GB 29690-2013Determination of marker residues of Nicarbazin in animal derived food by Liquid Chromatography-tandem Mass Spectrometric method (English PDF)

6.1 Preparation of the sample

6.1.1 Eggs

An appropriate amount of fresh or frozen egg white or test, peeled, and homogenized.

--- the test sample taken after homogenization, as the feed try.

--- blank sample taken after homogenization, as a blank sample.

--- blank sample taken after homogenization, adding a suitable concentration of the standard working solution, is added as a blank sample.

6.1.2 Chicken muscle

Fresh or frozen take appropriate blank or test tissue, minced, and homogenized.

--- the test sample taken after homogenization, as the feed try.

--- blank sample taken after homogenization, as a blank sample.

--- blank sample taken after homogenization, adding a suitable concentration of the standard working solution, is added as a blank sample.

Save 6.2 sample

Or less at -20 °C.

Determination Step 7

7.1 Extraction and purification

Sample Weigh 2g ± 0.02g, in 50mL centrifuge tubes, was added 10µg/mL4,4'- dinitro-stilbene working standard solution of urea suitable -D8 Amount of anhydrous sodium sulfate 2g, 8 mL of acetonitrile, vortexed 0.5min, ultrasonic 5min, 5000r/min centrifugal 10min, supernatant at 40 °C dry nitrogen, was added 75% aqueous methanol saturated with hexane 1mL, scroll 10s, plus 1.0 mL of 75% aqueous methanol, sufficient Vortexed, allowed to stand in a water bath at 40 °C 5min, 2000r/min centrifugal 5min, the supernatant and the lower layer, membrane filtration, for liquid chromatography - tandem Mass Spectrometry.

7.2 Preparation of standard curve

The precise amount of 10µg/mL4,4'- dinitro-stilbene working standard solutions of urea and 10µg/mL4,4'- dinitro-stilbene standard urea -D8 Working fluid amount, diluted with 75% aqueous methanol solution, were formulated as 4,4'-dinitro diphenyl urea concentration -D8 are 100ng/mL and 4,4'-Dinitro-stilbene 2,10,20,50,200 and urea concentration is 500ng/mL series control solution for liquid chromatography - tandem mass spectrometry.

A characteristic ion mass chromatogram peak area for the vertical axis, the standard solution at abscissa, the standard curve. Seeking regression equation and correlation coefficient.

7.3 Determination

7.3.1 LC Conditions

7.3.1.1 Column. C18 (150mm × 2.1mm, particle size 5µm), or equivalent person.

7.3.1.2 Mobile phase. methanol 0.1mol/L ammonium acetate solution (75 25, volume ratio).

7.3.1.3 flow rate. 0.2mL/min.

7.3.1.4 Column temperature. 30 °C.

7.3.1.5 Injection volume. 20µL.

7.3.2 MS conditions

7.3.2.1 ion source. electrospray ionization source.

7.3.2.2 Scan mode. negative ion scan.

7.3.2.3 Detection mode. multiple reaction monitoring.

7.3.2.4 ionization voltage. 3.0kV.

7.3.2.5 source temperature. 110 °C.

7.3.2.6 atomizing temperature. 350 °C.

7.3.2.7 Cone gas flow rate. 50L/h.

7.3.2.8 atomizing gas flow rate. 450L/h.

7.3.2.9 qualitative and quantitative Cone voltage of ion collision energy and are shown in Table 1.

Table 1 DNC and the internal standard qualitative and quantitative Cone voltage of ion collision energy

drug

Qualitative ion pair m/z Quantitative ion pair m/z Cone voltage Collision energy eV DNC 300.9 > 136.9 300.9 > 106.9 300.9 > 136.9 20 DNC-D8 309.0 > 141.0 309.0 > 141.0 20 18

7.3.3 Assay

Take a sample solution and control solution, as a single or multi-point calibration, according to the internal standard method, the peak area ratio calculation. Sample solution and control solution 4,4'-dinitro-4,4'-stilbene urea and urea-nitro-stilbene -D8 peak area ratio should be within the linear range of the detection instrument. Sample solution Compared the relative abundance of ions in solution and the standard solution was added blank relative abundance of ions, in accordance with Table 2. And blank control solution the features added ion mass chromatogram of the sample solution is given in Appendix A.

