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Determination of Albendazole and metabolites residues in aquatic products by High Performance Liquid Chromatographic method
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Basic data
| Standard ID | GB 29687-2013 (GB29687-2013) |
| Description (Translated English) | Determination of Albendazole and metabolites residues in aquatic products by High Performance Liquid Chromatographic method |
| Sector / Industry | National Standard |
| Classification of Chinese Standard | C53 |
| Classification of International Standard | 67.020 |
| Word Count Estimation | 11,170 |
| Quoted Standard | GB/T 6682; GB/T 1.1-2000 |
| Adopted Standard | GB/T 6682; GB/T 1.1-2000 |
| Regulation (derived from) | China Food & Drug Administration [2013] No. 234, November, 1, 2013 |
| Issuing agency(ies) | Ministry of Agriculture of the People's Republic of China, National Health and Family Planning Commission of the People's Republic of China |
| Summary | This standard specifies the Aquatic albendazole and metabolites (2 amino albendazole sulfone, albendazole sulfoxide, albendazole sulfone) residue detection sample preparation, high performance liquid phase chromatographic method. |
GB 29687-2013: Determination of Albendazole and metabolites residues in aquatic products by High Performance Liquid Chromatographic method
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Determination of Albendazole and metabolites residues in aquatic products by High Performance Liquid Chromatographic method
National Standards of People's Republic of China
National Food Safety Standard
Albendazole and its metabolites in aquatic products
Residues in HPLC
Published 2013-09-16
2014-01-01 implementation
Ministry of Agriculture, People's Republic of China
National Health and Family Planning Commission People's Republic of China released
National Food Safety Standard
Albendazole and its metabolites in aquatic products
Residues in HPLC
1 Scope
This standard specifies albendazole residues and metabolites (2-albendazole sulfone, sulfoxide albendazole, albendazole sulfone) in Aquatic
The method of sample preparation and determination by HPLC detection.
This standard applies to aquatic Residue albendazole and metabolites (2-albendazole sulfone, sulfoxide albendazole, albendazole sulfone)
Determination.
2 Normative references
The following documents for the application of this document is essential. For dated references, only applies to the version dated paper
Pieces. For undated references, the latest edition (including any amendments) applies to this document.
GB/T 6682 Water for analytical laboratory specifications and test methods
SC/T 3016 aquatic sampling
Principle 3
In a sample and the residual metabolite albendazole, extracted with ethyl acetate, hexane degreasing, back-extracted with ethyl acetate, HPLC -
Fluorescence detector, external standard.
4 Reagents and materials
The following reagents used, unless otherwise stated are analytical reagents, water as a water line with GB/T 6682 provisions.
4.1 Preparation of 2-amino-sulfone albendazole, albendazole sulfoxide, albendazole sulfone and albendazole reference. content of ≥99%.
4.2 hexane. chromatography.
4.3 Acetonitrile. chromatographically pure.
4.4 Methanol. HPLC grade.
4.5 ethyl acetate. chromatography.
4.6 dichloromethane. chromatography.
4.7 phosphoric acid.
4.8 twelve disodium hydrogen phosphate water.
4.9 heptane sulfonate. chromatography.
4.10 ammonium acetate.
4.11 20% methanol solution. Methanol 80mL, dissolved and diluted to 100mL with water.
4.12 0.05mol/L ammonium acetate solution. Take ammonium acetate 3.85g, dissolved and diluted with water to 1000mL.
4.13 0.04mol/L of sodium heptane - solution of phosphoric acid. phosphoric acid 2.7 mL take, mixing with water, added 8.08 g sodium heptane, dissolved in water and
Diluted to 1000mL.
4.14 3% phosphoric acid solution. Take phosphate 1.75mL, dissolve and dilute to 100mL with water.
4.15 0.02mol/L solution of disodium hydrogen phosphate. disodium-hydrogen phosphate 0.716 g, dissolve and dilute to 100mL with water, 3% phosphoric acid solution
PH was adjusted to 8.5, now with the current.
4.16 100μg/mL albendazole, albendazole sulfoxide, albendazole sulfone, and 2-amino albendazole sulfone standard stock solution. Weigh accurately A
Of benzene, albendazole albendazole sulfoxide and sulfones of each amino-acid and 10mg 11.5mg albendazole sulfone, respectively in 100mL
Flask, dissolved in methanol and diluted to the mark, formulated at a concentration of 100μg/mL standard stock solution. -18 ℃ below from light, there is
Valid for three months.
4.17 working standard solutions. were the precise amount of 100μg/mL2- amino albendazole sulfone, sulfoxide albendazole, albendazole sulfone, and A
Standard stock solution of benzene yl amount, in the same flask, dissolve with 80% methanol solution and dilute to volume, formulated at concentrations of 2-amino
Albendazole sulfone 1.0μg/mL, albendazole sulfoxide 2.0μg/mL, albendazole sulfone 0.2μg/mL and albendazole 5.0μg/mL of
Working standard solutions. 2 ℃ ~ 8 ℃ dark storage period of one month.
5 instruments and equipment
5.1 HPLC. with a fluorescence detector.
5.2 Analytical balance. a sense of volume 0.00001g.
5.3 Balance. a sense of the amount of 0.01g.
5.4 homogenizer.
5.5 centrifuge.
5.6 rotary evaporator.
5.7 Nitrogen blowing instrument.
5.8 pear-shaped bottle. 100mL.
5.9 Membrane. organic phase, 0.45μm.
Preparation and Storage of sample 6
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 ℃.
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 ℃ 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 ℃ 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 ℃, 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 ℃ 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 ℃.
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, 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).
X =
cS × A × V × 1000
AS × m
(1)
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)
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