GB 29691-2013Determination of Nicarbazin residues in edible tissues of chicken by High Performance Liquid Chromatographic method (English PDF)

6.1 Preparation of the sample

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 Extract

Sample Weigh 2.0g ± 0.02g, in 50mL centrifuge tube, acetonitrile was added 10mL, ultrasonic 5min, 4000r/min centrifugal 12min, the supernatant, heart-shaped flask in 50mL. The residue in acetonitrile was added 10mL, extraction was repeated once, the two supernatants were combined, added 3 mL of n-propanol, 60 °C rotary evaporated to near dryness. Was added 0.5 mL of acetonitrile, hexane 1mL, vortexed 3min, dissolved, to move with a plug 10mL centrifuge tube. With B Nitriles hexane 1mL 0.5mL repeated vortexing and once dissolved. The combined solution twice to 10mL stoppered centrifuge tube, was added n-hexane 2mL, vortex mixed 3min, 4000r/min centrifugal 5min, discarded the upper n-hexane solution. Coupled with hexane 2mL, extraction was repeated once, 4000r/min centrifugal 5min, the lower layer was taken and set aside.

7.2 Purification

C18 column with acetonitrile 10mL activated, taking stock solution through the column, the natural flow of dry, filtrate was collected. 4mL with the eluent, the eluate the filtrate and eluate were combined, rotary evaporated at 60 deg.] C to near dryness, the residue was dissolved in mobile phase was 2.0mL, membrane filtration, high performance liquid chromatography for Determination.

7.3 Preparation of standard curve

The precise amount of 100µg/mL4,4'- dinitro-stilbene amount of urea working standard solution, diluted with mobile phase, formulated at a concentration of 31.25, 62.5,125,250,500,1000,2000,4000 and 8000µg/L of the series of standard solution for HPLC assay. With measured Peak area for the vertical axis, corresponding to the concentration of standard solution as abscissa, the standard curve. Seeking regression equation and correlation coefficient.

7.4 Determination

7.4.1 Chromatographic conditions

7.4.1.1 Column. C18 (250mm × 4.6mm, particle size 5µm), or equivalent person.

7.4.1.2 Mobile phase. aqueous acetonitrile (5842, volume ratio).

7.4.1.3 flow rate. 1.0mL/min.

7.4.1.4 Detector. UV detector.

7.4.1.5 wavelength. 340nm.

7.4.1.6 Column temperature. 30 °C.

7.4.1.7 Injection volume. 20µL.

7.4.2 Assay

Take a sample solution and standard solutions corresponding, for single or multi-point calibration, by external standard method, the peak area is calculated. Standard solution and sample solution Solution of 4,4'-dinitro-stilbene urea response value should be within the linear range of the detection instrument. In the chromatographic conditions, the standard solutions and the empty White sample was added a sample solution by HPLC are given in Appendix A. FIG.

7.5 Blank test

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

Calculation and Expression of Results 8 the specimens were 4,4-dinitro diphenyl urea residues according to formula (1).

A × cS × V

AS × m

Where.

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

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

cS --- standard solution of 4,4'-dinitro-stilbene urea concentration, in micrograms per liter (µg/L);

V --- volume of the mobile phase the residue was dissolved in units of milliliters (mL);

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

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 in parallel, to retain the three decimal places.

9.1 Sensitivity

The detection limit of the method was 20µg/kg, the limit of quantitation of 100µg/kg.

9.2 Accuracy

This method of adding 100µg/kg ~ 400µg/kg on recovery levels of 70% to 110%.

9.3 Precision

The relative standard deviation of the method <=15%, inter-assay relative standard deviation <=20%.

Appendix A

Chromatogram

FIG A.1 4,4'- dinitro-stilbene chromatogram urea standard solution (200µg/L)

Figure A.2 chicken kidney tissue sample blank chromatogram

Figure A.3 blank Chicken kidney-dinitro-4,4'-stilbene urea sample chromatogram (200µg/L)