GB 29684-2013Determination of Erythromycin residues in aquatic products by Liquid Chromatography-tandem Mass Spectrometric method (English PDF)

6.1 Preparation of the sample

Product water test blank or edible 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

-20 °C below Save to 3 months.

Determination Step 7

7.1 Extract

Sample Weigh 5g ± 0.05g, in 50mL centrifuge tube, was added 200ng/mL erythromycin isotope internal standard working standard solution 50 L, Acetonitrile was added 15mL, immediately dispersed with stirring, ultrasonic 5min, shaking 5min, 4000r/min centrifugal 10min, supernatant, the other Centrifuge tube, the residue was added acetonitrile 5mL, extraction was repeated once, the two supernatants were combined, and sodium chloride in hexane 2g 15mL, shaking 5min, 4000r/min centrifugal 10min, the upper n-hexane solution was discarded, the lower layer was taken, in 100mL eggplant-shaped flask, to rotary evaporation at 40 °C Dry with phosphate buffer solution 5mL, dissolve the residue, hexane was added 5mL, mixing 1min, transferred to centrifuge tubes, 6000r/min centrifugal 5min, n-hexane layer was discarded, together with n-hexane 5mL, the operation is repeated once, and the lower layer was set aside.

7.2 Purification

5mL HLB column successively with methanol, water, phosphate buffered saline 5mL and 5mL activated, taking stock solution through the column with phosphate buffer Washed with 5mL centrifuge tube, the washing solution through the column, the stream of dry, washed with water 5mL methanol and 5mL of 40% was rinsed, drained, washed with methanol 8mL , Collecting eluate, in the eggplant-shaped flask, at 40 °C rotary evaporation to dryness, with 30% 1.0mL residue was dissolved in methanol, filter through Filter for liquid chromatography - tandem mass spectrometry.

7.3 Preparation of standard curve

The precise amount of 5µg/mL erythromycin standard working solution and 200ng/mL erythromycin isotope internal standard working standard solution amount, with 30% methanol solution and dissolving and diluting the standard Erythromycin isotopic concentration of 10ng/mL and the concentration of erythromycin 1,5,10, 25, 50 and 500ng/mL standard solution series for liquid chromatography - tandem mass spectrometry. A characteristic mass ion peak area 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 × 2.1mm, particle size 5µm), or equivalent person.

7.4.1.2 Mobile phase. A. methanol, B. 0.01 per mille aqueous acetic acid.

7.4.1.3 gradient elution. 0.0min ~ 1.0min, 30% A; 1.01min ~ 7min, 95% A; 7.01min ~ 17min; 30% A.

7.4.1.4 flow rate. 0.3mL/min.

7.4.1.5 Column temperature. 25 °C.

7.4.1.6 Injection volume. 25µL.

7.4.2 MS conditions

7.4.2.1 ion source. electrospray ionization source.

7.4.2.2 Scan mode. positive ion scan.

7.4.2.3 Detection mode. multiple reaction monitoring.

7.4.2.4 Spray voltage. 4700V.

7.4.2.5 ion transfer capillary temperature. 300 °C.

The collision-induced dissociation voltage source 7.4.2.6. 8V.

7.4.2.7 atomizing gas flow rate. 12.3L/h.

7.4.2.8 auxiliary gas flow rate. 1.7L/h.

7.4.2.9 qualitative, quantitative and collision energy of ions shown in Table 1.

Table 1 Qualitative and quantitative and collision energy of ions

The target compound

Qualitative ion pair m/z Quantitative ion pair m/z Collision energy eV Erythromycin 734.4 > 157.9 734.4 > 576.0 734.4 > 157.9 Erythromycin parity Internal standard prime 736.5 > 159.9 736.5 > 159.9 30

7.4.3 Assay

7.4.3.1 Qualitative determination

By retention time of sample chromatogram with retention times corresponding to the standard, characteristic peaks corresponding to the ion concentration of the standard color Characterized in contrast to qualitative ion peaks. Sample and standard deviation of the relative retention time of not more than 5%; relative abundance of ions in a sample wherein Consistent with the relative abundance of a considerable concentration of the standard solution, the relative abundance of the deviation does not exceed ± 20%, may be present in the sample is determined corresponding Was measured.

7.4.3.2 Quantitative determination

Take a sample and standard solutions, by external standard method, in order to quantify the peak area, the standard solution and sample solution erythromycin response value should be at the linear range of the detection equipment. In the above-described chromatography - mass spectrometry under conditions erythromycin standard solution added in the blank and wherein a sample solution ion mass Amount chromatogram 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 Erythromycin residues in a sample according to formula (1).

Where.

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

C --- erythromycin sample solution concentration unit nanograms per milliliter (ng/mL);

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

m --- try supply feed mass in grams (g).

Note. The blank value should be subtracted from the calculation results. Results are expressed as the arithmetic mean of the two measured parallel to three significant figures.

9.1 Sensitivity

The detection limit of the method was 0.5µg/kg, the limit of quantitation was 1µg/kg.

9.2 Accuracy

This method of adding 1µg/kg ~ 400µg/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

The ion mass chromatogram Description.

1 --- erythromycin wherein ion mass chromatogram (734.4 > 575.9);

2 --- erythromycin wherein ion mass chromatogram (734.4 > 159.9);

3 --- erythromycin isotopic ion mass chromatogram characteristic (736.50 > 159.9).

Figure A.1 erythromycin Erythromycin isotope standard and standard solution wherein ion mass chromatogram (25ng/mL)

Description.

1 --- erythromycin wherein ion mass chromatogram (734.4 > 575.9);

2 --- erythromycin wherein ion mass chromatogram (734.4 > 159.9);

3 --- erythromycin isotopic ion mass chromatogram characteristic (736.50 > 159.9).

Figure A.2 Grass edible tissue sample blank wherein the ion mass chromatogram

Description.

1 --- erythromycin wherein ion mass chromatogram (734.4 > 575.9);

2 --- erythromycin wherein ion mass chromatogram (734.4 > 159.9);

3 --- erythromycin isotopic ion mass chromatogram characteristic (736.50 > 159.9).

Figure A.3 Grass edible tissue sample blank add features erythromycin ion mass chromatogram (5µg/kg)

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