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GB 29692-2013: Determination of Quinolones residues in milk by High Performance Liquid Chromatographic method
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Determination of Quinolones residues in milk by High Performance Liquid Chromatographic method
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GB 29692-2013
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Basic data
| Standard ID | GB 29692-2013 (GB29692-2013) |
| Description (Translated English) | Determination of Quinolones residues in milk by High Performance Liquid Chromatographic method |
| Sector / Industry | National Standard |
| Classification of Chinese Standard | C53 |
| Classification of International Standard | 67.020 |
| Word Count Estimation | 17,180 |
| 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 Milk quinolones, fluoroquinolones, the residual amount of drug testing sample preparation and high-performance liquid chromatographic method. This standard applies to milk ciprofloxacin, danofloxacin so on. |
GB 29692-2013: Determination of Quinolones residues in milk 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 Quinolones residues in milk by High Performance Liquid Chromatographic method
National Standards of People's Republic of China
National Food Safety Standard
Determination of quinolones residues in milk
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 quinolones residues in milk
High performance liquid chromatography
method one
1 Scope
This method provides a sample preparation and high performance liquid chromatographic method Residues in Milk quinolone drug testing.
This method is applicable to milk ciprofloxacin, danofloxacin residues, enrofloxacin, sarafloxacin and difloxacin single or multiple drugs
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
Quinolones in a sample extracted with acetonitrile, and rotary evaporated to near dryness, dissolved in mobile phase. High Performance Liquid Chromatography - Fluorescence
Fixed, external standard.
4 Reagents and Materials
The following reagents used, unless otherwise stated who were of analytical reagent; water as a water line with GB/T 6682 provisions.
4.1 danofloxacin, enrofloxacin, ciprofloxacin hydrochloride, hydrochloric acid sarafloxacin stars and dihydrochloride difloxacin reference. content ≥99.0%.
4.2 phosphoric acid.
4.3 sodium hydroxide.
4.4 Acetonitrile. chromatographically pure.
4.5 triethylamine.
A saturated solution of sodium hydroxide 4.6. take sodium hydroxide appropriate amount, add water and shake to make into a saturated solution, after cooling, the setting of polyethylene plastic bottle, allowed to stand,
clarify.
4.7 5mol/L sodium hydroxide solution. saturated solution of sodium hydroxide to take 28mL, dissolve and dilute to 100mL with water.
4.8 0.03mol/L sodium hydroxide solution. Take 5mol/L sodium hydroxide solution 0.6mL, dissolve and dilute to 100mL with water.
4.9 0.05mol/L phosphoric acid solution triethylamine. phosphoric take 3.4mL, dissolved and diluted with water to 1000mL. Adjusted with triethylamine
pH to 2.4.
4.10 quinolones mixed standard stock solution. Precision Weigh danofloxacin reference substance 10mg, enrofloxacin, ciprofloxacin, sarafloxacin
And difloxacin reference substance 50mg, in 50mL volumetric flask, dissolve and dilute to volume with 0.03mol/L sodium hydroxide solution, formulated
Danofloxacin at a concentration of 0.2mg/mL, ciprofloxacin, enrofloxacin, sarafloxacin and difloxacin concentration of 1mg/mL quinolones
Standard mixing the drug stock solution. 2 ℃ ~ 8 ℃ storage period of three months.
4.11 quinolones working standard solutions. quinolones precise amount of mixed standard stock solution 1.0mL, in 100mL flask
In, diluted with mobile phase, danofloxacin formulated at a concentration of 2μg/mL, ciprofloxacin, enrofloxacin, sarafloxacin and difloxacin concentration
10μg/mL quinolones working standard solutions. 2 ℃ ~ 8 ℃, valid for 1 week.
5 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 oscillator.
5.5 centrifuge.
5.6 PTFE tube. 50mL.
5.7 heart-shaped bottle. 25mL.
5.8 filter. 0.45μm.
Preparation and Storage of sample 6
6.1 Preparation of the sample
An appropriate amount of fresh or frozen or blank test milk and mix well.
--- 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 2g ± 0.05g, in 50mL centrifuge tubes, add 100 L phosphoric acid, 4 mL of acetonitrile, vortexing, shaking speed 5min,
10000r/min centrifugal 10min, supernatant to another centrifuge tube, add n-hexane 5mL, scroll 1min, standing, and the lower layer in the serum
25mL heart-shaped bottle. 4 mL acetonitrile was added to the residue, extraction was repeated once, the supernatant was assigned with a n-hexane, the two extracts were combined,
Rotary evaporator at 50 deg.] C to a yellow oil droplets of only the remaining easy to dryness. The residue was dissolved with 1.0mL flow membrane filtration for HPLC
Determination chromatography.
