Search result: GB 29682-2013
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Determination of Penicillin residues in aquatic products by High Performance Liquid Chromatographic method
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GB 29682-2013
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| Standard ID | GB 29682-2013 (GB29682-2013) | | Description (Translated English) | Determination of Penicillins 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 | 10,175 | | Quoted Standard | GB/T 6682; GB/T 1.1-2000 | | Adopted Standard | GB/T 6682; GB/T 1.1-2000 | | 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 residues of penicillin drug testing sample preparation, high performance liquid chromatography method given. This standard applies to edible fish tissue penicillin G, oxacillin residues of drugs detection. |
GB 29682-2013
Determination of Penicillins residues in aquatic products by High Performance Liquid Chromatographic method
National Standards of People's Republic of China
National Food Safety Standard
Determination of residues in aquatic Penicillin
High performance liquid chromatography
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
Determination of residues in aquatic Penicillin
High performance liquid chromatography
1 Scope
This standard specifies the sample preparation and high performance liquid chromatographic method Seafood by penicillins residues detected.
This standard applies to edible fish tissues penicillin G, oxacillin, dicloxacillin, and nafcillin single or multiple drug residue
Allowance of detection.
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 sample of residual penicillin drugs, extracted with acetonitrile, an HLB column purified, 1,2,4-triazole derivatives and mercuric chloride solution, high performance liquid
Chromatography - UV determination, external standard.
4 Reagents and Materials
The following reagents used, unless otherwise stated are analytical reagents, water should be consistent with a water GB/T 6682 provisions.
4.1 penicillin G, oxacillin, dicloxacillin, and nafcillin Standard. content ≥98%.
4.2 methanol.
4.3 acetonitrile.
Sodium thiosulfate pentahydrate 4.4.
Sodium dihydrogen phosphate dihydrate 4.5.
Disodium hydrogen phosphate anhydrous 4.6.
4.7 ammonium sulfate.
4.8 sodium hydroxide.
4.9 n-hexane.
4.10 1,2,4-triazole.
4.11 mercuric chloride (Ⅱ).
4.12 HLB solid phase extraction column. 60mg/3mL, or equivalent person.
4.13 mobile phase A. sodium thiosulfate pentahydrate 3.9g, dissolved in water, adding sodium dihydrogen phosphate dihydrate 10.2g, disodium hydrogen phosphate anhydrous
4.9g, ammonium sulfate 6.8g, dissolved in water and diluted to 1000mL, membrane filtration.
4.14 mercuric chloride (Ⅱ) solution. Take mercuric chloride (Ⅱ) 0.27 g of, dissolved and diluted to 10mL with water, now with the current.
4.15 5mol/L sodium hydroxide solution. take sodium hydroxide 20g, dissolve and dilute to 100mL with water.
4.16 derivatizing reagents. 13.78 g 1,2,4-triazole-taking, 60mL water was dissolved, the solution was added mercuric chloride 10mL, with 5mol/L hydroxide
Sodium solution was adjusted to pH 9.0, diluted with water to 100mL. 2 ℃ ~ 8 ℃ dark storage period of three months.
4.17 1mg/mL Penicillin standard stock solution. Weigh accurately penicillin G, oxacillin, dicloxacillin, and nafcillin
The standards for each 10mg, respectively 10mL volumetric flask, dissolve and dilute to volume with water, is formulated at a concentration of 1mg/mL penicillin
G, oxacillin, dicloxacillin, and nafcillin standard stock solution, aliquoted, immediately stored in the dark at -20 deg.] C or less, a period
month. Repeated freezing and thawing should not use.
4.18 10μg/mL penicillin G standard working solution. precise amount of 1mg/mL penicillin G standard stock solution 1.0mL, in 100mL
Volumetric flask, dissolve and dilute to volume with water, is formulated at a concentration of 10μg/mL penicillin G standard working solution and packaging, to immediately at -20 ℃
Stored in the dark, valid for one month. Repeated freezing and thawing should not use.
4.19 10μg/mL oxacillin, dicloxacillin and nafcillin working standard solutions. precise amount of 1mg/mL West oxacillin
Lin, dicloxacillin and nafcillin 1.0 mL of each standard stock solution, to a 100mL volumetric flask, dissolve and dilute to volume with water, formulated
At a concentration of 10μg/mL oxacillin, dicloxacillin and nafcillin working standard solutions, dispensing, to immediately at -20 ℃
Stored in the dark, valid for one month. Repeated freezing and thawing should not use.
