GB 29683-2013Determination of Paracetamol residues in animal derived food by High Performance Liquid Chromatographic method (English PDF)

6.1 Preparation of the sample

Fresh or frozen blank or test tissue, fascia removed, 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

Sample Weigh 5g ± 0.05g, centrifuge tube with stopper, ethyl acetate was added 20mL vortex 2min, 6000r/min centrifugal 15min, the supernatant, in another centrifuge tube, the residue was added ethyl acetate 10mL, extraction was repeated once, the two supernatants were combined, 6000r/min from Heart 10min, the supernatant was evaporated to dryness in a water bath 45 °C, the residue was dissolved 3mL methanol, n-hexane was added 5mL, mixed, allowed to stand points Layer, hexane layer was discarded, the lower layer was taken, dried with nitrogen at 45 °C water bath, dissolve the residue was washed with water 6mL, 10000r/min centrifugal 10min, the supernatant was set aside.

7.2 Purification

HLB column eluted sequentially with methanol and 3mL 3mL water activation, taking stock solution through the column, controlling the flow rate of less than 2mL/min. 3mL water 5% methanol and 3mL rinse, 4 mL methanol, the eluate was collected, dried at 40 °C ~ 45 °C water bath nitrogen gas, the mobile phase 1.0mL the residue was dissolved, 10000r/min centrifuge 5min, the supernatant for HPLC.

7.3 Preparation of standard curve

The precise amount of 10µg/mL working solution of paracetamol standard amount, diluted with mobile phase, concentration of formulated 10,25,50,100, 200,400,800 and 1600µg/L of the series of standard solution for HPLC assay. Peak area was measured to the vertical axis, corresponding to the standard 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 (250mm × 4.6mm, particle size 5µm), or equivalent person.

7.4.1.2 Mobile phase. methanol 0.05mol/L ammonium acetate solution (20 80, volume ratio).

7.4.1.3 flow rate. 1.0mL/min.

7.4.1.4 UV detection wavelength. 250nm.

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, external standard method with peak area calculations. Standard solution and sample solution Acetaminophen in response shall be within the linear range of the detection instrument. In the chromatographic conditions, sample was added standard solution and blank HPLC chromatogram of 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 in a sample of the residual amount of acetaminophen drug according to formula (1).

X = A × cS × VAS × m (1) where.

--- for X-residue amount of the phenol compound to try drugs acetylamino micrograms per kilogram (µg/kg);

A --- sample solution acetaminophen peak area;

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

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

--- the AS standard working solution acetaminophen peak area;

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 of 3µg/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

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

Appendix A

Chromatogram

Figure A.1 acetaminophen chromatogram of the standard solution (10µg/L)

Figure A.2 in pig muscle blank sample chromatogram

Figure A.3 white pig muscle Add acetaminophen sample chromatogram (10µg/kg)