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Determination of Avermectin and Ivermectin residues in aquatic products by High Performance Liquid Chromatographic method
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Basic data
| Standard ID | GB 29695-2013 (GB29695-2013) |
| Description (Translated English) | Determination of Avermectin and Ivermectin 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 | 10,129 |
| 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 Avermectin, Ivermectin Residues sample preparation, high-performance liquid chromatographic method. This standard applies to edible fish tissue such as drugs avermectin residues detected. |
GB 29695-2013: Determination of Avermectin and Ivermectin residues in aquatic products by High Performance Liquid Chromatographic method
---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.
Determination of Avermectin and Ivermectin residues in aquatic products by High Performance Liquid Chromatographic method
National Standards of People's Republic of China
National Food Safety Standard
Aquatic products in the avermectin and ivermectin and more
HPLC residues
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
Aquatic products in the avermectin and ivermectin and more
HPLC residues
1 Scope
This standard specifies the sample preparation and high performance liquid chromatographic method aquatic avermectin and ivermectin residues.
Abamectin and ivermectin detecting residual amounts of edible fish is applicable to the organization's.
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
Principle 3
The specimens remaining avermectin and ivermectin, extracted with acetonitrile, degreasing hexane, basic alumina column purification, N- methylimidazole, and
Trifluoroacetic anhydride derivatization, high performance liquid chromatography - fluorescence determination, external standard.
4 Reagents and Materials
The following reagents used, unless otherwise stated were of analytical reagent; water as a water line with GB/T 6682 provisions.
4.1 avermectin standard. B1a content of ≥94%. Ivermectin Standard. B1a content of ≥92%.
4.2 Acetonitrile. chromatographically pure.
4.3 Methanol. HPLC grade.
4.4 n-hexane.
4.5 trifluoroacetic anhydride.
4.6 N- methylimidazole.
4.7 acetic acid. excellent pure.
4.8 over anhydrous sodium. 650 ℃ drying 4h, cool in a desiccator for use.
4.9 SPE cartridge of basic alumina. 1g/3mL, or equivalent person.
4.10 derivatizing agent A. N- methyl imidazole take 1mL, acetonitrile 1mL, mix, now with the current.
4.11 derivatizing agent B. trifluoroacetic anhydride take 1mL, acetonitrile 2mL, mix, now with the current.
4.12 0.4% acetic acid solution. 0.4mL harvest acid, dissolve and dilute to 100mL with water.
4.13 100μg/mL abamectin and ivermectin mixed standard stock solution. Weigh accurately abamectin and ivermectin each standard
10mg, in 100mL flask, dissolved in methanol and diluted to the mark, formulated at a concentration of 100μg/mL of Ivermectin and Abamectin
Element mixed standard stock solution. -18 ℃ below, valid for six months.
4.14 10μg/mL abamectin and ivermectin working standard solutions. precise amount of 100μg/mL abamectin and ivermectin mixed
Combined standard stock solution 10mL, in 100mL volumetric flask, dilute to the mark with methanol, formulated at a concentration of 10μg/mL abamectin and Iraq
Ivermectin working standard solutions. 2 ℃ ~ 8 ℃ storage period of three months.
5 Apparatus
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 vortex mixer.
5.5 ultrasonic cleaning.
5.6 centrifuge.
5.7 Nitrogen blowing instrument.
5.8 rotary evaporator.
5.9 SPE.
5.10 stopper glass centrifuge tube. 5mL.
5.11 over anhydrous sodium Column. Glass column (φ10mm × 150mm, No. 2 sand core) was charged with 5g of anhydrous sodium sulfate.
5.12 eggplant-shaped bottle. 50mL.
5.13 stopper polypropylene centrifuge tube. 50mL.
5.14 filter. 0.45μm.
Preparation and Storage of sample 6
6.1 Preparation of the sample
Fresh or thawed take appropriate 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
-18 ℃ below saved.
Determination Step 7
7.1 Preparation of standard curve
The precise amount of 1μg/mL abamectin and ivermectin working standard solutions amount, diluted with methanol to prepare a concentration of 0.1,
0.2, 0.5 and 2μg/mL of the series of standard solution from each centrifuge tube 0.1mL to 5mL, blowing at 45 ℃ ~ 55 ℃ water bath nitrogen
Dryness, treated according to the derivatization step. For HPLC. To measure peak area for the vertical axis, corresponding to the concentration of standard solution of abscissas
Standard, the standard curve. Seeking regression equation and correlation coefficient.
7.2 extract
Sample Weigh 5g ± 0.05g, in 50mL polypropylene centrifuge tube, acetonitrile was added 15mL, vortexed 1min, ultrasonic 30min,
4000r/min centrifugal 5min, supernatant to another 50mL polypropylene centrifuge tube. The residue was added acetonitrile 10mL, extraction was repeated
Once, the combined two extracts. In the extract was added hexane 10mL, vortexed thoroughly mixed 1min, 4000r/min centrifugal 5min, discarded
The upper hexane layer. The combined hexane extracts 10mL, extraction was repeated once, hexane layer was discarded, the standby.
7.3 Purification
Take stock solution, successively dried over anhydrous sodium over basic alumina column and a solid phase extraction column activated 10mL of acetonitrile, the filtrate was collected. Be filtered
The flow to do, rinsed with acetonitrile tube 10mL, transferred over anhydrous sodium sulfate and basic alumina column solid phase extraction, drying, and the combined filtrates
In the eggplant-shaped flask, at 40 ℃ ~ 50 ℃ rotary evaporated to dryness. The residue was dissolved in acetonitrile 3mL 2 times, and transferred to a glass stoppered 5mL
Centrifuge tubes, 45 ℃ ~ 55 ℃ water bath blown dry with nitrogen.
7.4 Derivatization
To the centrifuge tube was added 5mL derivatization reagents A100μL, stopper tightly, vortexed 30s, derivatizing reagents B plus
150 L, the stopper tightly, vortexed 30s, sealed dark, rt derived 15min, add 750 L of methanol, vortexed 30s, filtered,
For HPLC.
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 flow rate. 1.5mL/min.
7.5.1.3 Injection volume. 20μL.
7.5.1.4 Column temperature. 35 ℃.
7.5.1.5 detector. excitation wavelength 365nm, emission wavelength 475nm.
7.5.1.6 elution gradient shown in Table 1.
Table 1 Table elution gradient
time
min
Methanol
Acetonitrile
0.4% acetic acid
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
Ivermectin and Abamectin solution shall be within the linear response range of the detection instrument. In the chromatographic conditions, the standard solution and blank
HPLC sample solution was added in Appendix A. FIG.
7.6 blank test
But without addition of the sample, the same steps employed in parallel operation.
8 results calculated
Residues avermectin or ivermectin test compound according to formula (1).
X =
c × V
(1)
Where.
--- for the respective X-try feed avermectin or ivermectin remaining amount micrograms per kilogram (μg/kg);
Corresponding concentration of avermectin or ivermectin C --- sample solution, in units of nanograms per milliliter (ng/mL);
V --- total volume of sample solution, in milliliters (mL);
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 after the replicates, three significant figures.
9 method sensitivity, accuracy and precision
9.1 Sensitivity
The method detection limit of 2μg/kg, the limit of quantitation of 4μg/kg.
9.2 Accuracy
This method of adding 4μg/kg ~ 20μg/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
Chromatogram
Figure A.1 abamectin and ivermectin chromatogram of the standard solution (100ng/mL)
Figure A.2 tilapia musculature blank sample chromatogram
Figure A.3 tilapia musculature add abamectin and ivermectin blank sample chromatogram (2μg/kg)
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