GB 29693-2013Determination of Halofuginone 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

Weigh sample 4g ± 0.04g, in 100mL centrifuge tube, add trypsin 50mg, water was added 2 mL (liver) or 10mL water (Muscle tissue), vortex 1min, washed with 10% sodium carbonate solution to adjust the pH to 8.0, enzymatic 3h water bath at 40 °C. Removed, returned to room temperature, was added 10% sodium carbonate solution 2mL, vortexed 1min, combined with ethyl acetate 20mL, whirl 10s, 3min allowed to stand in an ice water bath, immediately 2000r/min centrifugal 2min, 100mL supernatant was transferred to a separatory funnel. Layer liquid extracted with ethyl acetate 20mL repeated once, Two supernatant were combined in the same separatory funnel, add 0.125mol/L ammonium acetate solution 10mL, shake for 1min, standing 2min, the collection the aqueous phase layer. Coupled 0.125mol/L ammonium acetate solution 10mL, extraction was repeated once, the two aqueous phases were combined, transferred to heart-shaped flask, at 38 °C Rotary evaporator, the residue divisible ethyl acetate and set aside.

7.2 Purification

HLB 3mL column successively with methanol, water and 3mL 0.125mol/L ammonium acetate 3mL activation, taking stock solution through the column, the flow rate control 1mL/min, washed with toluene and 2mL, squeezed, eluting with 3 mL of methanol, collect the eluate, dry nitrogen at 38 °C, mobile phase the residue was dissolved 1.0mL, mix, membrane filtration, for HPLC.

7.3 Preparation of matrix-matched standard curve

Halofuginone standard stock solution amount precise amount of 10µg/mL, and dissolved in methanol and diluting the concentration of 50,100,300, 500 and 1000µg/L standard solution series, from each 1.0 mL, were added to the extracted, the purification step of processing the blank sample eluate in a water bath at 38 °C nitrogen blow, the residue was dissolved with 1.0mL flow for HPLC. To the measured peak area Ordinate, 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 (150mm × 3.9mm, particle size 4µm), or equivalent person.

7.4.1.2 mobile phase. acetonitrile -0.25mol/L ammonium acetate buffer - water.

7.4.1.3 flow rate. 1.0mL/min.

7.4.1.4 Column temperature. 30 °C.

7.4.1.5 Detection wavelength. 243nm.

7.4.1.6 Injection volume. 25µL.

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 Liquid halofuginone response value should be within the linear range of the detection instrument. In the chromatographic conditions, high performance liquid control solution and the sample solution FIG chromatography are given in Appendix A.

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 the specimens halofuginone residual quantity according to formula (1).

Where.

--- for the residual amount of X-try feed halofuginone, in units of micrograms per kilogram (µg/kg);

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

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

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

NOTE. The results should be subtracted from the value of the blank sample, Results are expressed as the arithmetic mean of measured parallel to three significant figures.

9.1 Sensitivity

The method detection limit of 25µg/kg; limit of quantification was 50µg/kg.

9.2 Accuracy

This method of adding 50µg/kg ~ 500µg/kg on recovery levels of 80% to 110%.

9.3 Precision

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

Appendix A

Chromatogram

Figure A.1 halofuginone chromatogram standard solution (100ng/mL)

Figure A.2 Chicken liver tissue sample blank chromatogram

Figure A.3 blank Chicken liver tissue sample chromatogram halofuginone added (100ng/g)