7.2 Preparation of standard curve
The precise amount of working standard solutions quinolones amount, diluted with mobile phase to prepare a concentration of ciprofloxacin, enrofloxacin, salad
Gatifloxacin and difloxacin as 5,10,50,100,300 and 500μg/L, the concentration of danofloxacin 1,2,10,20,60 and 100μg/L in series
Standard solution for HPLC assay. To measure peak area for the vertical axis, corresponding to the concentration of standard solution of abscissa, the standard curve
line. Seeking regression equation and correlation coefficient.
7.3 Determination
7.3.1 Chromatographic conditions
7.3.1.1 Column. C18 (250mm × 4.6mm, particle size 5μm), or equivalent person.
7.3.1.2 Mobile phase. 0.05mol/L phosphoric acid - triethylamine acetonitrile (90 10, volume ratio) to membrane filtration.
7.3.1.3 flow rate. 1.8mL/min.
7.3.1.4 Detection wavelength. excitation wavelength 280 nm; emission wavelength of 450nm.
7.3.1.5 Column temperature. 30 ℃.
7.3.1.6 Injection volume. 20μL.
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 ciprofloxacin, danofloxacin, enrofloxacin, sarafloxacin and difloxacin response value should be within the linear range of the detection instrument. above
Under the chromatographic conditions described below, the standard solution and the sample solution was added tissue white HPLC Appendix A. FIG.
7.4 Blank test
But without addition of the sample, the measurement step is performed in exactly the same operation in parallel.
8 and the result of the calculation expression
Quinolones residues in a sample (μg/kg) according to formula (1).
X =
c × V
(1)
Where.
--- for the respective X-try feed quinolone drug residues micrograms per kilogram (μg/kg);
C --- corresponding quinolone concentration, in units of nanograms per milliliter (ng/mL) in the sample solution;
V --- The residue was dissolved in mobile phase 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 measured parallel to three significant figures.
9 detection sensitivity, accuracy and precision
9.1 Sensitivity
This method ciprofloxacin, enrofloxacin, sarafloxacin and difloxacin detection limit of 5μg/kg, the limit of quantitation of 10μg/kg; danofloxacin
Star detection limit of 1μg/kg, the limit of quantitation of 2μg/kg.
9.2 Accuracy
This method of adding 10μg/kg ~ 100μg/kg on recovery levels of 60% to 100%.
9.3 Precision
The relative standard deviation of the method ≤15%, inter-assay relative standard deviation ≤20%.
Method Two
10 range
This method provides a sample preparation and high performance liquid chromatographic method 11 quinolones detected Residues in Milk.
This method is applicable to milk enrofloxacin, ciprofloxacin, danofloxacin, sarafloxacin, difloxacin, norfloxacin, ofloxacin, Pei
Sparfloxacin, lomefloxacin, flumequine and oxolinic acid residues in a single or multiple drugs.
11 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 12
Quinolones of the specimens with 10% trichloroacetic acid - acetonitrile extraction, reversed phase polymeric SPE column purification, dissolved in mobile phase, high
Performance liquid - Fluorescence method, external standard.
13 Reagents and materials
All of the following reagents, unless otherwise stated who are analytical reagents; water as a water line with GB/T 6682 provisions.
13.1 enrofloxacin, ciprofloxacin, danofloxacin mesylate, sarafloxacin, difloxacin, norfloxacin, ofloxacin, methanesulfonic acid Pei
Sparfloxacin, lomefloxacin hydrochloride, flumequine and oxolinic acid reference. Content ≥98.0%.
13.2 Acetonitrile. chromatographically pure.
13.3 Methanol. HPLC grade.
13.4 ethanol.
13.5 acetic acid.
13.6 citric acid.
13.7 ammonium acetate.
13.8 triethylamine.
13.9 sodium hydroxide.
13.10 TCA.
13.11 SPE column reverse phase polymer. Strata-X filler, 60mg/3mL, or equivalent person.
13.12 10% solution of acetic acid. acetic acid of 10mL, dissolve and dilute to 100mL with water.
13.13 0.5mol/L sodium hydroxide solution. take sodium hydroxide 2g, dissolve and dilute to 100mL with water.
13.14 10% trichloroacetic acid solution. Take trichloroacetic 100g, dissolved and diluted with water to 1000mL.
-10 13.15% trichloroacetic acid in acetonitrile. acetonitrile take 10mL, washed with 10% trichloroacetic acid solution was dissolved and diluted to 100mL.
