GB 29689-2013Determination of Thiamphenicol 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 and mix well.

--- 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 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, in 50mL centrifuge tubes, ethyl acetate was added 20mL, oscillation 10min, 4000r/min centrifugal 5min, the supernatant in the heart-shaped flask, the residue was added ethyl acetate 20mL, extraction was repeated once, the two extracts were combined, rotating at 45 °C Evaporated to near dryness, water was added to a 5mL flask heart-shaped, so that the ultrasonic 5min fully dissolved, transferred to 50mL centrifuge tubes. Add water to chicken 5mL After repeated dissolution vial heart go same centrifuge tube. 20mL hexane plus shaking 5min, 4000r/min centrifugal 2min, and the lower layer Liquid reserve.

7.2 Purification

C18 column eluted sequentially with acetonitrile and water 5mL 5mL activation. Take stock solution through the column, controlling the flow rate 1mL/min, squeezed. 5mL acetonitrile , Collecting eluate, dry nitrogen at 45 °C, the mobile phase the residue was dissolved 1.0mL, membrane filtration, for HPLC.

7.3 Preparation of standard curve

The precise amount of 10µg/mL thiamphenicol appropriate amount of working standard solution, diluted with mobile phase to prepare a concentration of 20,50,125,250 and 500µg/L of the series of standard solution for HPLC assay. To measure peak area for the vertical axis, the standard solution concentration corresponding to a cross Axis, the standard curve. Seeking regression equation and correlation coefficient.

7.4 High Performance Liquid Chromatography

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 (20 80, by volume), filtered through a filter membrane prior to use ultrasound.

7.4.1.3 flow rate. 1.0mL/min.

7.4.1.4 Detection wavelength. 225nm.

7.4.1.5 Column temperature. 30 °C.

7.4.1.6 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 Thiamphenicol fluid response of the instrument to be within the linear range of detection. In the chromatographic conditions, the standard solution and the blank test tissue was added FIG HPLC sample solution 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 Residues thiamphenicol the specimens according to formula (1).

Where.

--- for X-try Residues feed thiamphenicol micrograms per kilogram (µg/kg);

--- C concentration in the sample solution thiamphenicol, in units of nanograms per milliliter (ng/mL);

V --- volume of the mobile phase the residue was dissolved 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 measured parallel to three significant figures.

9.1 Sensitivity

The detection limit of the method was 10µg/kg, the limit of quantitation of 10µg/kg.

9.2 Accuracy

This method of adding 10µg/kg ~ 100µg/kg on recovery levels of 70% to 110%.

9.3 Precision

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

Appendix A

Chromatogram

Figure A.1 thiamphenicol chromatogram of the standard solution (50µg/L)

Figure A.2 milk blank sample chromatogram

Figure A.3 milk sample blank add thiamphenicol chromatogram (10µg/kg)