GB 29694-2013Determination of Sulfonamides residues in animal derived food by High Performance Liquid Chromatographic method (English PDF)

6.1 Preparation of the sample

Fresh or thawed 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 5g ± 0.05g, polytetrafluoroethylene in 50mL centrifuge tubes, ethyl acetate was added 20mL, whirl 2min, 4000r/min Centrifuge 5min, supernatant to l00mL heart-shaped flask, the residue was added ethyl acetate 20mL, extraction was repeated once, the two extracts were combined.

7.2 Purification

Heart-shaped flask was added 0.1mol/L hydrochloric acid solution 4mL, concentrated by rotary evaporation to at least 40 °C 3mL, transferred to 10mL centrifuge tubes in. With 0.1mol/L hydrochloric acid solution was washed 2mL heart-shaped bottle, go in the same centrifuge tube. Heart-shaped flask and then washed 3mL hexane, n-hexyl Go alkoxy same centrifuge tube, vortexed 30s, 3000r/min centrifugal 5min, discarded hexane. Again washed with n-hexane heart-shaped 3mL Bottles, full turn in the same centrifuge tube, vortexed 30s, 3000r/min centrifugal 5min, discarded hexane, and the lower layer was reserved.

MCX column washed with methanol and 2mL 0.1mol/L hydrochloric acid solution 2mL activated, taking stock solution through the column, controlling the flow rate 1mL/min.

Sequentially with 0.1mol/L hydrochloric acid solution and 50% methanol 1mL 2mL acetonitrile, and eluted with 4 mL eluate, the eluate was collected, 40 °C dry nitrogen, 0.1% formic acid in acetonitrile was added to dissolve the residue 1.0mL, membrane filtration, high performance liquid chromatography for the determination.

7.3 Preparation of standard curve

The precise amount of 10µg/mL sulfonamides working standard solutions amount, diluted with 0.1% formic acid in acetonitrile, the concentration of formulated 10,50,100,250,500,2500 and 5000µg/L in a mixed series of standard solutions for HPLC assay. To the measured peak area 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 LC reference condition

7.4.1.1 Column. ODS-3C18 (250mm × 4.5mm, particle size 5µm), or equivalent person.

7.4.1.2 Mobile phase. 0.1% formic acid in acetonitrile, gradient elution shown in Table 1.

7.4.1.3 flow rate. 1mL/min.

7.4.1.4 Column temperature. 30 °C.

7.4.1.5 Detection wavelength. 270nm.

7.4.1.6 Injection Volume. 100µL.

Table 1 Mobile phase gradient elution

time

min

0.1% formic acid

Acetonitrile

0.0 8317

5.0 8317

10.0 80 20

22.3 60 40

22.4 10 90

30.0 10 90

31.0 8317

48.0 8317

7.4.2 Assay

Take a sample solution and a control solution corresponding, for single or multi-point calibration, by external standard method, the peak area is calculated. Sample solution and control solution Solution sulfonamides response value should be within the linear range of the detection instrument. In the chromatographic conditions, high control solution and the sample solution Performance liquid chromatography, see Appendix A. FIG.

7.5 Blank test

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

8 and the result of the calculation expression Residues in the specimens of sulfonamides according to formula (1).

Where.

--- for the residual amount of X-try the corresponding sulfonamides feed micrograms per kilogram (µg/kg);

Corresponding sulfonamides --- C concentration in the sample solution, in micrograms per milliliter (µg/mL);

The residue was dissolved --- V 0.1% formic acid in acetonitrile by volume 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 after the replicates, three significant figures.

9.1 Sensitivity

The detection limit of the method of pig and chicken muscle tissue was 5µg/kg, the limit of quantitation of 10µg/kg; detection limit of pig and chicken liver for 12µg/kg, the limit of quantitation of 25µg/kg.

9.2 Accuracy

Back to the method of muscle tissue in the 10µg/kg ~ 200µg/kg, in the liver tissue was added 25µg/kg ~ 200µg/kg concentration level the yield was 60% to 120%.

9.3 Precision

The relative standard deviation of the method <=l5%, inter-assay relative standard deviation <=20%.

Appendix A

Chromatogram

Description.

1 --- sulfacetamide;

2 --- sulfapyridine;

3 --- oxazole sulfonamides;

4 --- sulfamerazine;

5 --- sulfamethazine;

6 --- sulfamonomethoxine pyridazine;

7 --- benzoyl sulfonamide;

8 --- Sulfamonomethoxine;

Sulfa. 9 --- chloropyridazine;

10 --- sulfamethoxazole;

. 11 --- sulfisoxazole;

12 --- sulfadiazine methoxy pyridazine;

13 is --- pyrazole sulfonamides.

Figure A.1 chromatogram