GB 29702-2013Determination of Trimethoprim residues in aquatic products 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

-18 °C below saved.

Determination Step 7

7.1 Extract

Sample Weigh 5g ± 0.05g, in 50mL stoppered centrifuge tube, add chloroform 15mL, methanol 14mL, 0.1mol/L sulfuric acid solution Solution 6mL, vortexed 2min, 4000r/min centrifugal 3min, 150mL supernatant to a separatory funnel. Methanol was added to the residue and 14mL 0.1mol/L sulfuric acid solution, 6 mL, extraction was repeated once, the two supernatants were combined in a separatory funnel, was added 2mol/L hydroxide Solution of potassium 2mL, dichloromethane 30mL, shake for 2min, standing layer, the lower layer was taken to the eggplant-shaped flask, added to a separatory funnel with dichloromethane 30mL extraction was repeated once, the lower two combined solution at 40 °C rotary evaporated to near dryness, washed with 5% acetic acid solution to dissolve the residue 6mL, spare.

7.2 Purification

MCX cation exchange column washed with methanol and 5% acetic acid solution 6mL 6mL activated, taking stock solution passed through the column, washed with 5% acetic acid solution and 6mL 6mL methanol, and eluted with ammonia in methanol 15mL, at 40 °C rotary evaporated to near dryness and dissolved with mobile phase 1.0mL the residue was filtered through a filter for the Determination.

7.3 Preparation of standard curve

The precise amount of trimethoprim working standard solution amount, diluted with mobile phase to prepare a concentration of 50,200,500,1000,2000, 5000µg/L standard solutions for HPLC assay. To measure peak area for the vertical axis, corresponding to the concentration of standard solution of abscissas Standard, the standard curve. Seeking regression equation and correlation coefficient.

7.4 Determination

7.4.1 Chromatographic conditions

7.4.1.1 Column. ZORBAX-C18 column (4.6mm, particle size 5µm 250mm ×), or equivalent person.

7.4.1.2 Mobile phase. 0.5% perchloric acid methanol solution (30 70, volume ratio).

7.4.1.3 flow rate. 1.0mL/min.

7.4.1.4 Column temperature. 35 °C.

7.4.1.5 Injection volume. 20µL.

7.4.1.6 Detection wavelength. 230nm.

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 Trimethoprim response was linear within the range of values to be detected by the 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.

9.1 Sensitivity

The limit of quantification was 20µg/kg.

9.2 Accuracy

This method of adding 20µg/kg ~ 1000µg/kg on recovery levels of 70% to 110%.

9.3 Precision of the method

<=15% relative standard deviation in this method and inter - batch relative standard deviation of <=15%.

Appendix A

Chromatogram

Figure A.1 trimethoprim chromatogram of the standard solution (200µg/kg)

Figure A.2 in Muscle tissue sample blank chromatogram

Figure A.3 in Muscle tissue sample blank add chromatogram trimethoprim (200µg/kg)