GB 29695-2013Determination of Avermectin and Ivermectin 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 Preparation of standard curve

The precise amount of 1µg/mL abamectin and ivermectin working standard solutions amount, diluted with methanol to prepare a concentration of 0.1, 0.2, 0.5 and 2µg/mL of the series of standard solution from each centrifuge tube 0.1mL to 5mL, blowing at 45 °C ~ 55 °C water bath nitrogen Dryness, treated according to the derivatization step. For HPLC. 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.

7.2 Extract

Sample Weigh 5g ± 0.05g, in 50mL polypropylene centrifuge tube, acetonitrile was added 15mL, vortexed 1min, ultrasonic 30min, 4000r/min centrifugal 5min, supernatant to another 50mL polypropylene centrifuge tube. The residue was added acetonitrile 10mL, extraction was repeated Once, the combined two extracts. In the extract was added hexane 10mL, vortexed thoroughly mixed 1min, 4000r/min centrifugal 5min, discarded the upper hexane layer. The combined hexane extracts 10mL, extraction was repeated once, hexane layer was discarded, the standby.

7.3 Purification

Take stock solution, successively dried over anhydrous sodium over basic alumina column and a solid phase extraction column activated 10mL of acetonitrile, the filtrate was collected. Be filtered the flow to do, rinsed with acetonitrile tube 10mL, transferred over anhydrous sodium sulfate and basic alumina column solid phase extraction, drying, and the combined filtrates in the eggplant-shaped flask, at 40 °C ~ 50 °C rotary evaporated to dryness. The residue was dissolved in acetonitrile 3mL 2 times, and transferred to a glass stoppered 5mL Centrifuge tubes, 45 °C ~ 55 °C water bath blown dry with nitrogen.

7.4 Derivatization

To the centrifuge tube was added 5mL derivatization reagents A100µL, stopper tightly, vortexed 30s, derivatizing reagents B plus

150 L, the stopper tightly, vortexed 30s, sealed dark, rt derived 15min, add 750 L of methanol, vortexed 30s, filtered, for HPLC.

7.5 Determination

7.5.1 Chromatographic conditions

7.5.1.1 Column. C18 (250mm × 4.6mm, particle size 5µm), or equivalent person.

7.5.1.2 flow rate. 1.5mL/min.

7.5.1.3 Injection volume. 20µL.

7.5.1.4 Column temperature. 35 °C.

7.5.1.5 detector. excitation wavelength 365nm, emission wavelength 475nm.

7.5.1.6 elution gradient shown in Table 1.

Table 1 Table elution gradient

time

min

Methanol

Acetonitrile

0.4% acetic acid

7.5.2 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 Ivermectin and Abamectin solution shall be within the linear response range of the detection instrument. In the chromatographic conditions, the standard solution and blank HPLC sample solution was added in Appendix A. FIG.

7.6 Blank test

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

9.1 Sensitivity

The method detection limit of 2µg/kg, the limit of quantitation of 4µg/kg.

9.2 Accuracy

This method of adding 4µg/kg ~ 20µg/kg on recovery levels of 70% to 120%.

9.3 Precision

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

Appendix A

Chromatogram

Figure A.1 abamectin and ivermectin chromatogram of the standard solution (100ng/mL)

Figure A.2 tilapia musculature blank sample chromatogram

Figure A.3 tilapia musculature add abamectin and ivermectin blank sample chromatogram (2µg/kg)

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