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Determination of Sulfonamides residues in animal derived food by High Performance Liquid Chromatographic method
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Basic data
| Standard ID | GB 29694-2013 (GB29694-2013) |
| Description (Translated English) | Determination of Sulfonamides 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 | 10,146 |
| 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 sulfa drug residues detected sample preparation, high-performance liquid chromatographic method. This standard applies to pig, chicken muscle, liver tissue the detection of drug residues. |
GB 29694-2013: Determination of Sulfonamides residues in animal derived food 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 Sulfonamides residues in animal derived food by High Performance Liquid Chromatographic method
National Standards of People's Republic of China
National Food Safety Standard
Animal food 13 sulfonamides 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
Animal food 13 sulfonamides residues in
HPLC
1 Scope
This standard specifies the sample preparation and high performance liquid chromatographic method in animal food sulfonamides of Residues.
Sulfacetamide muscle and liver tissue This standard applies to the pigs and chickens, sulfapyridine, sulfanilamide oxazole, sulfamerazine, sulfadiazine
Methyl pyrimidine, pyridazine sulfamonomethoxine, sulfanilamide benzoyl, sulfamonomethoxine, sulfanilamide chloropyridazine, sulfamethoxazole, sulfisoxazole, sulfamethazine
Pyridazine and oxygen detection pyrazole sulfonamides single or a plurality of drug residues.
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
Sulfonamides residues in a sample, and extracted with ethyl acetate, 0.1mol/L hydrochloric acid solution was solvent switched, hexane degreasing, MCX column
Purification by high performance liquid chromatography - UV method, 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 sulfacetamide, sulfapyridine, pyridazine sulfamonomethoxine, sulfanilamide benzoyl, sulfamonomethoxine, sulfanilamide chloropyridazine, sulfamethoxazole, sulfonamide iso
Oxazole, sulfonamides dimethoxy pyridazine, pyrazole sulfonamides reference. content ≥99%; sulfa yl, sulfamerazine, sulfamethazine. Content
≥98%.
4.2 ethyl acetate. chromatography.
4.3 Acetonitrile. chromatographically pure.
4.4 Methanol. HPLC grade.
4.5 hydrochloric acid.
4.6 n-hexane.
4.7 formic acid. HPLC grade.
4.8 ammonia.
4.9 MCX Column. 60mg/3mL, or equivalent person.
4.10 0.1% formic acid solution. Take acid 1mL, dissolved and diluted with water to 1000mL.
4.11 0.1% formic acid in acetonitrile. 0.1% formic acid take 830mL, dissolved in acetonitrile and diluted to 1000mL.
4.12 Eluent. aqueous ammonia take 5mL, dissolved in methanol and diluted to 100mL.
4.13 0.1mol/L hydrochloric acid solution. Take 0.83mL of hydrochloric acid, dissolve and dilute to 100mL with water.
4.14 50% methanol in acetonitrile. Methanol 50mL, dissolved in acetonitrile and diluted to 100mL.
4.15 100μg/mL sulfonamides mixed standard stock solution. Weigh accurately sulfonamides respective standards 10mg, to a 100mL flask
, Washed with acetonitrile, dissolved and diluted to the mark, formulated at a concentration of 100μg/mL sulfonamides mixed standard stock solution. -20 ℃ less
, Valid for six months.
4.16 10μg/mL sulfonamides working standard solutions. precise amount of 100μg/mL sulfonamides mixed standard stock solution
5.0 mL, in 50mL volumetric flask, dilute to the mark with acetonitrile, formulated at a concentration of 10μg/mL sulfonamides working standard
liquid. -20 ℃ below, valid for six months.
5. Apparatus
5.1 HPLC. with UV detector or diode array 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 meter.
5.5 centrifuge.
5.6 homogenizer.
5.7 rotary evaporator.
5.8 Nitrogen blowing instrument.
5.9 SPE.
5.10 heart-shaped bottle. 100mL.
5.11 PTFE tube. 50mL.
5.12 Membrane. organic phase, 0.22μm.
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
Sample Weigh 5g ± 0.05g, polytetrafluoroethylene in 50mL centrifuge tubes, ethyl acetate was added 20mL, whirl 2min, 4000r/min
Centrifuge 5min, supernatant to l00mL heart-shaped flask, the residue was added ethyl acetate 20mL, extraction was repeated once, the two extracts were combined.
