GB 29681-2013Determination of Levamisole residues in milk by High Performance Liquid Chromatographic method (English PDF)

6.1 Preparation of the sample

An appropriate amount of fresh or frozen or blank test milk, homogeneous mixing.

--- 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, a centrifuge tube, carbonate buffer was added 5mL, ethyl acetate was added 10mL, mix, 6000r/min Centrifugal 10min, the supernatant was eggplant-shaped flask, together with 10mL ethyl acetate and extracted once, twice combined supernatants rotating at 50 °C water bath Evaporated to dryness, the residue dissolved in carbonate buffer was 5mL standby.

7.2 Purification

3mL C18 column successively with water, methanol and 3mL 3mL activated carbonate buffer, taking stock solution through the column, 3mL water rinsed, dried A Alcohol 5mL, collecting eluate, dry nitrogen at 50 deg.] C water bath, the residue was dissolved in mobile phase was 1.0mL, membrane filtration, high performance liquid supply Phase Chromatography.

7.3 Preparation of standard curve

Accurately taken 10µg/mL working standard solution amount levamisole, diluted with mobile phase to prepare a concentration of 10,20,50,100,200, 400 and 800µg/L series of working standard solution for HPLC assay. Peak area was measured to the vertical axis, corresponding to the standard solution Concentration as the abscissa, the standard curve. Seeking regression equation and correlation coefficient.

7.4 Determination

7.4.1 LC Conditions

7.4.1.1 Column. C18 (l50mm × 4.6mm, particle size 5µm), or equivalent person.

7.4.1.2 Mobile phase. 0.02mol/L sodium dihydrogen phosphate buffer solution of diethylamine in methanol (70 30, volume ratio).

7.4.1.3 flow rate. 1mL/min.

7.4.1.4 Detection wavelength. 220nm.

7.4.1.5 Injection volume. 50µL.

7.4.1.6 Column temperature. 30 °C.

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 Liquid levamisole response value should be within the linear range of the detection instrument. In the chromatographic conditions, sample was added standard solution and blank solution HPLC liquid 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 Residue (µg/kg) in a sample according to the class of levamisole formula (1).

X = A × cS × VAS × m (1) where.

--- for X-try levamisole feed residues, micrograms per kilogram (µg/kg);

A --- peak area of sample solution levamisole;

cS --- standard working solution concentration of levamisole, micrograms per liter (µg/L);

V --- The residue was dissolved in mobile phase volume in milliliters (mL);

--- the AS standard working solution of the peak area of levamisole;

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 2.5µg/kg, the limit of quantitation of 5µg/kg.

9.2 Accuracy

This method of adding 5µg/kg ~ 20µg/kg on recovery levels of 70% to 110%.

9.3 Precision

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

Appendix A

Chromatogram

Figure A.1 levamisole chromatogram standard solution (20µg/L)

Figure A.2 milk blank sample chromatogram

Figure A.3 milk sample blank add levamisole chromatogram (20µg/kg)