Table 2 sample solution ion relative abundance tolerance range%

Tolerance relative abundance > 50 ± 20 20 ~ 50 ± 25 10 ~ 20 ± 30 <=10 ± 50

7.4 Blank test

But without addition of the sample, the same steps employed in parallel operation.

8 and the result of the calculation expression Single-point calibration.

AA'iScSciS

AiSASc'iS

Calibration or standard curve by.

A'iS =

c'iS

to obtain a and b b, then c = ciS (A AiS-

DNC remaining amount in a sample according to formula (3) is calculated.

Where.

C --- sample solution of 4,4'-dinitro-stilbene urea concentration, in units of nanograms per milliliter (ng/mL);

A --- sample solution of 4,4'-dinitro diphenyl urea of average peak area;

A'iS --- control solution of 4,4'-dinitro-stilbene urea -D8 peak area;

cS --- control solution of 4,4'-dinitro-stilbene urea concentration, in units of nanograms per milliliter (ng/mL);

--- CIS averaged concentration in the sample solution of 4,4'-dinitro diphenyl urea -D8, expressed in nanograms per milliliter (ng/mL);

--- AiS sample solution of 4,4'-dinitro-stilbene urea -D8 peak area;

--- the AS control solution of 4,4'-dinitro-stilbene urea peak area;

c'iS --- control solution of 4,4'-dinitro diphenyl urea -D8 average concentration, expressed in nanograms per milliliter (ng/mL);

--- try feed for X-4,4'-nitro-diphenyl urea residues are, in micrograms per kilogram (µg/kg);

V --- volume of the residue was dissolved in 75% aqueous methanol, in milliliters (mL);

m --- try supply feed mass in grams (g).

Note. The blank value should be subtracted from the results, expressed as the arithmetic mean of the measurement result measured parallel to three significant figures.

9.1 Sensitivity

The detection limit of the method was 0.5µg/kg, the limit of quantitation was 1µg/kg.

9.2 Accuracy

The method of the present egg sample is added in a concentration of 1µg/kg ~ 10µg/kg, chicken muscle sample concentration of water added in 1µg/kg ~ 300µg/kg on recovery levels of 80% to 120%.

9.3 Precision

The relative standard deviation of the method <=20%, inter-assay relative standard deviation <=20%.

Appendix A

Chromatogram

Description.

1 --- DNC wherein ion mass chromatogram (300.9 > 106.9);

2 --- DNC wherein ion mass chromatogram (300.9 > 136.9);

3 --- DNC-D8 wherein ion mass chromatogram (309.0 > 141.0);

4 --- Blank Organization ion chromatogram (TIC).

FIG A.1 4,4'- dinitro-stilbene urea standard solution and internal standard ion mass chromatogram characterized (4ng/mL)

Figure A.2 chicken muscle tissue sample blank wherein the ion mass chromatogram

Description.

1 --- DNC wherein ion mass chromatogram (300.9 > 106.9);

2 --- DNC wherein ion mass chromatogram (300.9 > 136.9);

3 --- DNC-D8 wherein ion mass chromatogram (309.0 > 141.0);

4 --- Blank Organization ion chromatogram (TIC).

Figure A.2 (continued)

Figure A.3 blank Chicken muscle-dinitro-4,4'-stilbene ureido wherein the sample and the internal standard ion mass chromatogram (2ng/g)

Description.

1 --- DNC wherein ion mass chromatogram (300.9 > 106.9);

2 --- DNC wherein ion mass chromatogram (300.9 > 136.9);

3 --- DNC-D8 wherein ion mass chromatogram (309.0 > 141.0);

4 --- Blank Organization ion chromatogram (TIC).

Figure A.3 (CONTINUED)