13.16 10% aqueous methanol. Methanol 10mL, dissolved and diluted to 100mL with water.
13.17 citric acid/ammonium acetate buffer. Take citric acid 10.56g, ammonium acetate, 7.87g, dissolved in water and diluted to 1000mL, with triethyl
Amine pH adjusted to 4.0, filter membrane.
Acetonitrile 13.18 - citric acid/ammonium acetate buffer. acetonitrile take 8mL, dissolved in citric acid/ammonium acetate buffer and diluted to 100mL.
13.19 1mg/mL norfloxacin, ofloxacin, enrofloxacin, sarafloxacin and difloxacin standard stock solution. Weigh accurately Connaught fluoride sand
Star, ofloxacin, enrofloxacin, sarafloxacin and difloxacin reference substance amount, respectively 10mL volumetric flask, acetic acid solution 200μL
It was dissolved, diluted with methanol to the mark, formulated at a concentration of 1mg/mL standard stock solution quinolones of. Or less at -20 ℃ with
Valid for six months.
13.20 1mg/mL ciprofloxacin, lomefloxacin, pefloxacin and danofloxacin standard stock solution. accurately weighed ciprofloxacin hydrochloride,
Lomefloxacin, pefloxacin mesylate and danofloxacin mesylate reference standard amount, respectively 10mL volumetric flask, dissolve 200μL water,
Dilute with methanol, formulated at a concentration of 1mg/mL standard stock solution quinolones of. -20 ℃ below, valid for 6
Months.
13.21 1mg/mL flumequine and oxolinic acid standard stock solution. accurately weighed flumequine and oxolinic acid reference standard amount, respectively, in an amount of 10mL
Flask, was dissolved with 400μL of sodium hydroxide solution, dilute to the mark with methanol, formulated at a concentration of 1mg/mL standard quinolones
Stock solution. -20 ℃ below, valid for six months.
13.22 quinolones working standard solutions. the precise amount of the amount of standard stock solution quinolones, in volumetric flask, dissolved in water and
And dilute to volume, formulated as norfloxacin, ciprofloxacin, ofloxacin, pefloxacin, lomefloxacin, enrofloxacin, sarafloxacin, difluoromethyl sand
Stars and quinoline acid concentration 8μg/mL, flumequine concentration of 4μg/mL, danofloxacin working standard solutions at a concentration of 2.4μg/mL of.
2 ℃ ~ 8 ℃ storage period of one month.
14 instruments and equipment
14.1 HPLC. with fluorescence detector.
14.2 Analytical Balance. a sense of volume 0.00001g.
14.3 balance. a sense of the amount of 0.01g.
14.4 homogenizer.
14.5 vortex mixers.
14.6 ultrasonic bath.
14.7 high-speed refrigerated centrifuge.
14.8 blown dry with nitrogen concentrators.
14.9 SPE.
14.10 filters. 0.45μm.
14.11 polypropylene centrifuge tube.
Sample Preparation and Storage of 15
15.1 Preparation of samples
An appropriate amount of fresh or frozen or blank test milk and mix well.
--- 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 solution, is added as a blank sample.
Save 15.2 sample
Or less at -20 ℃.
Determination Step 16
16.1 Preparation of standard curve
The precise amount of an appropriate amount of working standard solution, diluted with water, vortex mix the danofloxacin concentrations 15,30,60,120,240 and
480μg/L, the concentration of flumequine 25,50,100,200,400 and 800μg/L, the concentration of other quinolones 50,100,200,400,
800 and 1600μg/L of the series of standard solution for HPLC assay. Peak area for the vertical axis, corresponding to the concentration of the standard solution
Abscissa, the standard curve. Seeking regression equation and correlation coefficient.
16.2 extract
Weigh the sample 1g ± 0.02g, in 15mL polypropylene centrifuge tube, was added acetonitrile - 10% trichloroacetic acid solution 5mL, Vortex, super
Sound 5min, at 4 ℃ 8000r/min 6min centrifugation, the supernatant and set aside.
16.3 Purification
SPE column was washed successively with water and methanol and 3mL 3mL activation, taking stock solution through the column, controlling the flow rate 1mL/min, washed with 10% aqueous methanol
3mL rinsed, drained, eluting with 3mL methanol and sucked dry. The eluate was collected, dried under nitrogen 60 ℃ ~ 70 ℃, with acetonitrile - citric acid/acetate
The residue was dissolved ammonium formate buffer 500μL was vortexed, the following 4 ℃ 12000r/min 6min centrifugation, the supernatant for HPLC
Chromatography.
