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Delivery: <= 3 days. True-PDF full-copy in English will be manually translated and delivered via email. GB 5009.204-2014: National Food Safety Standard -- Determination of acrylamide in food Status: Valid GB 5009.204: Historical versions
Basic dataStandard ID: GB 5009.204-2014 (GB5009.204-2014)Description (Translated English): National Food Safety Standard -- Determination of acrylamide in food Sector / Industry: National Standard Classification of Chinese Standard: X04 Classification of International Standard: 67.040 Word Count Estimation: 13,168 Date of Issue: 12/1/2014 Date of Implementation: 5/1/2015 Older Standard (superseded by this standard): GB/T 5009.204-2005 Regulation (derived from): National Health and Family Planning Committee Announcement 2014 No. 19 Issuing agency(ies): National Health and Family Planning Commission of the People's Republic of China Summary: This Standard specifies the method for the determination of acrylamide in foods. This Standard applies to thermal processing (such as fried, broiled, baked, etc.) Determination of acrylamide in food. GB 5009.204-2014: National Food Safety Standard -- Determination of acrylamide in food---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.(National Food Safety Standard Determination of acrylamide in food) National Standards of People's Republic of China National Food Safety Standard Determination of acrylamide in food Issued on. 2014-12-01 2015-05-01 implementation ForewordThis standard replaces GB/T 5009.204-2005 "foods Determination of acrylamide content by gas chromatography-mass spectrometry (GC-MS) method." This standard compared with GB/T 5009.204-2005, the main changes are as follows. - Increasing the first method using stable isotope dilution liquid chromatography - mass spectrometry/mass spectrometry; - Gas chromatography - mass spectrometry to the external standard method using stable isotope dilution method. National Food Safety Standard Determination of acrylamide in food1 ScopeThis standard specifies the method for the determination of acrylamide in food. This standard applies to thermal processing (such as fried, broiled, baked, etc.) Determination of acrylamide in food. The first method of stable isotope dilution liquid chromatography - mass spectrometry/mass spectrometry Principle 2 The standard application stable isotope dilution technique, adding acrylamide 13C3 labeled internal standard solution in the sample, with water as the extraction solvent, After solid phase extraction column or matrix solid-phase dispersion extraction purification, liquid chromatography - mass spectrometry/mass spectrometry in multiple reaction monitoring (MRM) or anti-choice It should monitor (SRM) to detect internal standard.3 Reagents and materialsNote. Unless otherwise indicated, the methods used were of analytical grade reagents and water as a water GB/T 6682 regulations. 3.1 Reagents 3.1.1 formic acid (HCOOH). chromatography. 3.1.2 Methanol (CH3OH). chromatography. 3.1.3 n-hexane (n-C6H14). AR, redistilled after use. 3.1.4 ethyl acetate (CH3COOC2H5). AR, redistilled after use. 3.1.5 anhydrous sodium sulfate (Na2SO4). 400 ℃, baked 4 h. 3.1.6 Ammonium sulfate [(NH4) 2SO4]. 3.1.7 diatomite. ExtrelutTM 20 or equivalent. 3.2 Standard 3.2.1 acrylamide (CH2 = CHCONH2) standards (purity > 99%). 3.2.2 13C3- acrylamide ( CH2 = CH CONH2,) standards (purity > 98%). 3.3 preparation of standard solution 3.3.1 preparation of standard solution of acrylamide 3.3.1.1 acrylamide standard stock solution (1000 mg/L). Weigh accurately acrylamide standards, dissolved in methanol and set the volume so that propionate Acrylamide concentration of 1000 mg/L, set -20 ℃ refrigerator. 