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GB 29685-2013: Determination of Lincomycin, Clindamycin and Spectinomycin residues in animal derived food by Gas Chromatography Mass Spectrometric method
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GB 29685-2013	English	259	Add to Cart	3 days [Need to translate]	Determination of Lincomycin, Clindamycin and Spectinomycin residues in animal derived food by Gas Chromatography Mass Spectrometric method

GB 29685-2013: Determination of Lincomycin, Clindamycin and Spectinomycin residues in animal derived food by Gas Chromatography Mass Spectrometric method

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Determination of Lincomycin, Clindamycin and Spectinomycin residues in animal derived food by Gas Chromatography Mass Spectrometric method National Standards of People's Republic of China National Food Safety Standard Animal foods lincomycin, clindamycin, and Grand View Determination of neomycin 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 Animal foods lincomycin, clindamycin, and Grand View Determination of neomycin residues in gas chromatography - mass spectrometry

1 Scope

This standard specifies the animal food lincomycin, clindamycin and spectinomycin Residues sample preparation and gas chromatography - mass spectroscopy Given method. This standard applies to pigs, cattle and chicken muscles and kidneys as well as milk and eggs lincomycin, clindamycin and spectinomycin single or multiple Detecting a residual amount of the drug.

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 lincomycin, clindamycin and spectinomycin, extracted with phosphate buffer, 3% trichloroacetic acid precipitated protein, ion pairs Reversed-phase SPE column purification principle, N, O- bis (trimethylsilyl) trifluoroacetamide derivative, 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. Lincomycin hydrochloride 4.1 Standard. content ≥99%; clindamycin hydrochloride and spectinomycin hydrochloride standard. content ≥95%. 4.2 N, O- bis (trimethylsilyl) trifluoroacetamide. Acetate 4.3. excellent pure. 4.4 Methanol. HPLC grade. 4.5 Acetonitrile. chromatographically pure. 4.6 hexane. chromatography. 4.7 potassium dihydrogen phosphate. pure class distinctions. 4.8 phosphoric acid. pure class. 4.9 Potassium hydroxide. pure class. 4.10 TCA. pure class distinctions. 4.11 sodium dodecyl sulfate. HPLC grade. 4.12 HLB solid phase extraction column. 60mg/3mL, or equivalent person. The extract was 4.13. Take 1.36 g of potassium dihydrogen phosphate, dissolved and diluted to 1000mL with water, adjusted to pH 4.0 with phosphoric acid. 4.14 3% trichloroacetic acid solution. Take trichloroacetic 3.0g, dissolve and dilute to 100mL with water. 4.15 2mol/L potassium hydroxide solution. Take potassium hydroxide 11.6g, dissolve and dilute to 100mL with water. 4.16 sodium dodecyl sulfate solution. Take sodium dodecyl sulfate 2.78g, dissolved and diluted to 50mL with water. 4.17 sodium dodecyl sulphate buffer. Take 2.78 g sodium dodecyl sulfate, dissolved in water, adding acetic acid 1mL, diluted with water 500mL. 4.18 1mg/mL lincomycin, clindamycin and spectinomycin standard stock solution. Weigh accurately lincomycin, clindamycin and spectinomycin Each standard 10mg, respectively 10mL volumetric flask, dissolved in methanol and diluted to the mark, formulated at a concentration of 1mg/mL of Lincomycin Su, clindamycin and spectinomycin standard stock solution. 2 ℃ ~ 8 ℃ from light, valid for 6 months. 4.19 100μg/mL standard working solution. precise amount of 1mg/mL lincomycin, clindamycin and spectinomycin standard stock solution of each 1.0 mL, respectively 10mL volumetric flask, dilute to the mark with methanol, formulated at a concentration of 100μg/mL lincomycin, clindamycin, and Spectinomycin standard working solution. 