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Determination of Cyromazine and Melamine residues in animal derived food by Ultra Performance Liquid Chromatography-tandem Mass Spectrometric method
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Basic data
| Standard ID | GB 29704-2013 (GB29704-2013) |
| Description (Translated English) | Determination of Cyromazine and Melamine residues in animal derived food by Ultra Performance Liquid Chromatography-tandem Mass Spectrometric method |
| Sector / Industry | National Standard |
| Classification of Chinese Standard | C53 |
| Classification of International Standard | 67.020 |
| Word Count Estimation | 12,145 |
| 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 cyromazine, melamine residues of metabolite detection sample preparation and ultra performance liquid chromatography tandem mass spectrometry. |
GB 29704-2013: Determination of Cyromazine and Melamine residues in animal derived food by Ultra Performance Liquid Chromatography-tandem Mass Spectrometric method
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Determination of Cyromazine and Melamine residues in animal derived food by Ultra Performance Liquid Chromatography-tandem Mass Spectrometric method
National Standards of People's Republic of China
National Food Safety Standard
Animal foods metabolite of cyromazine and melamine
Determination of remaining performance liquid chromatography - tandem mass spectrometry
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 cyromazine and melamine residues metabolite assay
Ultra performance liquid chromatography - tandem mass spectrometry
1 Scope
This standard specifies the sample preparation and animal foods UPLC cyromazine and melamine metabolite of Residues - string
With mass spectrometry.
This standard applies to chicken muscle, kidney and eggs cyromazine residues and metabolites melamine.
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 cyromazine and melamine metabolites, extracted with 3% trichloroacetic acid solution, MCX purification column, ultra-high performance liquid chromatography
Spectrum - tandem mass spectrometry, 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 cyromazine and melamine reference. content ≥98.0%.
4.2 TCA.
4.3 Acetonitrile. chromatographically pure.
4.4 Methanol. HPLC grade.
4.5 formic acid.
4.6 ammonia.
4.7 ammonium acetate.
4.8 MCX SPE. 60mg/3mL, or equivalent person.
4.93% trichloroacetic acid solution. Take trichloroacetic 3g, dissolve and dilute to 100mL with water.
4.10 2% formic acid aqueous solution. Take acid 2mL, dissolve and dilute to 100mL with water.
4.11 5% ammoniated methanol. aqueous ammonia to take 5mL, dissolved in methanol and diluted to 100mL.
4.12 50% aqueous acetonitrile. acetonitrile take 50mL, dissolve and dilute to 100mL with water.
4.13 0.1mol/L ammonium acetate solution. Take ammonium acetate 7.71g, dissolved and diluted with water to 1000mL.
4.14 1mg/mL cyromazine, melamine standard stock solution. Weigh accurately cyromazine and melamine reference 10mg, respectively,
Brown 10mL volumetric flask, dissolved with 50% aqueous acetonitrile and dilute to volume, formulated at a concentration of 1mg/mL of the standard reservoir cyromazine
Preparation of standard stock solution and melamine solution. 2 ℃ ~ 8 ℃ storage period of three months.
4.15 10μg/mL cyromazine and melamine working standard solutions. cyromazine precise amount of standard stock solution 1mg/mL and
1.0 mL of each stock standard solution of melamine, to a 100mL volumetric flask, dissolved in acetonitrile and dilute to volume, formulated at a concentration of
10μg/cyromazine and melamine mL of working standard solutions. 2 ℃ ~ 8 ℃ storage period of three months.
5. Apparatus
5.1 ultra performance liquid chromatography - tandem mass spectrometer. Distribution ionization source.
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 centrifuge.
5.6 Nitrogen blowing instrument.
5.7 centrifuge tube. 50mL.
Microporous filter head 5.8. 0.2μm.
Preparation and Storage of sample 6
6.1 Preparation of the sample
6.1.1 eggs
Fresh or frozen egg white or test, peeled, 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.
6.1.2 chicken muscle meat and kidney
Fresh or frozen 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 Preparation of matrix-matched standard curve
The precise amount of 10μg/mL cyromazine and melamine working standard solutions amount, diluted with 95% acetonitrile in water to prepare a concentrated
It is a series of mixed standard solution 2,5,10,20,50 and 100ng/mL and 1.0 mL from each, are extracted by dissolution, purification and drying
6 parts of the blank sample residue was filtered, for performance liquid chromatography - tandem mass spectrometry. A characteristic ion mass chromatogram peak area for the vertical
Coordinate matrix-matched control solution concentration as the abscissa, the standard curve. Seeking regression equation and correlation coefficient.
7.2 extract
Sample Weigh 2g ± 0.02g, in 50mL centrifuge tube, 3% trichloroacetic acid solution was added 20mL, vortexed 30s, ultrasonic
10min, allowed to stand for 10min, 10000r/min centrifugal 10min, the supernatant was set aside.