3.3.1.2 acrylamide intermediate solution (100 mg/L). Pipette acrylamide standard stock solution 1 mL, diluted with methanol to 10 mL, so Acrylamide concentration of 100 mg/L, set -20 ℃ refrigerator. 3.3.1.3 acrylamide working solution Ⅰ (10 mg/L). Pipette acrylamide intermediate solution 1 mL, with 0.1% formic acid solution was diluted to 10 mL, acrylamide concentration of 10 mg/L. Pro formulated with time. 3.3.1.4 acrylamide working solution Ⅱ (1 mg/L). Pipette acrylamide working solution Ⅰ1 mL, with 0.1% formic acid solution was diluted to 10 mL ,, acrylamide concentration of 1 mg/L. Pro formulated with time. 3.3.2 C3- acrylamide internal standard solution 3.3.2.1 13C3- acrylamide internal standard stock solution (1000 mg/L). Weigh accurately C3- acrylamide standards, dissolved in methanol and Volume, so 13C3- acrylamide concentration of 1 000 mg/L, set -20 ℃ refrigerator. 3.3.2.2 internal standard working solution (10 mg/L). Pipette internal standard stock solution 1 mL, diluted with methanol to 100 mL, so 13C3- acryloyl Amine concentration of 10 mg/L, set -20 ℃ refrigerator. 3.3.3 standard curve working solutions Take six 10 mL flask, respectively Pipette 0.1 mL, 0.5 mL, 1 mL of acrylamide working solution II (1 mg/L) and 0.5 mL, 1 mL and 3 mL of acrylamide working solution I (10 mg/L) and the internal standard working solution (10 mg/L) 0.1 mL, diluted with 0.1% formic acid Explanation to the mark. Standard solution concentration of acrylamide in the series were 10 μg/L, 50 μg/L, 100 μg/L, 500 μg/L, 1000 μg/L, 3000 μg/L, the internal standard at a concentration of 100 μg/L. Pro formulated with time.4 instruments and equipment4.1 Liquid chromatography - mass spectrometry/mass spectrometry (LC-MS/MS). 4.2 HLB solid phase extraction column. 6 mL, 200 mg, or equivalent. 4.3 Bond Elut-Accucat SPE. 3 mL, 200 mg, or equivalent. 4.4 tissue grinder. 4.5 rotary evaporator. 4.6 nitrogen concentrator. 4.7 oscillator. 4.8 Glass column. column length 30 cm, column diameter 1.8 cm. 4.9 vortex mixer. 4.10 ultrapure water system. 4.11 Analysis of balance. a sense of the amount of 0.1 mg. 4.12 Centrifuge. Speed ≤10000 r/m. Step 5 Analysis 5.1 Sample Preparation 5.1.1 Sample Extraction Take 50 g sample, the pulverizer, -20 ℃ cryopreservation. Accurately weighed sample 1 g ~ 2 g (accurate to 0.001 g), was added 10 mg/L C3- acrylamide internal standard working solution of 10 μL (or 20 μL), equivalent to 100 ng (or 200 ng) of C3- acrylamide internal standard. After the addition of ultrapure water 10 mL, shaken for 30 min, at 4000 r/m centrifuged 10 min, the supernatant to be purified. 5.1.2 Sample purification NOTE. Optionally the following methods for purification. 5.1.2.1 Matrix Solid Phase Extraction Method (Option 1). Ammonium sulfate was added to the supernatant samples extracted in 15 g, oscillation 10 min, so that Its fully dissolved at 4000 r/m centrifuged 10 min, the supernatant 10 mL, spare. The supernatant was insufficient as 10 mL, then with saturated ammonium sulfate Complement. Take a clean glass column, at the bottom of a small glass wool and compacted fill, followed by filling 10 g of anhydrous sodium sulfate, 2 g of diatomaceous earth. Weigh 5 g Celite ExtrelutTM supernatant after 20 and the sample stirring evenly into a chromatography column. Rinse with 70 mL of n-hexane, control flow Speed of 2 mL/min, n-hexane eluent was discarded. Elution with 70 mL ethyl acrylamide, controlled flow rate of 2 mL/min, collecting B Ethyl elution solution, and reduced pressure on a rotary evaporator to near dryness at 45 ℃ water bath, the residue was washed with ethyl acetate evaporating flask three times (each 1 mL), And transferred to a test tube was added 1 mL of 0.1% formic acid solution, vortexing. After a stream of nitrogen blown off the upper organic phase was added 1 mL Hexane, vortexing at 3500 r/m centrifuged 5 min, take the lower aqueous phase was 0.22 μm membrane filter the aqueous phase until the LC-MS/MS assay. 