer on the homogeneous extraction 2 min, centrifugation, take the supernatant in another centrifuge tube, standing, take the next layer of dichloromethane Layer 10 mL. Repeat the extraction once, merge the two extracts, at 40oC nitrogen blowing near dry, with methanol volume to 2 mL, vortex, membrane, for Determination of Ultra - High Pressure Liquid Chromatography by Tandem Mass Spectrometry.

7.2 Determination

7.2.1 Liquid Chromatographic Reference Conditions

A) Column. C18, 1.8 µm, 2.1 x 50 mm or equivalent;

B) 0.1% formic acid aqueous solution of methanol, gradient elution see Table 1;

C) Flow rate. 0.5 mL/min;

D) Injection volume. 5 µL;

E) Column temperature. room temperature;

Table 1 Liquid Chromatography Gradient Elution Procedure

Time (min) 0.1% Formic acid Aqueous solution (%) Methanol (%)

7.2.2 Mass spectrometry reference conditions

A) ion source. electrospray ion source B) Scan mode. negative ion mode C) Detection method. Multiple reaction monitoring MRM D) the rate. the unit mass resolution E) Refer to A.1 for other reference mass spectrometry conditions

7.2.3 Chromatographic determination and confirmation

According to the determined liquid chromatography - tandem mass spectrometry conditions, the sample and blank matrix plus standard working solution, the response value should be detected in the instrument in the linear range, the peak area of the chromatogram was quantified by the external standard method, and the negative ion mode was scanned. Under the above chromatographic conditions, 2-methyl-4-chloro and 2-methyl-4-chlorobutyric acid of the retention time of about 1.90 min and 2.10 min. 2 Selective ion flow chart of the standard solution of methyl 4-chloro and 2-chlorobutyric acid is shown in Appendix B.1 and attached Record B.2.

If the sample solution with the standard working solution in the selective ion chromatogram at the same retention time there is a chromatographic peak that can be produced according to the two Group of ions between the abundance ratio, to confirm it.

Under the same experimental conditions, the retention time of the test substance in the sample and the corresponding working solution in the standard retention time deviation of ± 2.5% and the relative abundance of the qualitative ions of each component in the sample spectrum and the relative abundance of the qualitative ions in the standard working solution are not allowed when the range specified in Table 2 is specified, it may be determined that such a test substance is present in the sample.

The maximum allowable error of relative ion abundance when using gas chromatography - mass spectrometry

7.3 Blank test

Relative abundance (base) 50% 20% to 50% 10% to 20% <=10% Permissible relative deviations ± relative to ± relative permissible relative permissible Except for the addition of samples, according to the above conditions and procedures for determination.

10.1 Quantitation limits

The quantitative line of this method is. 0.01 mg/kg.

10.2 Recovery rate

In the beef, pork sausage, fish, chicken and beef canned samples were added 2 a 4 chlorine, 2 a 4 chlorobutyric acid, add the concentration and its back the yield is given in Appendix E.

Appendix A

(Informative)

Mass spectrometry parameters A. Liquid Chromatography-Mass Spectrometry/Mass Spectrometry Parameters A) Air curtain gas pressure (CUR). 15 Psi (nitrogen);

B) Ion source spray voltage (IS). - 4500V;

C) atomization temperature (TEM). 550 oC;

D) Atomizing gas pressure (GAS1). 55 Psi (nitrogen);

E) Heating auxiliary gas (GAS2). 80 Psi (nitrogen);

F) collision air flow ratio (CAD). 7;

G) other parameters in Table A.

Table A MCPA and MCPA Select ion and mass spectrometry parameters

No. Compound Reference Retention Time Qualitative Ion Pair Discharge Voltage (DP)/V Collision Gas Energy (CE)/V * For quantitative ion pairs

1) Non-commercial notices. The parameters listed in A are done on the API 4000 Plus mass spectrometer/mass spectrometer. The experimental instruments listed here are for the purpose of providing Test, does not involve commercial purposes, to encourage standard users to try different models of equipment.

Appendix B

(Informative)

Figure B.1 MCPA Standard LC-MS/MS MRM Chart

Figure B.2 MCPB Standard LC-MS/MS MRM Chart

Appendix C

(Normative appendix) Laboratory repeatability requirements

Table C.1 Laboratory repeatability requirements

Measured component content Appendix D (Normative appendix) Inter-laboratory reproducibility requirements

Table D.1 Inter-laboratory reproducibility requirements

Measured component content Appendix E (Informative) Concentration and recovery of MCPA and MCPB in different matrices

Table E.1 Concentration and recovery of MCPA and MCPB in different matrices

Average recovery (%) MCPA MCPB Add concentration (mg/kg) Add concentration (mg/kg) 0.01 0.05 0.1 0.01 0.05 0.1 Beef 91.5 101.7 93.8 87.5 100 105.2 Pork sausage 107.6 98.2 102.4 77.1 78.9 95.6 Fish 94.8 84.3 82.4 93.3 82.7 82.3 Chicken 94.8 97.0 99.5 90.7 95.5 95.9 Beef canned 91.0 89.6 93.9 95.5 99.9 98.0.