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GB 29693-2013: Determination of Halofuginone residues in animal derived food by High Performance Liquid Chromatographic method
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Determination of Halofuginone residues in animal derived food by High Performance Liquid Chromatographic method
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GB 29693-2013
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Basic data
| Standard ID | GB 29693-2013 (GB29693-2013) |
| Description (Translated English) | Determination of Halofuginone residues in animal derived food by High Performance Liquid Chromatographic method |
| Sector / Industry | National Standard |
| Classification of Chinese Standard | C53 |
| Classification of International Standard | 67.020 |
| Word Count Estimation | 10,156 |
| 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 animal-derived food halofuginone residue detection sample preparation, HPLC methods. This standard applies to chicken muscle and liver tissue halofuginone residues detected. |
GB 29693-2013: Determination of Halofuginone residues in animal derived food by High Performance Liquid Chromatographic method
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Determination of Halofuginone residues in animal derived food by High Performance Liquid Chromatographic method
National Standards of People's Republic of China
National Food Safety Standard
Determination Changshan residues in animal foods ketone
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 Changshan residues in animal foods ketone
High performance liquid chromatography
1 Scope
This standard specifies the sample preparation and high performance liquid chromatographic method in Animal Food Residues of halofuginone.
Detection of the residual amount of halofuginone This standard applies to the chicken muscle and liver tissues.
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
Halofuginone remaining in a sample, by trypsin digestion, extracted with ethyl acetate, liquid-liquid extraction, an HLB, purified by high performance liquid chromatography - Purple
Determination outside, 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 halofuginone hydrobromide reference. content ≥99.6%.
4.2 Methanol. HPLC grade.
4.3 ethyl acetate. chromatography.
4.4 Acetonitrile. chromatographically pure.
Anhydrous sodium carbonate 4.5.
4.6 Trypsin. 1000U/mg.
4.7 ammonium acetate.
4.8 acetic acid.
4.9 toluene.
4.10 HLB solid phase extraction column. 500mg/6mL, or equivalent person.
4.11 0.25mol/L ammonium acetate buffer. Take 19.27 g ammonium acetate, 30mL acetic acid was added, dissolved and diluted to 1000mL with water, with ethyl
Acid adjusted to pH 4.3.
4.12 0.125mol/L ammonium acetate buffer. Take 0.25mol/L ammonium acetate buffer 500mL, dissolved in water and diluted to 1000mL.
4.13 10% sodium carbonate solution. anhydrous sodium carbonate 10g, dissolve and dilute to 100mL with water.
4.14 mobile phase. acetonitrile take 250mL, 0.25mol/L ammonium acetate buffer 150mL and 600 mL of water, mixing, pH was adjusted with glacial acetic acid
To 4.3, membrane filtration, now with the current.
4.15 200μg/mL halofuginone standard stock solution. Weigh accurately halofuginone hydrobromide standard 10mg, brown in 50mL flask with
0.25mol/L ammonium acetate buffer solution and diluted to the mark, formulated at a concentration of 200μg/mL standard stock solution of halofuginone. seal,
Wrapped in aluminum foil, 2 ℃ ~ 8 ℃ from light, valid for 6 months.
4.16 10μg/mL standard stock solution halofuginone. precise amount of 200μg/mL standard stock solution 0.5mL halofuginone brown amount in 10mL
Flask, dissolve and dilute to volume with water, is formulated at a concentration of 10μg/mL standard stock solution halofuginone. Sealed, wrapped in aluminum foil, 2 ℃ ~
8 ℃ dark preservation, valid for six months.
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 centrifuge.
5.5 homogenizer.
5.6 a vortex.
5.7 rotary evaporator.
5.8 Nitrogen blowing instrument.
5.9 SPE.
5.10 constant temperature water bath.
5.11 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
Or less at -20 ℃.
