GB 29709-2013Determination of Azaperone and metabolite in animal derived food by High Performance Liquid Chromatographic method (English PDF)

6.1 Preparation of the sample

Fresh or frozen tissue white, the 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, a centrifuge tube, acetonitrile was added 10mL, vortexed 1min, ultrasonic 2min, 5000r/min centrifugal 5min, the supernatant, the residue was added 10mL of acetonitrile extraction was repeated once, the two supernatants were combined. 40 °C rotary evaporated to dryness in a water bath. use 2% formic acid were dissolved 5mL aqueous residue was added 4 mL of n-hexane, mixed, 5000r/min centrifugal 5min, n-hexane layer was discarded, the standby.

7.2 Purification

MCX cartridge 3mL successively with methanol, water and 2% aqueous formic acid 3mL 3mL activation, taking stock solution through the column, the flow rate is less than the control 1mL/min, with 2% aqueous formic acid and methanol 3mL 3mL rinsed, drained, washed with 5% ammoniated methanol 4mL. The eluent was collected in 40 °C nitrogen blow, the residue was dissolved in mobile phase was 1.0mL, Vortex for HPLC.

7.3 Preparation of standard curve

The precise amount of 1µg/mL working standard solutions amount, diluted with methanol, and formulated to a concentration of 10,20,50,100,200,500 1000µg/L mixed standard solution series. Each concentration was repeated 3 times for HPLC, to measure the average peak area obtained for the vertical Coordinates, the corresponding standard concentration as the abscissa, the standard curve. Seeking regression equation and correlation coefficient.

7.4 Determination

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. acetonitrile 5mmol/L ammonium acetate solution (50 50, volume ratio).

7.4.1.3 flow rate. 1mL/min.

7.4.1.4 UV detection wavelength. 250nm.

7.4.1.5 Column temperature. 40 °C.

7.4.1.6 Injection volume. 50µL.

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 Azapirone and liquid nitrogen piperidin alcohols response value should be within the linear range of the detection instrument. In the chromatographic conditions, the standard solution and blank add HPLC chromatograms of the sample solution in Appendix A.

7.5 Blank test

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

Calculation and Expression of Results 8 the specimens azapirone alcohol residues and piperazine nitrogen (µg/kg) according to formula (1) to (3) Calculated.

Single-point calibration.

cSA

Calibration or standard curve by. AS = acS b to obtain a and b, then c = Ab (2) X = cV (3) where.

C --- try to supply feed concentration in the solution and the appropriate azapirone metabolite, in micrograms per liter (µg/L);

cS --- standard working solution in a concentration corresponding azapirone and metabolites, in micrograms per liter (µg/L);

A --- area of the respective sample azapirone and metabolite peaks;

--- the AS standard working solution in the corresponding area of the azapirone and metabolite peaks;

--- for X-try respective feed azapirone and metabolite residues micrograms per kilogram (µg/kg);

V --- The residue was dissolved in mobile phase volume 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 2µg/kg, the limit of quantitation of 5µg/kg.

9.2 Accuracy

This method in the muscle, tissue sebum 5µg/kg ~ 60µg/kg, liver and kidney tissues at 5µg/kg ~ 100µg/kg addition concentration on recovery levels of 60% to 110%.

9.3 Precision

<=20% relative standard deviation in this method and inter - batch relative standard deviation of <=20%.

Appendix A

Chromatogram

Figure A.1 azapirone and metabolite standards solution chromatogram (10µg/L)

Figure A.2 in pig muscle blank sample chromatogram

Figure A.3 in pig muscle and blank add azapirone metabolite sample chromatogram (5µg/kg)