5.1.2.2 Solid phase extraction (SPE) (Option 2). Add 5 mL of n-hexane in the supernatant samples extracted shaken extraction 10 min, in 10000 r/m centrifuged 5 min, the organic phase was removed, then extracted once with 5 mL of n-hexane was repeated, with 6 mL water quickly through 0.45 μm aqueous phase Membrane filter, to be performed HLB solid phase extraction (SPE) process. HLB solid phase extraction column prior to use successively with 3 mL of methanol, 3 mL of water activation. Take the above filtrate 5 mL HLB solid phase extraction column effluent was collected and eluted with 4 mL of 80% aqueous methanol, collect all the elution Liquid, and combined with the effluent to be subjected to Bond Elut-Accucat solid-phase extraction (SPE); Bond Elut-Accucat followed by solid phase extraction column 3 mL of methanol, after 3mL water-activated, on all the sample eluent HLB solid phase extraction (SPE), the outflow by gravity, collect all Effluent, under nitrogen stream effluent was concentrated to near dryness with 0.1% formic acid solution to volume 1.0 mL, until LC-MS/MS assay. 5.2 Instrument Reference conditions 5.2.1 Chromatographic conditions The column was Atlantis C18 column (5 μm, 2.1 mm ID × 150 mm) or an equivalent post. Pre-column. C18 guard column (5 μm, 2.1 mm ID × 30 mm) or an equivalent post. Mobile phase. 0.1% formic acid in methanol (10.90 by volume). Flow rate. 0.2 mL/min. Injection volume. 25 μL. Column temperature. 26 ℃. 5.2.2 MS parameters 5.2.2.1 triple quadrupole tandem mass spectrometer Detection mode. multiple reaction monitoring (MRM). Ionization. Cationic electrospray ionization (ESI). Capillary voltage. 3 500 V. Cone voltage. 40 V. RF Lens 1 Voltage. 30.8 V. Ion source temperature. 80 ℃. Desolvation temperature. 300 ℃. Ion collision energy. 6 eV. Acrylamide. precursor ion m/z 72, ion m/z 55, ion m/z 44. C3 acrylamide. precursor ion m/z 75, ion m/z 58, ion m/z 45. Quantitative ion. acrylamide m/z 55,13C3 acrylamide m/z 58. 5.2.2.2 Ion Trap Tandem Mass Spectrometer Detection. Select reaction monitoring (SRM). Ionization. Cationic electrospray ionization (ESI). Spray voltage. 5 000 V. Heated capillary temperature. 300 ℃. Sheath gas. N2,40 Arb. Auxiliary gas. N2,20 Arb. Collision-induced dissociation (CID). 10 V. Collision energy. 40 V. Acrylamide. precursor ion m/z 72, ion m/z 55, ion m/z 44. C3 acrylamide. precursor ion m/z 75, ion m/z 58, ion m/z 45. Quantitative ion. acrylamide m/z 55,13C3 acrylamide m/z 58. Draw 5.3 standard curve The standard series working solution were injected into the liquid chromatography - mass spectrometry/mass spectrometry system to determine the corresponding acrylamide and the internal standard peak area to Acrylamide series of each standard working solution sample concentration (μg/L) as the abscissa, acrylamide (m/z 55) and the internal standard 13C3 acrylamide (M/z 58) peak area ratio of the vertical axis, the standard curve. 5.4 Determination of the sample solution The sample solution was injected into the liquid chromatography - mass spectrometry/mass spectrometry system, measured acrylamide (m/z 55) and 13C3 acrylamide internal standard (m/z 58) The peak area ratio according to the standard curve to obtain an acrylamide test solution sample concentration (μg/L), at least twice the number of measurements in parallel. 