2 ℃ ~ 8 ℃ from light, one week period. 5. Apparatus 5.1 Gas chromatography - mass spectrometry. with the 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 freezing high-speed centrifuge. 5.5 electric heated water bath. 5.6 vortex mixer. 5.7 homogenizer. 5.8 PTFE tube. 50mL. Preparation and Storage of sample 6 6.1 Preparation of the sample 6.1.1 pork, beef and chicken muscle, liver and kidney tissue 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. 6.1.2 milk and eggs An appropriate amount of fresh or frozen test blank or milk and eggs, 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 The following tissue samples stored at -20 ℃. Eggs and milk sample or less 2 ℃ ~ 8 ℃ stored. Determination Step 7 7.1 Preparation of matrix-matched standard curve Were the precise amount of 100μg/mL lincomycin, clindamycin and spectinomycin appropriate amount of working standard solution, diluted with methanol and formulated into concentrated 50,100,200,400,800,1600 degrees and 3200μg/L of a mixed series of standard solution from each 1.0 mL, extracted separately dissolved, Purifying and drying the residue of the sample blank, thoroughly mixed, dried at room temperature, was added N, O- bis (trimethylsilyl) trifluoroacetamide and 500μL 100 L acetonitrile, vortex mixed for 1min, sealed at 75 deg.] C incubator IH derivatization reaction, dry nitrogen at room temperature, was added n-hexane solution 0.5mL The residue solution was vortexed, gas chromatography - mass spectrometry. To measure 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 muscle and kidney tissue Sample Weigh 5g ± 0.05g, polytetrafluoroethylene in 50mL centrifuge tube, the extract was added 10mL, shaking 5min, 5000r/min Centrifugal 10min, supernatant to another centrifuge tube, extract the residue was added 10mL, extraction was repeated once, the two supernatants were combined, plus 3% trichloroacetic acid solution 5mL, mix, 8000r/min centrifugal 10min, the supernatant was 2mol/L potassium hydroxide solution adjusted to pH 5.8 ± 0.2, sodium dodecyl sulfate solution was added 2mL, vortexed, allowed to stand for 15min, standby. 7.2.2 milk and eggs organization Sample Weigh 5g ± 0.05g, polytetrafluoroethylene in 50mL centrifuge tube, the extract was added 10mL, shaking 5min, 5000r/min Centrifugal 10min, supernatant to another centrifuge tube, extract the residue was added 10mL, extraction was repeated once, the two supernatants were combined, add n Hexane 5mL, mixing, 5000r/min centrifugal 15min, the lower layer were washed with 2mol/L potassium hydroxide solution adjusted to pH 5.8 ± 0.2, add Sodium dodecyl sulfate solution 2mL, vortexed, allowed to stand for 15min, standby. 7.3 Purification HLB 3mL column successively with methanol, water and sodium dodecyl sulfate 3mL 3mL activation buffer, taking stock solution through the column, rinsed with water Twice 3 mL, eluted with 4mL of methanol, collect the eluate, at blowing nitrogen at 40 ℃ to nearly dry, dry at room temperature, was added N, O- bis (C Butyldimethylsilyl) trifluoroacetamide and acetonitrile 500μL 100 L, vortex mixed for 1min, sealed, derived from the reaction 75 deg.] C incubator IH, chamber Temperature nitrogen blow, the residue was dissolved with n-hexane 0.5mL, vortexed, gas chromatography - mass spectrometry. 7.4 Determination 7.4.1 Chromatographic conditions 7.4.1.1 Column. capillary column Rtx-5 (5% phenyl methyl polysiloxane, 30m × 0.25mm), or equivalent person. 7.4.1.2 Inlet temperature. 280 ℃. 7.4.1.3 Injection mode. splitless. 7.4.1.4 Injection volume. 1μL. 7.4.1.5 modes. constant current. 7.4.1.6 Carrier gas. helium (99.999%). 7.4.1.7 column flow rate. 1mL/min. Table 1 7.4.1.8 oven ramp program. Table 1 Column temperature heating procedure Starting temperature Termination temperature Heating rate ℃/min Hold Time min total time min 2.0 2.0 19.5 7.4.2 MS conditions 7.4.2.1 Ion source temperature.200 ℃. 