GB 29686-2013Determination of Avilamycin residues in edible tissues of swine by Liquid Chromatography-tandem Mass Spectrometric method (English PDF)

6.1 Preparation of the sample

Fresh or frozen 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

Or less at -20 °C.

Determination Step 7

7.1 Extract

Muscle or fat was weighed sample 2g ± 0.05g, liver or kidney sample 1g ± 0.05g, in 15mL centrifuge tube, and acetone 4 mL, vortexed 5min, at 6000r/min centrifugal 10min, supernatant at 50mL round bottom flask. The residue was added acetone 4mL, the extraction was repeated twice, and the combined supernatant three times, at 60 deg.] C rotary evaporated to dryness, the residue was dissolved with 1mol/L sodium hydroxide solution, 4 mL, Go 50mL centrifuge tube, at 70 deg.] C water bath for 2h, extracted with 85% phosphoric acid adjusted to pH 1.0, was added 4 mL of ethyl acetate, were mixed and 6000r/min centrifugal 10min, supernatant to another centrifuge tube, was repeated twice, and the combined supernatant three times, plus 2µg/mL dichloromethoxy Benzoic acid 0.5 mL internal standard (except for the blank sample), the standby mix.

7.2 Purification

10mL ethyl acetate and extracted with an alumina column activated, taking stock solution passed through the column, 5mL each with n-hexane, ethyl acetate and methanol 5mL 5mL, and eluted twice with an eluent, 7 mL each, were collected eluent at 50 °C rotary evaporated to dryness, residue dissolved in methanol 1.0mL it was vortexed for liquid chromatography - tandem mass spectrometry.

7.3 Preparation of standard curve

The precise amount of 10µg/mL avilamycin marker residue amount DIA working standard solution, diluted with methanol, formulated at a concentration of 5, 50,100,200,400 and 800µg/L standard solution series for liquid chromatography - tandem mass spectrometry. Ion mass peak characteristics to surface Volume of the ordinate, the abscissa the concentration of the standard solution, the standard curve. Seeking regression equation and correlation coefficient.

7.4 Determination

7.4.1 LC Conditions

7.4.1.1 Column. C18 (150mm × 4.6mm, particle size 5µm), or equivalent person.

7.4.1.2 Mobile phase. methanol-water (50 50, volume ratio).

7.4.1.3 flow rate. 0.6mL/min.

7.4.1.4 Injection amount. 10µg/L.

7.4.1.5 Column temperature. 40 °C.

7.4.2 MS conditions

7.4.2.1 ion source. APCI.

7.4.2.2 Scan mode. negative ion scan.

7.4.2.3 Detection mode. selective reaction monitoring.

7.4.2.4 evaporation temperature. 400 °C.

7.4.2.5 Ion transfer tube temperature. 275 °C.

7.4.2.6 Sheath Gas Pressure. 25arb.

7.4.2.7 Auxiliary gas pressure. 5arb.

7.4.2.8 selected reaction monitoring optimization parameters in Table 1.

Table 1 avilamycin selected reaction monitoring of optimization parameters

Drug Name

Relative retention time min Parent ion Mass number Qualitative and ion collision energy eV Quantitative ion and collision energy eV Avilamycin Residues DIA marker 7.10 249 249 > 189 (23) 249 > 205 (16) 249 > 205 (16) Dicamba 8.80 219 219 > 159 (18) 219 > 145 (15) 219 > 175 (8)

7.4.3 Assay

7.4.3.1 Qualitative determination

By retention time of sample chromatogram with retention times of the respective standards, wherein each of the ion peaks and the corresponding concentration of the standard Wherein each of the ion peaks qualitative contrast. Sample and standard deviation of the relative retention time of not more than 5%; with a sample wherein ions Consistent with the abundance relative abundance fairly mixed standard solution concentration, relative abundance does not exceed a predetermined deviation Table 2, the sample can be stored is determined the respective analyte.

The maximum allowable deviation relative ion abundance qualitative determination of Table 2% the relative ion abundances > 50 20 ~ 50 10 ~ 20 <=10 Permissible relative deviation ± 20 ± 25 ± 30 ± 50 Ratio for the sample and standard solutions try quantitative ion area of single-point calibration for quantification.

7.4.3.2 Quantitative determination

Take a sample and standard solutions, the external standard method to avilamycin peak area, the standard solution and the sample solution response values are it should be in the linear range of the detection instrument. In the above-described chromatography - mass spectrometry under conditions avilamycin added standard solution and the blank sample solution Laid Intrinsic ion mass chromatogram given 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 Residues in the specimens avilamycin marker residue (DIA) according to formula (1).

A × cS × ci × ASi × V AS × cSi × Ai × m (1) where.

--- for the residual amount of X-try DIA compound, in units of micrograms per kilogram (µg/kg);

A --- The sample solution DIA peak area;

Standard solution concentration cS --- DIA, expressed in nanograms per milliliter (ng/mL);

--- CI concentration in the sample solution in the standard units of nanograms per milliliter (ng/mL);

Peak area of the internal standard solution ASi --- standard substance;

V --- the final volume of the sample solution, in milliliters (mL);

--- the AS standard solution of DIA peak area;

Concentration of a standard solution of cSi --- standard material, in units of nanograms per milliliter (ng/mL);

Peak area of the internal standard in the sample solution --- AI thereof;

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

This limit of detection in muscle and adipose tissue was 10µg/kg, the limit of quantitation of 20µg/kg; subject in liver and kidney tissues LOD was 20µg/kg, the limit of quantitation of 50µg/kg.

9.2 Accuracy

This method of adding 20µg/kg ~ 600µg/kg on recovery levels of 70% to 120%.

9.3 Precision

The relative standard deviation of the method <=20%, inter-assay relative standard deviation <=20%.

Appendix A

The ion mass chromatogram

Figure A.1 standard solution wherein ion mass chromatogram (DIA5µg/L, dicamba 1µg/mL)

Figure A.2 characterized in porcine kidney blank sample ion mass chromatogram

Figure A.3 pig kidney sample blank add avilamycin wherein ion mass chromatogram (50µg/kg)