16.4 Determination
16.4.1 chromatographic conditions
16.4.1.1 Column. C18 (250mm × 4.6mm, particle size 5μm), or equivalent person.
16.4.1.2 mobile phase. acetonitrile - citric acid/ammonium acetate buffer gradient shown in Table 1.
Table 1 gradient elution change settings
time
min
flow
mL/min
Acetonitrile
Citric acid/ammonium acetate buffer
curve
0.01 2.0 892--
30.0 2.0 55 459
32.0 2.0 892--
35.0 2.0 892--
16.4.1.3 Column temperature. 50 ℃.
16.4.1.4 injection volume. 20μL.
16.4.1.5 fluorescence detector. wavelength change procedure is provided in Table 2.
16.4.1.6 5min backward delay the next sample.
Table 2 fluorescence wavelength variation setting program
time
min
Excitation wavelength Ex
nm
Emission wavelength Em
nm
23.4 312 366
32.0 278 465
16.4.2 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
Quinolones was the response value should be within the linear range of the detection instrument. In the chromatographic conditions, the standard solution and blank add organizations
HPLC of the sample solution was added respectively in Appendix B. FIG.
16.5 Blank test
But without addition of the sample, the measurement step is performed in exactly the same operation in parallel.
17 and the result of the calculation expression
17.1 standard calibration curve
The concentration and the peak area corresponding to the standard curve regression analysis, then according to equation (2).
X =
Ab
(2)
Where.
--- for the respective X-try feed quinolone drug residues micrograms per kilogram (μg/kg);
A --- try for a respective feed quinolone peak area in the sample solution measured after treatment quinolones;
B --- intercept regression equation of the standard curve;
--- A standard curve slope of the regression equation.
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.
17.2-point calibration
Quinolone residues in a sample (μg/kg) according to formula (3) is calculated.
Xi = cS ×
Ai
AS ×
(3)
Where.
--- XI for the residual amount of the corresponding quinolone compound try micrograms per kilogram (μg/kg);
Standard solution concentration cS --- quinolones respective units of nanograms per milliliter (ng/mL);
--- AI in the sample solution corresponding quinolones peak area;
--- the AS standard solution corresponding quinolones peak area;
V --- The residue was dissolved with acetonitrile - citric acid/ammonium acetate buffer volume in milliliters (mL);
m --- try supply feed mass in grams (g).
18 detection sensitivity, accuracy and precision
18.1 Sensitivity
This method norfloxacin, ofloxacin, ciprofloxacin, pefloxacin, lomefloxacin, enrofloxacin, sarafloxacin, difloxacin and quinolin
Acid detection limit of 25μg/kg, the limit of quantitation of 50μg/kg; danofloxacin detection limit of 7.5μg/kg, the limit of quantitation of 15μg/kg; flumequine
The detection limit was 12.5μg/kg, the limit of quantitation of 25μg/kg.
18.2 Accuracy
This method norfloxacin, ofloxacin, ciprofloxacin, pefloxacin, lomefloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid
In the 50μg/kg ~ 200μg/kg, danofloxacin at 15μg/kg ~ 60μg/kg, Flumequine Add 25μg/kg ~ 100μg/kg concentration
On recovery levels of 60% to 110%.
18.3 Precision
The relative standard deviation of the method ≤15%, inter-assay standard deviation aligned ≤20%.
Appendix A
Chromatogram
Figure A.1 chromatogram quinolones control solution (20μg/L)
Figure A.2 milk blank sample chromatogram
Description.
1 --- ciprofloxacin;
2 --- danofloxacin;
3 --- enrofloxacin;
4 --- Sarafloxacin;
5 --- difloxacin.
Figure A.3 milk sample blank add quinolone chromatogram (100μg/kg)
Appendix B
Chromatogram
Figure B.1 quinolones matrix-matched standard solution chromatogram
(Norfloxacin NOR, ofloxacin OFL, ciprofloxacin CIPRO, pefloxacin PEF, lomefloxacin LOME, enrofloxacin ENRO, sarafloxacin
SARA, Difloxacin concentrations DIF OXA and oxolinic acid was 100μg/L, danofloxacin DANO of 30μg/L, flumequine FLU of 50μg/L)
Figure B.2 milk blank sample chromatogram
Figure B.3 milk sample blank add quinolone chromatograms
(NOR, OFL, CIPRO, PEF, LOME, ENRO, SARA, DIF and OXA of 25μg/kg, DANO of 7.5μg/kg, FLU
As 12.5μg/kg)
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