Determination Step 7
7.1 extract
Weigh sample 4g ± 0.04g, in 100mL centrifuge tube, add trypsin 50mg, water was added 2 mL (liver) or 10mL water
(Muscle tissue), vortex 1min, washed with 10% sodium carbonate solution to adjust the pH to 8.0, enzymatic 3h water bath at 40 ℃. Removed, returned to room temperature, was added
10% sodium carbonate solution 2mL, vortexed 1min, combined with ethyl acetate 20mL, whirl 10s, 3min allowed to stand in an ice water bath, immediately
2000r/min centrifugal 2min, 100mL supernatant was transferred to a separatory funnel. Layer liquid extracted with ethyl acetate 20mL repeated once,
Two supernatant were combined in the same separatory funnel, add 0.125mol/L ammonium acetate solution 10mL, shake for 1min, standing 2min, the collection
The aqueous phase layer. Coupled 0.125mol/L ammonium acetate solution 10mL, extraction was repeated once, the two aqueous phases were combined, transferred to heart-shaped flask, at 38 ℃
Rotary evaporator, the residue divisible ethyl acetate and set aside.
7.2 Purification
HLB 3mL column successively with methanol, water and 3mL 0.125mol/L ammonium acetate 3mL activation, taking stock solution through the column, the flow rate control
1mL/min, washed with toluene and 2mL, squeezed, eluting with 3 mL of methanol, collect the eluate, dry nitrogen at 38 ℃, mobile phase
The residue was dissolved 1.0mL, mix, membrane filtration, for HPLC.
7.3 Preparation of matrix-matched standard curve
Halofuginone standard stock solution amount precise amount of 10μg/mL, and dissolved in methanol and diluting the concentration of 50,100,300,
500 and 1000μg/L standard solution series, from each 1.0 mL, were added to the extracted, the purification step of processing the blank sample eluate
In a water bath at 38 ℃ nitrogen blow, the residue was dissolved with 1.0mL flow for HPLC. To the measured peak area
Ordinate, corresponding to the concentration of standard solution as abscissa, the standard curve. Seeking regression equation and correlation coefficient.
7.4 Determination
7.4.1 Chromatographic conditions
7.4.1.1 Column. C18 (150mm × 3.9mm, particle size 4μm), or equivalent person.
7.4.1.2 mobile phase. acetonitrile -0.25mol/L ammonium acetate buffer - water.
7.4.1.3 flow rate. 1.0mL/min.
7.4.1.4 Column temperature. 30 ℃.
7.4.1.5 Detection wavelength. 243nm.
7.4.1.6 Injection volume. 25μL.
7.4.2 Assay
Take a sample solution and a control solution corresponding, for single or multi-point calibration, by external standard method, the peak area is calculated. Sample solution and control solution
Liquid halofuginone response value should be within the linear range of the detection instrument. In the chromatographic conditions, high performance liquid control solution and the sample solution
FIG chromatography are given in 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
The specimens halofuginone residual quantity according to formula (1).
X =
c × V
(1)
Where.
--- for the residual amount of X-try feed halofuginone, in units of micrograms per kilogram (μg/kg);
C --- halofuginone concentration in the sample solution, in micrograms per milliliter (μg/mL);
V --- The residue was dissolved in mobile phase volume in milliliters (mL);
m --- try supply feed mass in grams (g).
NOTE. The results should be subtracted from the value of the blank sample, Results are expressed as the arithmetic mean of measured parallel to three significant figures.
9 detection sensitivity, accuracy and precision
9.1 Sensitivity
The method detection limit of 25μg/kg; limit of quantification was 50μg/kg.
9.2 Accuracy
This method of adding 50μg/kg ~ 500μg/kg on recovery levels of 80% to 110%.
9.3 Precision
The relative standard deviation of the method ≤15%, inter-assay relative standard deviation ≤20%.
Appendix A
Chromatogram
Figure A.1 halofuginone chromatogram standard solution (100ng/mL)
Figure A.2 Chicken liver tissue sample blank chromatogram
Figure A.3 blank Chicken liver tissue sample chromatogram halofuginone added (100ng/g)
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