7.3 Purification
MCX column washed with methanol and 3mL 3mL water activation, 10mL take stock solution through the column, 3mL plus 2% formic acid, methanol 3
mL rinsed, drained, with 5% ammoniated methanol 3mL elution, drained, eluate dry nitrogen 50 ℃, with 95% aqueous acetonitrile
The residue was dissolved 1.0mL, membrane filtration, ultra for liquid chromatography - tandem mass spectrometry.
7.4 Determination
7.4.1 Chromatographic conditions
7.4.1.1 Column. BEHHILIC column (50mm × 2.1mm, particle size 1.7μm), or equivalent person.
7.4.1.2 Mobile phase. A. acetonitrile; B. 0.1mol/L ammonium acetate solution.
7.4.1.3 Gradient. 0min ~ 2.5min, 95% A linear gradient to 80% A; 2.5min ~ 4min, maintaining 95% A.
7.4.1.4 flow rate. 0.3mL/min.
7.4.1.5 Column temperature. 30 ℃.
7.4.1.6 Injection volume. 10μL.
7.4.2 MS conditions
7.4.2.1 ion source. electrospray ionization source.
7.4.2.2 Scan mode. positive ion scan.
7.4.2.3 Detection mode. multiple reaction monitoring.
7.4.2.4 ionization voltage. 3.0kV.
7.4.2.5 source temperature. 110 ℃.
7.4.2.6 atomizing temperature. 350 ℃.
7.4.2.7 Cone gas flow rate. 50L/h.
7.4.2.8 atomizing gas flow rate. 650L/h.
7.4.2.9 qualitative, cone voltage and collision energy of ions and the corresponding quantified. see Table 1.
Table 1 Qualitative and quantitative ion pair and the corresponding cone voltage and collision energy
drug
Qualitative ion pair
m/z
Quantitative ion pair
m/z
Cone voltage
Collision energy
eV
Cyromazine
166.9 > 84.6
166.9 > 124.8
166.9 > 84.6 35
Melamine
126.9 > 84.7
126.9 > 67.7
126.9 > 84.7 30
twenty two
7.4.3 Assay
Take a sample solution and a matrix matching the corresponding standard solution, as a single or multi-point calibration, external standard method. Standard solution and the sample solution
Peak area cyromazine and melamine should be within the linear range of the detection instrument. Ion in the sample solution with the matrix matching the relative abundance of
Compared with a standard relative abundance of ions in solution, in accordance with Table 2. Chromatographic quality standard solution and sample solution of ions of each feature
Figure in Appendix A.
Table 2 sample ions in the solution relative abundance tolerance range
The relative abundance
Tolerance
> 50 ± 20
20 ~ 50 ± 25
10 ~ 20 ± 30
≤10 ± 50
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 cyromazine and melamine residues (μg/kg) according to formula (1).
X =
AcSV1V3
ASV2m
(1)
Where.
--- for X-try respective feed cyromazine residues or melamine, in micrograms per kilogram (μg/kg);
A --- respective sample solution cyromazine or melamine peak area;
--- respective concentrations cS cyromazine or melamine, units of nanograms per milliliter (ng/mL) matrix-matched standard solution;
Vl --- extract of a total volume of liquid in milliliters (mL);
--- V2 through the solid phase extraction column with the extract volume in milliliters (mL);
V3 --- volume of the residue was dissolved in milliliters (mL);
--- the AS matrix-matched standard solution corresponding cyromazine or melamine 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 detection sensitivity, accuracy and precision
9.1 Sensitivity
The detection limit of the method was 1μg/kg, limit of quantification was 2.5μg/kg.
9.2 Accuracy
This method of adding 2.5μg/kg ~ 100μg/kg on recovery levels of 70% to 120%.
9.3 Precision
≤20% relative standard deviation in this method and inter - batch relative standard deviation of ≤20%.
Appendix A
Chromatogram
a)
b)
c)
d)
Figure A.1 cyromazine and melamine standard solution wherein ion mass chromatogram (10ng/mL)
a)
b)
c)
d)
e)
f)
g)
h)
Figure A.2 chicken kidney tissue sample blank wherein the ion mass chromatogram
a)
b)
c)
d)
Description.
1 --- cyromazine wherein ion mass chromatogram (166.9 > 84.6);
2 --- cyromazine wherein ion mass chromatogram (166.9 > 124.8);
3 --- Melamine wherein ion mass chromatogram (126.9 > 67.7);
4 --- Melamine wherein ion mass chromatogram (126.9 > 84.7).
Figure A.3 blank Add chicken kidney tissue sample features of cyromazine and melamine-ion mass chromatogram (10ng/g)
...