5.5 Mass Spectrometry Respectively, the sample and standard series working solution into the liquid chromatography - mass spectrometry/mass spectrometer, record the total ion chromatogram and mass spectra (see Appendix A Figure A.1 ~ Figure A.2) and acrylamide and the internal standard peak area, retention time and fragment ion abundance qualitative requirements detected prop When acrylamide peak signal to noise ratio (S/N) is greater than 3, and the retention time of the standard solution is retained in the test sample of the title compound in the target compound Between consistent while being monitored peak abundance of the respective ion abundance ratio of the standard solution of the test sample of the title compound in a ratio of the target compound Caused, deviation allowed in Table 1. The maximum permissible relative ion abundances in Table 1 when measured deviation qualitative Relative ion abundances (Baseline peak%) Permissible relative deviation (RSD) > 50% ± 20% > 20% ~ 50% ± 25% > 10% ~ 20% ± 30% ≤10% ± 50%6 expression analysisAcrylamide content in the sample according to the formula (1) internal standard method. fA (1) Where. X-- sample content of acrylamide in micrograms per kilogram (μg/kg); A-- sample Acrylamide (m/z 55) peak with 13C3 acrylamide internal standard (m/z 58) peak area ratio of the peaks corresponding Acrylamide mass, in units of nanograms (ng); Within f-- sample standard amount of scaling factors (when the internal standard 10 μL f = 1 or when the internal standard 20 μL f = 2); Sample size M-- internal standard when, in grams (g). The calculation result of the arithmetic mean of two under the same condition of independent determination results indicated that the results of three significant figures (or One after the decimal point).7 precisionTwo independent determination results under the absolute difference in repeatability condition must not exceed 20% of the arithmetic mean.8 OtherQuantitative limit of 10 μg/kg. The second method of stable isotope dilution gas chromatography - mass spectrometry Principle 9 The standard application stable isotope dilution technique, adding acrylamide 13C3 labeled internal standard solution in the sample, with water as the extraction solvent, Sample extract matrix solid-phase dispersion extraction purification, after bromine reagent derived, using gas chromatography - tandem mass spectrometer ion multiple reaction monitoring (MRM) or gas chromatography - mass spectrometry in selected ion monitoring (SIM) to detect internal standard. 10 Reagents and materials Note. Unless otherwise indicated, the methods used were of analytical grade reagents, ultra-pure water. 10.1 Reagents 10.1.1 hexane (n-C6H14). AR, redistilled after use. 10.1.2 ethyl acetate (CH3COOC2H5). AR, redistilled after use. 10.1.3 anhydrous sodium sulfate (Na2SO4). 400 ℃, baked 4 h. 10.1.4 ammonium sulfate [(NH4) 2SO4]. 10.1.5 sodium thiosulfate (Na2S2O3.5H2O). 10.1.6 bromine (Br2). 10.1.7 hydrobromide (HBr). content > 48.0%. 10.1.8 potassium bromide (KBr). 10.1.9 ultrapure water conductivity (25 ℃) ≤0.01 mS/m. 10.1.10 bromine reagents. 10.1.11 diatomite. ExtrelutTM 20 or equivalent. 10.2 reagent preparation 10.2.1 saturated bromine water. 100 mL of ultrapure water, placed in 200 mL brown reagent bottle, 8 mL of bromine, 4 ℃ dark place 8 h, the upper saturated aqueous bromine. 10.2.2 bromine reagent. Weigh potassium bromide 20.0 g, add ultrapure water 50 mL, completely dissolved, then add 1.0 mL and 16.0 mL hydrobromide Saturated bromine water, shake, diluted with ultrapure water to 100 mL, 4 ℃ stored. 