7.2 Purification
Heart-shaped flask was added 0.1mol/L hydrochloric acid solution 4mL, concentrated by rotary evaporation to at least 40 ℃ 3mL, transferred to 10mL centrifuge tubes
in. With 0.1mol/L hydrochloric acid solution was washed 2mL heart-shaped bottle, go in the same centrifuge tube. Heart-shaped flask and then washed 3mL hexane, n-hexyl
Go alkoxy same centrifuge tube, vortexed 30s, 3000r/min centrifugal 5min, discarded hexane. Again washed with n-hexane heart-shaped 3mL
Bottles, full turn in the same centrifuge tube, vortexed 30s, 3000r/min centrifugal 5min, discarded hexane, and the lower layer was reserved.
MCX column washed with methanol and 2mL 0.1mol/L hydrochloric acid solution 2mL activated, taking stock solution through the column, controlling the flow rate 1mL/min.
Sequentially with 0.1mol/L hydrochloric acid solution and 50% methanol 1mL 2mL acetonitrile, and eluted with 4 mL eluate, the eluate was collected,
40 ℃ dry nitrogen, 0.1% formic acid in acetonitrile was added to dissolve the residue 1.0mL, membrane filtration, high performance liquid chromatography for the determination.
7.3 Preparation of standard curve
The precise amount of 10μg/mL sulfonamides working standard solutions amount, diluted with 0.1% formic acid in acetonitrile, the concentration of formulated
10,50,100,250,500,2500 and 5000μg/L in a mixed series of standard solutions for HPLC assay. To the measured peak area
The vertical axis, corresponding to the concentration of standard solution as abscissa, the standard curve. Seeking regression equation and correlation coefficient.
7.4 Determination
7.4.1 LC reference condition
7.4.1.1 Column. ODS-3C18 (250mm × 4.5mm, particle size 5μm), or equivalent person.
7.4.1.2 Mobile phase. 0.1% formic acid in acetonitrile, gradient elution shown in Table 1.
7.4.1.3 flow rate. 1mL/min.
7.4.1.4 Column temperature. 30 ℃.
7.4.1.5 Detection wavelength. 270nm.
7.4.1.6 Injection Volume. 100μL.
Table 1 Mobile phase gradient elution
time
min
0.1% formic acid
Acetonitrile
0.0 8317
5.0 8317
10.0 80 20
22.3 60 40
22.4 10 90
30.0 10 90
31.0 8317
48.0 8317
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
Solution sulfonamides response value should be within the linear range of the detection instrument. In the chromatographic conditions, high control solution and the sample solution
Performance liquid chromatography, see Appendix A. FIG.
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
Residues in the specimens of sulfonamides according to formula (1).
X =
c × V
(1)
Where.
--- for the residual amount of X-try the corresponding sulfonamides feed micrograms per kilogram (μg/kg);
Corresponding sulfonamides --- C concentration in the sample solution, in micrograms per milliliter (μg/mL);
The residue was dissolved --- V 0.1% formic acid in acetonitrile by volume 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 detection sensitivity, accuracy and precision
9.1 Sensitivity
The detection limit of the method of pig and chicken muscle tissue was 5μg/kg, the limit of quantitation of 10μg/kg; detection limit of pig and chicken liver for
12μg/kg, the limit of quantitation of 25μg/kg.
9.2 Accuracy
Back to the method of muscle tissue in the 10μg/kg ~ 200μg/kg, in the liver tissue was added 25μg/kg ~ 200μg/kg concentration level
The yield was 60% to 120%.
9.3 Precision
The relative standard deviation of the method ≤l5%, inter-assay relative standard deviation ≤20%.
Appendix A
Chromatogram
Description.
1 --- sulfacetamide;
2 --- sulfapyridine;
3 --- oxazole sulfonamides;
4 --- sulfamerazine;
5 --- sulfamethazine;
6 --- sulfamonomethoxine pyridazine;
7 --- benzoyl sulfonamide;
8 --- Sulfamonomethoxine;
Sulfa. 9 --- chloropyridazine;
10 --- sulfamethoxazole;
. 11 --- sulfisoxazole;
12 --- sulfadiazine methoxy pyridazine;
13 is --- pyrazole sulfonamides.
Figure A.1 chromatogram
...