7.4.2.2 Interface Temperature. 280 ℃. 7.4.2.3 Mass collected (the SIM). lincomycin and clindamycin scanning ion. 126 (quantitative ion), 127,73. Spectinomycin scanning ion. 145 (quantitative ion), 171,187,201. 7.4.3 Assay 7.4.3.1 qualitative determination By retention time and the retention time of the standard sample chromatogram, wherein each of the ion peaks corresponding standard concentration for each chromatographic In contrast to the qualitative characteristics of ion peaks. The relative abundance of the sample ions characteristic; relative deviation of retention time with the standard sample is not more than 5% Relative abundance consistent with a considerable degree of mixed standard solution concentration, relative abundance does not exceed a predetermined deviation Table 2, it can be determined present in the sample with The object to be measured. The maximum allowable deviation relative ion abundance qualitative determination of Table 2 The relative ion abundances > 50% > 20-50% of > 10% to 20% ≤10% Permissible relative deviation ± 10% ± 15% ± 20% ± 50% Ratio for the sample and standard solutions try quantitative ion area of single-point calibration for quantification. 7.4.3.2 quantitative determination Take a sample and standard solutions, by external standard method, in order to quantify the peak area of the standard solution and sample solution lincomycin, clindamycin, and large Spectinomycin response value should be within the linear range of the detection instrument. In the above-described chromatography - mass spectrometry under conditions, lincomycin, clindamycin and mildew Grand Su added standard solution and blank sample solution ion mass chromatogram characterized in Appendix A. 7.5 Blank test But without addition of the sample, the same steps employed in parallel operation. Calculation and Expression of Results 8 Single-point calibration. c = cSA AS (1) Matrix-matched standard curve calibrated by. AS = acS b, to obtain a and b, then c = Ab (2) The specimens lincomycin, clindamycin residues and spectinomycin (3) is calculated according to the formula. X = cV (3) Where. C --- try to supply feed solution corresponding lincomycin, clindamycin, or spectinomycin concentration, in micrograms per liter (μg/L); cS --- respective standard solution lincomycin, clindamycin, or spectinomycin concentration, in micrograms per liter (μg/L); A --- try to supply feed solution of the peak area of the corresponding derivative of lincomycin, clindamycin, or spectinomycin; Peak area of the AS derivatives --- respective standard solution lincomycin, clindamycin, or spectinomycin; --- for the respective X-try feed lincomycin, clindamycin residues or spectinomycin micrograms per kilogram (μg/kg); The residue was dissolved V --- 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 The detection limit of the method, lincomycin and clindamycin 15μg/kg, spectinomycin as 25μg/kg. This method Lin quantitative limits and clindamycin in muscle, milk and eggs is 20μg/kg, the quantitative limits for the kidney 50μg/kg; spectinomycin limit of quantitation was 50μg/kg. 9.2 Accuracy This method clindamycin 20μg/kg ~ 200μg/kg, in the lincomycin at 20μg/kg ~ 3000μg/kg and spectinomycin 50μg/kg ~ 4000μg/kg the levels recoveries of 70% to 110%. 9.3 Precision ≤15% relative standard deviation in this method and inter - batch relative standard deviation of ≤20%.

Appendix A

Ion chromatogram Figure A.1 ion chromatogram of the standard solution derivative (Lincomycin and clindamycin 100μg/L, spectinomycin 250μg/L) Figure A.2 in pig muscle ion chromatogram of blank sample Figure A.3 blank in pig muscle added lincomycin, clindamycin and spectinomycin ion chromatogram of the sample (Lincomycin and clindamycin 20μg/kg, spectinomycin 50μg/kg) ...