GB 29687-2013Determination of Albendazole and metabolites residues in aquatic products by High Performance Liquid Chromatographic method (English PDF)

6.1 Preparation of the sample

An appropriate amount of fresh or frozen fish, to the scales, peeled, taken along the back muscles; shrimp, to the head, peeled, taking part of the muscle. 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 Shrimp, crabs

7.1.1 Extract

Sample Weigh 2g ± 0.02g, in 50mL centrifuge tubes, ethyl acetate was added 15mL, homogeneous 30s, shaking 5min, 4000r/min Centrifugal 10min, the supernatant was 100mL pear-shaped flask, the residue was set aside. Another a 50mL centrifuge tubes, ethyl acetate was added 15mL, washed Homogenizer 30s, the residue in the washing liquid, shaken 5min, 4000r/min centrifugal 10min, the supernatant was combined to 100mL pear shaped flask, at 35 °C rotary evaporated to dryness and 1.0mL 20% methanol solution was dissolved the residue.

7.1.2 Degreasing

The above solution was transferred to 10mL centrifuge tube, again pear-shaped flask was added n-hexane 1mL, washed with n-hexane into a centrifuge tube, vibration Swing 1min, 4000r/min centrifugal 5min, n-hexane layer was discarded, and then n-hexane was added 1mL, operation was repeated twice, and the lower layer was reserved.

7.1.3 Purification

Stock solution was added 0.04mol/L phosphoric acid 1mL, mix, add 1.5 mL of dichloromethane, mixed 1min, 4000r/min centrifugal 5min, Methylene chloride was taken in 10mL centrifuge tube, together with 1.5 mL of dichloromethane, extraction was repeated twice, three times with dichloromethane were combined in 10mL Centrifuge tube, 35 °C dry nitrogen; with 0.02mol/L of disodium hydrogen phosphate solution 1.0mL dissolve the residue, ethyl acetate was added 2mL, mixed Together 1min, 4000r/min centrifugal 5min, ethyl acetate was transferred to another 10mL centrifuge tube, followed by adding disodium hydrogen phosphate was added Ethyl acetate 2mL, extraction was repeated twice, ethyl acetate three times were combined, dried nitrogen at 40 °C, with 20% methanol - water 1.0mL residue was dissolved Retentate was filtered through a filter, for liquid chromatography.

7.1.4 Preparation of matrix-matched standard solution

Weigh 5 parts blank sample 2g ± 0.02g, in 50mL centrifuge tube, working standard solutions were added and 0,10,25,100

200 L, mixing, extraction by grease removal, purification steps, the substrate concentration of standard solution are. 2-amino albendazole sulfone 0, 0.01, 0.025,0.1 and 0.2µg/mL, and albendazole sulfoxide 0,0.02,0.05,0.2 0.4µg/mL, albendazole sulfone 0,0.002,0.005, 0.02 and 0.04µg/mL, and albendazole 0,0.05,0.125,0.5 1µg/mL, for high performance liquid chromatography. To the measured peak area Ordinate, corresponding to the concentration of standard solution as abscissa, the standard curve. Seeking regression equation and correlation coefficient.

7.2 Fish

7.2.1 Extract

Sample Weigh 2g ± 0.02g, in 50mL centrifuge tubes, ethyl acetate was added 15mL, homogeneous 30s, shaking extraction 5min, 4000r/min centrifugal 10min, the supernatant was 100mL pear-shaped flask, the residue was set aside. Another a 50mL centrifuge tube was added ethyl acetate 15mL, washing homogenizer 30s, this residue was poured into the above-described shaking extraction 5min, 4000r/min centrifugal 10min, the supernatant was combined to 100mL pear-shaped flask by rotary evaporation to dryness, was added 1.0mL 20% methanol solution of the residue was dissolved in 35 °C under reduced pressure.

7.2.2 Degreasing

The above solution was transferred to 10mL centrifuge tube, again pear-shaped flask was added n-hexane 1mL, washed with n-hexane into a centrifuge tube, shaken 1min, 4000r/min centrifugal 5min, n-hexane layer was removed, hexane was added 1mL, law operation was repeated twice. Lower solution through the organic phase 0.45µm filter for liquid chromatography.