5. Apparatus
5.1 HPLC. with UV detector.
5.2 Analytical balance. a sense of volume 0.00001g.
5.3 Balance. a sense of the amount of 0.01g.
5.4 freezing high-speed centrifuge.
5.5 temperature water bath.
5.6 vortex mixer.
5.7 SPE.
5.8 tissue homogenizer.
5.9 nitrogen blow device.
5.10 centrifuge tube. 50mL.
5.11 filter. 0.45μm.
Preparation and Storage of sample 6
6.1 Preparation of the sample
To the scales of fish, peeled, taken along the back muscle tissue, muscle or skin take fish 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 ℃.
Determination Step 7
7.1 Preparation of standard curve
They were the precise amount of 10μg/mL penicillin G standard working solution and 10μg/mL oxacillin, dicloxacillin and nafcillin mixed
Standard working solution combined amount, diluted with mobile phase to prepare a penicillin G concentration of 20,50,100,200,400,800 and 1600μg/L, oxacillin West
Lin, dicloxacillin and nafcillin concentrations 100,200,400,800,1600,3200 and 6400μg/L series penicillins
Mixed standard solution, 500 L depicting, by derivatization steps for HPLC assay. Peak area was measured to the vertical axis, corresponding to the standard
Standard solution concentration as the abscissa, the standard curve. Seeking regression equation and correlation coefficient.
7.2 extract
Sample Weigh 5g ± 0.05g, in 50mL centrifuge tube, acetonitrile was added 15mL, mix, add hexane 5mL, Vortex,
6000r/min centrifugal 15min, n-hexane layer was discarded, the lower layer was taken, to another centrifuge tube. The residue was combined with acetonitrile in 10mL,
Hexane 5mL, Vortex, 6000r/min centrifugal 15min, n-hexane layer was discarded, the lower layer was twice combined, rotary evaporated at a water bath at 40 ℃
Dry, the residue was dissolved 5mL water, alternate.
7.3 Purification
HLB column eluted sequentially with methanol and 3mL 3mL water activation, taking stock solution through the column, 1mL rinsed with water, acetonitrile 3mL elution,
Eluent to dry at 45 ℃ ~ 50 ℃ water bath with nitrogen. The residue was dissolved in mobile phase was 1.0mL, Vortex, spare.
7.4 Derivatization
500 L stock solution was accurately taken at 1.5mL polypropylene centrifuge tube, add 500 L derivatization reagent, Vortex, at 65 deg.] C water
The reaction bath was 10min, rapid cooling on ice at 4 ℃ 10000r/min centrifugal 10min, the supernatant for HPLC assay.
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 Mobile phase. acetonitrile Mobile phase A (65 35, volume ratio).
7.5.1.3 flow rate. 1mL/min.
7.5.1.4 UV detection wavelength. 325nm.
7.5.1.5 Column temperature. 30 ℃.
7.5.1.6 Injection volume. 50μL.
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
Liquid penicillins response value should be within the linear range of the detection instrument. In the chromatographic conditions, the standard solution and the blank test was added
HPLC sample solution is given in Appendix A. FIG.
7.6 Blank test
But without addition of the sample, the same steps employed in parallel operation.
8 and the result of the calculation expression
Residues of the specimens Penicillins (μg/kg) according to formula (1).
X =
A × cS × V
AS × m
(1)
Where.
--- for the respective X-feed penicillin try residual amount of drug, in micrograms per kilogram (μg/kg);
A --- sample solution corresponding penicillins derivative peak area;
Corresponding penicillins concentration cS --- standard working solution, in micrograms per liter (μg/L);
The total volume of the resulting solution was derivatized V ---, milliliters (mL);
--- the AS standard working solution in the corresponding penicillins derivative peak area;
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 test method sensitivity, accuracy and precision
9.1 Sensitivity
The limit of detection of penicillin G is 3μg/kg, the limit of quantitation of 10μg/kg, oxacillin, dicloxacillin, nafcillin
The detection limit of 10μg/kg, the limit of quantitation of 50μg/kg.
9.2 Accuracy
This method of adding 10μg/kg ~ 600μg/kg on recovery levels of 70% to 110%.
9.3 Precision
≤20% relative standard deviation in this method and inter - batch relative standard deviation of ≤20%.
Appendix A
Chromatogram
Figure A.1 penicillins chromatogram of the standard solution
(Penicillin G20μg/L, oxacillin, dicloxacillin and nafcillin each of 100μg/L)
Figure A.2 carp muscle tissue sample blank chromatogram
Figure A.3 carp muscle tissue sample blank add penicillins chromatograms
(Penicillin G10μg/kg, oxacillin, dicloxacillin and nafcillin 50μg/kg)
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