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GB 29691-2013: Determination of Nicarbazin residues in edible tissues of chicken by High Performance Liquid Chromatographic method
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GB 29691-2013: Determination of Nicarbazin residues in edible tissues of chicken by High Performance Liquid Chromatographic method

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Determination of Nicarbazin residues in edible tissues of chicken by High Performance Liquid Chromatographic method National Standards of People's Republic of China National Food Safety Standard Determination of nicarbazin residues in edible tissues of chicken High performance liquid chromatography Published 2013-09-16 2014-01-01 implementation Ministry of Agriculture, People's Republic of China National Health and Family Planning Commission People's Republic of China released National Food Safety Standard Determination of nicarbazin residues in edible tissues of chicken High performance liquid chromatography

1 Scope

This standard specifies the edible chicken tissues nicarbazin identified residue of 4,4'-stilbene-nitro urea Residues high and sample preparation Performance liquid chromatographic method. This standard applies to chicken muscle, liver and kidney tissues identified nicarbazin residue nitro stilbene 4,4' urea Residues Determination.

2 Normative references

The following documents for the application of this document is essential. For dated references, only applies to the version dated paper Pieces. For undated references, the latest edition (including any amendments) applies to this document. GB/T 6682 Water for analytical laboratory specifications and test methods Principle 3 Remaining in a sample of 4,4'-dinitro diphenyl urea were extracted with acetonitrile, hexane degreasing, C18 column purification eluted aqueous acetonitrile, efficient Liquid chromatography, external standard.

4 Reagents and materials

The following reagents used, unless otherwise indicated, are analytical reagents, water is in line with a water GB/T 6682 provisions. 4.1 4,4-dinitro-stilbene urea reference. content of ≥99%. 4.2 N, N- dimethylformamide. 4.3 Acetonitrile. chromatographically pure. 4.4 Methanol. HPLC grade. 4.5 hexane. chromatography. 4.6 n-propanol. 4.7 C18 solid phase extraction column. 500mg/3mL, or equivalent person. 4.8 Eluent. acetonitrile take 70mL, dissolve and dilute to 100mL with water. 4.9 1mg/mL4,4'- dinitro diphenyl ureas are standard stock solution. Weigh accurately 4,4'-dinitro-stilbene urea reference 25mg, in 25mL volumetric flask, dissolve and dilute to volume with dimethylformamide, prepared at a concentration of 4,4-dinitro 1mg/mL are two Phenylurea standard stock solution. -20 ℃ below, valid for six months. 4.10 100μg/mL4,4'- urea-dinitro-stilbene standard working solution. precise amount of 1mg/mL4,4'- standard urea-dinitro-stilbene reservoir 1.0 mL was prepared, in 10mL volumetric flask, dissolved in methanol and diluted to the mark, formulated at a concentration of 4,4-dinitro 100μg/mL of Stilbene urea working standard solution. The following 2 ℃ ~ 4 ℃, valid for 1 week. 5. Apparatus 5.1 HPLC. with UV detector. 5.2 Analytical balance. a sense of volume 0.00001g. 5.3 Balance. a sense of the amount of 0.01g. 5.4 homogenizer. 5.5 rotary evaporator. 5.6 vortex mixer. 5.7 ultrasonic cleaning. 5.8 centrifuge. 5.9 heart-shaped bottle. 50mL. 5.10 stoppered centrifuge tube. 10mL, 50mL. 5.11 Membrane. organic phase, 0.45μm. Preparation of sample material 6 and stored 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 ℃. 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 ℃ 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 ℃. 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). X = A × cS × V AS × m (1) 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 sensitivity detection method, accuracy and precision 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) ...