7.2.3 Preparation of standard solution mixed matrix

Weigh 5 parts blank sample 2g ± 0.02g, in 50mL centrifuge tube, working standard solutions were added 10,25,100 and 200 L, Mixing, by extraction, degreasing steps, the substrate concentration of standard solution are. 2-amino albendazole sulfone and 0,0.01,0.025,0.1 0.2µg/mL, and albendazole sulfoxide 0,0.02,0.05,0.2 0.4µg/mL, and albendazole sulfone 0,0.002,0.005,0.02 0.04µg/mL, and albendazole 0,0.05,0.125,0.5 1µg/mL, for high performance liquid chromatography. In the measured peak area of the vertical axis, Standard solution concentration corresponding to the abscissa, the standard curve. Seeking regression equation and correlation coefficient.

7.3 Determination

7.3.1 Chromatographic conditions

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

7.3.1.2 Detection wavelength. excitation wavelength. 290nm, emission wavelength. 320nm.

7.3.1.3 Column temperature. 30 °C.

7.3.1.4 flow rate. 1.0mL/min.

7.3.1.5 Injection volume. 30µL.

7.3.1.6 Mobile phase. gradient program in Table 1.

Table 1 gradient elution program

time

min

Acetonitrile

Methanol

0.05mol/L ammonium acetate 0.00 10882 30.0 401 743 32.0 502 030 40.0 502 030 40.1 10882 45.0 10882

7.3.2 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 albendazole, sulfoxide, albendazole sulfoxide and 2-amino albendazole sulfone response value should be within the linear range of the detection instrument.

In the chromatographic conditions, the standard solution and the blank of FIG tissue HPLC sample solution was added respectively in Appendix A.

7.4 Blank test

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

8 and the result of the calculation expression Test compound and metabolite albendazole residues according to formula (1).

cS × A × V × 1000

AS × m

Where.

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

--- matrix cS standard solution concentration of standard solution and the corresponding metabolite albendazole, in micrograms per milliliter (µg/mL);

A --- the sample solution and the metabolite albendazole corresponding peak area;

V --- sample constant volume in milliliters (mL);

Matrix --- the AS standard solution and the peak area of the metabolite albendazole respective standard solution;

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 The method of sensitivity, accuracy and precision

9.1 Sensitivity

The detection limit of the method. albendazole is 10µg/kg, 2- amino albendazole sulfone 2.5µg/kg, albendazole sulfoxide 5µg/kg, A Reached yl phenyl sulfone 0.5µg/kg.

The limit of quantification of the method. albendazole is 25µg/kg, 2- amino albendazole sulfone 5µg/kg, albendazole sulfoxide 10µg/kg, A Reached yl phenyl sulfone 1µg/kg.

9.2 Accuracy

This method of albendazole in 25µg/kg ~ 500µg/kg, 2- amino albendazole sulfone 5µg/kg ~ 100µg/kg, sub albendazole Sulfone 10µg/kg ~ 200µg/kg albendazole sulfone and recoveries in the level of concentration 1µg/kg ~ 20µg/kg 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

Description.

Albendazole sulfone. 1 --- 2-amino;

2 --- albendazole sulfoxide;

3 --- albendazole sulfone;

4 --- albendazole.

Figure A.1 its metabolite albendazole mixed standard solution chromatogram

(Albendazole 0.05µg/mL, 2- amino albendazole sulfone 0.01µg/mL, albendazole sulfoxide 0.02µg/mL and albendazole sulfone 0.0024µg/mL)

Figure A.2 carp muscle tissue sample blank chromatogram

Description.

Albendazole sulfone. 1 --- 2-amino;

2 --- albendazole sulfoxide;

3 --- albendazole sulfone;

4 --- albendazole.

Figure A.3 carp muscle tissue sample blank add albendazole and metabolites chromatogram

(Albendazole 62.5µg/kg, 2- amino albendazole 12.5µg/kg, albendazole sulfoxide 25µg/kg, albendazole sulfone 2.5µg/kg)