GB 29682-2013Determination of Penicillin residues in aquatic products by High Performance Liquid Chromatographic method (English PDF)

6.1 Preparation of the sample

To the scales of fish, peeled, taken along the back muscle tissue, muscle or skin take fish 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

Or less at -20 °C.

Determination Step 7

7.1 Preparation of standard curve

They were the precise amount of 10µg/mL penicillin G standard working solution and 10µg/mL oxacillin, dicloxacillin and nafcillin mixed Standard working solution combined amount, diluted with mobile phase to prepare a penicillin G concentration of 20,50,100,200,400,800 and 1600µg/L, oxacillin West Lin, dicloxacillin and nafcillin concentrations 100,200,400,800,1600,3200 and 6400µg/L series penicillins Mixed standard solution, 500 L depicting, by derivatization steps 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.

7.2 Extract

Sample Weigh 5g ± 0.05g, in 50mL centrifuge tube, acetonitrile was added 15mL, mix, add hexane 5mL, Vortex, 6000r/min centrifugal 15min, n-hexane layer was discarded, the lower layer was taken, to another centrifuge tube. The residue was combined with acetonitrile in 10mL, Hexane 5mL, Vortex, 6000r/min centrifugal 15min, n-hexane layer was discarded, the lower layer was twice combined, rotary evaporated at a water bath at 40 °C Dry, the residue was dissolved 5mL water, alternate.

7.3 Purification

HLB column eluted sequentially with methanol and 3mL 3mL water activation, taking stock solution through the column, 1mL rinsed with water, acetonitrile 3mL elution, Eluent to dry at 45 °C ~ 50 °C water bath with nitrogen. The residue was dissolved in mobile phase was 1.0mL, Vortex, spare.

7.4 Derivatization

500 L stock solution was accurately taken at 1.5mL polypropylene centrifuge tube, add 500 L derivatization reagent, Vortex, at 65 deg.] C water the reaction bath was 10min, rapid cooling on ice at 4 °C 10000r/min centrifugal 10min, the supernatant for HPLC assay.

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 Mobile phase. acetonitrile Mobile phase A (65 35, volume ratio).

7.5.1.3 flow rate. 1mL/min.

7.5.1.4 UV detection wavelength. 325nm.

7.5.1.5 Column temperature. 30 °C.

7.5.1.6 Injection volume. 50µL.

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 Liquid penicillins response value should be within the linear range of the detection instrument. In the chromatographic conditions, the standard solution and the blank test was added HPLC sample solution is given in Appendix A. FIG.

7.6 Blank test

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

8 and the result of the calculation expression Residues of the specimens Penicillins (µg/kg) according to formula (1).

A × cS × V

AS × m

Where.

--- for the respective X-feed penicillin try residual amount of drug, in micrograms per kilogram (µg/kg);

A --- sample solution corresponding penicillins derivative peak area;

Corresponding penicillins concentration cS --- standard working solution, in micrograms per liter (µg/L);

The total volume of the resulting solution was derivatized V ---, milliliters (mL);

--- the AS standard working solution in the corresponding penicillins derivative 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 limit of detection of penicillin G is 3µg/kg, the limit of quantitation of 10µg/kg, oxacillin, dicloxacillin, nafcillin the detection limit of 10µg/kg, the limit of quantitation of 50µg/kg.

9.2 Accuracy

This method of adding 10µg/kg ~ 600µg/kg on recovery levels of 70% 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 penicillins chromatogram of the standard solution

(Penicillin G20µg/L, oxacillin, dicloxacillin and nafcillin each of 100µg/L)

Figure A.2 carp muscle tissue sample blank chromatogram

Figure A.3 carp muscle tissue sample blank add penicillins chromatograms

(Penicillin G10µg/kg, oxacillin, dicloxacillin and nafcillin 50µg/kg)