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Determination of 17β-Estradiol, Estriol and Ethinylestradiol Residues in Milk and Milk Products by Gas Chromatography-Mass Spectrometric method
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Basic data
| Standard ID | GB 29698-2013 (GB29698-2013) |
| Description (Translated English) | Determination of 17��-Estradiol, Estriol and Ethinylestradiol Residues in Milk and Milk Products by Gas Chromatography-Mass Spectrometric method |
| Sector / Industry | National Standard |
| Classification of Chinese Standard | C53 |
| Classification of International Standard | 67.020 |
| Word Count Estimation | 11,127 |
| 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, dairy products estrogen residues in drug testing sample preparation, gas chromatography mass spectrometry. This standard applies to milk and dairy products. |
GB 29698-2013: Determination of 17β-Estradiol, Estriol and Ethinylestradiol Residues in Milk and Milk Products by Gas Chromatography-Mass Spectrometric method
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Determination of 17β-Estradiol, Estriol and Ethinylestradiol Residues in Milk and Milk Products by Gas Chromatography-Mass Spectrometric method
National Standards of People's Republic of China
National Food Safety Standard
Milk and dairy products 17β- estradiol, estriol,
Determination of ethinyl estradiol residues in gas chromatography - 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
Milk and dairy products 17β- estradiol, estriol,
Determination of ethinyl estradiol residues in gas chromatography - mass spectrometry
1 Scope
This standard specifies the GC sample preparation and drug residues in milk and dairy products in an amount detectable estrogen - mass spectrometry.
This standard applies to milk and milk samples detected 17β- estradiol, estriol, ethinylestradiol 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
Estrogen remaining in a sample, a solvent mixture of ethyl acetate and acetonitrile extraction, solid-phase extraction column, derivatized silylating agent, the mold plasma
Formula Gas Chromatography - Mass Spectrometry, 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 17β- estradiol, estriol, ethinyl estradiol. content ≥98.0%.
4.2 disulfide erythritol.
4.3 N- methyl trimethylsilyl trifluoroacetyl ammonia.
4.4 trimethylsilane.
4.5 acetonitrile.
4.6 ethyl acetate.
4.7 methanol.
4.8 toluene.
4.9 n-hexane.
4.10 hydrochloric acid.
4.11 sodium hydroxide.
4.12 C18 solid phase extraction column. LC-C18,500mg/3mL, or equivalent person.
4.13 silica gel solid phase extraction column. LC-Si, 500mg/3mL, or equivalent person.
4.14 95% hexane in ethyl acetate. n-hexane to take 95mL, dissolved with ethyl acetate and dilute to 100mL.
4.15 hexane 70% ethyl acetate. hexane to take 70mL, dissolved with ethyl acetate and dilute to 100mL.
4.16 1mol/L sodium hydroxide solution. take sodium hydroxide 40g, dissolved and diluted with water to 1000mL.
4.17 5mol/L hydrochloric acid solution. Take concentrated hydrochloric acid 48mL, dissolve and dilute to 100mL with water.
4.18 derivatizing agent. take dithio erythritol 0.01g, dissolved in N- methyl trimethylsilyl trifluoroacetyl ammonia (MSTFA) 5mL, in
Adding trimethylsilyl subsurface 10 L, mix, 2 ℃ ~ 8 ℃ overnight, dark moisture sealed. Derivatizing agent should be colorless, if
Brown-red color change occurs, etc., indicate reagent failure.
4.19 1mg/mL17β- estradiol, estriol, ethinylestradiol standard stock solution. Weigh accurately 17β- estradiol, estriol, ethinyl estradiol standard
Each 10mg, respectively 10mL volumetric flask, dissolved in methanol and diluted to the mark, dubbed the concentration of the standard stock solution 1mg/mL of.
-20 ℃ below, valid for six months.
4.20 10mg/L17β- estradiol, estriol, ethinylestradiol working standard solutions. precise amount of 1mg/mL17β- estradiol, estriol
Ethinyl estradiol and 1.0 mL of each standard stock solution, to a 100mL volumetric flask, dilute to the mark with methanol, formulated at a concentration of 10mg/L in a mixed
Working standard solution, 2 ℃ ~ 8 ℃ storage period of one month.
5. Apparatus
5.1 Gas chromatography - mass spectrometry. EI source.
5.2 Analytical balance. a sense of volume 0.00001g.
5.3 Balance. a sense of the amount of 0.01g.
5.4 Nitrogen blowing instrument.
5.5 SPE.
5.6 homogenizer.
5.7 vortex mixer.
5.8 centrifuge.
5.9 oven.
5.10 pH meter.
5.11 rotary concentrator.
5.12 filter. 0.22μm.
6 Sample Preparation and Storage
6.1 Preparation of the sample
An appropriate amount of fresh or frozen blank or test sample, mixed, 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
Save 2 ℃ ~ 8 ℃.
Determination Step 7
7.1 Preparation of standard curve
The precise amount of 10mg/L working standard solutions amount, diluted with methanol, and formulated to a concentration of 10,50,100,200,500
1000μg/L series of working standard solution, blown dry with nitrogen at 40 ℃ water bath, treated according to the derivatization step, process gas chromatography - mass spectrometry. With
Measured peak area for the vertical axis, corresponding to the concentration of standard solution as abscissa, the standard curve. Seeking regression equation and correlation coefficient.
7.2 extract
7.2.1 Liquid Sample
Sample Weigh 10g ± 0.05g, in 50mL centrifuge tube, add acetonitrile 5mL, ethyl acetate 15mL, vortexed 3min,
8000r/min centrifugal 5min, the supernatant was collected in another 50mL centrifuge tube, the residue was extracted repeatedly once, twice combined supernatants at
At 40 ℃ rotary evaporated to near dryness, dissolved in 1mol/L sodium hydroxide solution three times 6mL, 50mL transferred to another centrifuge tube, add
20mL hexane vortexed 1min, 8000r/min centrifugal 3min, extract the lower layer was collected, adjusted to pH with 5mol/L hydrochloric acid solution
To 5.0 to 5.2, the standby.
7.2.2 solid samples
Sample Weigh 10g ± 0.05g, in 50mL centrifuge tubes, ethyl acetate was added 15mL, vortexed 3min, 8000r/min centrifugal
5min, supernatant was collected in another 50mL centrifuge tube, the residue was extracted repeatedly once, twice supernatants were combined, rotary evaporated to 40 deg.] C water bath
Near dryness, dissolved in 1mol/L sodium hydroxide solution 6mL three times, transferred to another centrifuge tube 50mL, 20mL hexane plus vortex transducer
Swing 1min, 8000r/min centrifugal 3min, lower layer was collected extracts were washed with 5mol/L hydrochloric acid solution was adjusted to pH 5.0 to 5.2, the standby.
7.3 Purification
C18 column and washed with 5mL methanol and 5mL water activated, taking stock solution through the column, 5mL water rinsed, drained, eluting with methanol 5mL,
The eluate was collected, dried in a water bath at 40 ℃ nitrogen. The residue was dissolved with n-hexane to 95% ethyl acetate 5mL, through with hexane 5mL
The activated silica gel, n-hexane was added 5mL rinsed, drained, and then 5mL of 70% ethyl acetate-hexane, collecting eluate,
Water bath at 40 ℃ blown dry with nitrogen.
7.4 Derivatization
The residue was added toluene and 100μL of each derivatization reagent were dissolved, mixed, sealing, derived 60min, cooled, gas phase in an oven at 80 ℃
Chromatography - Mass Spectrometry.
7.5 Determination
7.5.1 Chromatographic conditions
7.5.1.1 column HP-5MS capillary column (30m × 0.25mm, film thickness 0.25μm), or equivalent person.
7.5.1.2 The carrier gas was Helium, constant flow 1.0mL/min.
7.5.1.3 injector temperature 220 ℃.
7.5.1.4 Injection volume. 1μL, splitless.
7.5.1.5 column starting temperature of 100 deg.] C (holding 1min), at a heating rate of 20 ℃/min was raised to 200 ℃ (holding 3min), and then to
Heating rate 20 ℃/min was raised to 260 ℃ (holding 5min), and then at a heating rate of 20 ℃/min was raised to 280 deg.] C (holding 5min).
7.5.2 MS conditions
7.5.2.1 ion source (EI) temperature.200 ℃.
7.5.2.2 EM Voltage. tuning voltage is higher than 200V.
7.5.2.3 electron energy. 70eV.
7.5.2.4 GC/MS transfer line temperature. 280 ℃.
7.5.2.5 Quadrupole temperature. 160 ℃.
7.5.2.6 selected ion monitoring (SIM) .( m/z) 232,285,326,416 (17β- estradiol); 311,345,414,504 (estriol); 285,
300,425,440 (ethinyl estradiol).
7.5.3 Assay
7.5.3.1 qualitative determination
By retention time of sample chromatogram with retention times of the respective standards, wherein each of the ion peaks and the corresponding concentration of the standard
Wherein each of the ion peaks qualitative contrast. Sample and standard deviation of the relative retention time of not more than 5%; with a sample wherein ions
Relative abundance consistent with the abundance of an equivalent concentration of a mixed standard solution, the relative abundance of the deviation does not exceed specified in Table 1, the sample can be stored is determined
The respective analyte.
The maximum allowable error relative ion abundances% Table 1 Qualitative diagnosis
Relative abundance > 50 20 ~ 50 10 ~ 20 ≤10
Tolerance ± 10 ± 15 ± 20 ± 50
Quantitative determination 7.5.3.2
Take appropriate sample solution and standard solutions appropriate to do single-point or multi-point calibration, by external standard method, in order to quantify the peak area, sample and standard solutions
Liquid 17β- estradiol, estriol and ethinyl estradiol response value shall be within the linear range of the detection instrument. In the chromatographic conditions, the standard
Solution, add blank sample and blank sample chromatogram and mass spectrum given in Appendix A.
7.6 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
The sample of 17β- estradiol, estriol remaining amount, calculated as ethinyl estradiol formula (1).
X =
A × cS × V
AS × m
(1)
Where.
--- for X-try feed corresponding 17β- estradiol, estriol, ethinylestradiol residues micrograms per kilogram (μg/kg);
A --- Sample peak area corresponding 17β- estradiol, estriol and ethinyl estradiol; and
cS --- standard solution corresponding 17β- estradiol, estriol and ethinyl estradiol concentration, in micrograms per liter (μg/L);
The residue was dissolved V --- volume in milliliters (mL);
--- the AS standard solution corresponding 17β- estradiol, estriol and ethinyl estradiol 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 method detection limit of 0.5μg/kg, the limit of quantitation was 1.0μg/kg.
9.2 Accuracy
This method of adding 1μg/kg ~ 10μg/kg on recovery levels of 60% to 120%.
9.3 Precision
The relative standard deviation of the method ≤15%, inter-assay relative standard deviation ≤20%.
Appendix A
Chromatogram
Description.
ES --- 17β- estradiol derivatives;
--- the EES ethinyl estradiol derivative;
EST --- estriol derivatives.
FIG A.1 17β- estradiol, ethinylestradiol, estriol chromatogram of the standard solution (100μg/L)
Figure A.2 milk blank sample chromatogram
Description.
ES --- 17β- estradiol derivatives;
--- the EES ethinyl estradiol derivative;
EST --- estriol derivatives.
Figure A.3 blank add milk 17β- estradiol, ethinyl estradiol and estriol sample chromatogram (1μg/kg)
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