10.2.3 sodium thiosulfate solution (0.1 mol/L). Weigh sodium thiosulfate 2.48 g, add ultrapure water 50 mL, completely dissolved, ultra Water was diluted to 100 mL, 4 ℃ stored. 10.2.4 saturated ammonium sulfate solution. Weigh 80 g of ammonium sulfate crystals were added to ultrapure water 100 mL, sonication, at room temperature. 10.3 Standards 10.3.1 acrylamide (CH2 = CHCONH2) standards. purity > 99%. 10.3.2 13C3- acrylamide (13CH2 = CH CONH2,) standards. purity > 98%. Preparation of standard solution of 10.4 10.4.1 acrylamide and the internal standard solution. with 3.3.1 and 3.3.2. 10.4.2 standard curve working solution. take five 10 mL volumetric flask, respectively Pipette 0.1 mL, 0.5 mL, 2 mL solution of acrylamide work Liquid Ⅱ (1 mg/L) and 1 mL and 0.5 mL of working solution of acrylamide Ⅰ (1 mg/L) and 0.5 mL of internal standard working solution (1 mg/L), Dilute to the mark with ultrapure water. Series of standard solutions of acrylamide concentrations of 10 μg/L, 50 μg/L, 200 μg/L, 500 μg/L, 1000 μg/L, the internal standard at a concentration of 50 μg/L. Pro formulated with time. 11 instruments and equipment 11.1 Gas chromatography - quadrupole mass spectrometer (GC-MS). 11.2 Column. DB-5ms column (30 m × 0.25 mm id × 0.25 μm) or equivalent columns. 11.3 tissue grinder. 11.4 rotary evaporator. 11.5 nitrogen concentrator. 11.6 oscillator. 11.7 Glass Column. column length 30 cm, column diameter 1.8 cm. 11.8 vortex mixer. 11.9 ultra-pure water equipment. 11.10 analytical balance. a sense of the amount of 0.1 mg. 11.11 centrifuge. speed ≤10 000 r/m. 12 analysis steps 12.1 Sample Preparation 12.1.1 Sample Extraction Take 50 g sample, the pulverizer, -20 ℃ cryopreservation. Accurately weighed sample 2 g (accurate to 0.001 g), was added 10.0 mg/L C3- acrylamide standard solution 10 μL (or 20 μL), equivalent to 100 ng (or 200 ng) of C3- acrylamide internal standard, then add Super Water 10 mL, after shaking 30 min, at 4 000 r/m centrifuged 10 min, the supernatant spare. 12.1.2 sample clean-up It was added to the supernatant samples extracted in ammonium sulfate 15 g, oscillation 10 min, to fully dissolve at 4 000 r/m centrifuged 10 min, The supernatant was 10 mL, spare. The supernatant was insufficient as 10 mL, then with saturated ammonium sulfate supplement. Take a clean glass column, at least in the bottom of the fill Xu glass wool, pressed successively filled anhydrous sodium sulfate 10 g, ExtrelutTM 20 diatomite 2 g. Weigh 5g ExtrelutTM 20 and diatomaceous earth Supernatant samples above spare stirring evenly into a chromatography column. Rinse with 70 mL of n-hexane, controlling the flow rate of 2 mL/min, abandoned Go hexane eluent. With 70 mL ethyl acetate, to control the flow rate of 2 mL/min, collecting a solution of ethyl acetate and 45 ℃ Water bath rotary evaporator under reduced pressure to near dryness, the residue was washed with ethyl acetate evaporating flask three times (each 1 mL), and transferred to a 1 mL of Ultrapure water tubes vortexed. After a stream of nitrogen blown off the upper organic phase was added 1 mL of n-hexane, vortexed at 3500 r/m Centrifugation 5 min, take the lower aqueous phase alternate derivative. 12.1.3 Derivatives Derived sample. Bromine reagent in the sample extract 1 mL, vortexing, 4 ℃ for at least 1 h after the addition of 0.1 mol/L sulfur Sodium thiosulfate solution of about 100 μL, vortexing to remove excess derivatizing agent; 2 mL ethyl acetate was added, vortexed 1 min, in 4000 r/m centrifuged 5 min, draw the upper organic phase was transferred to a test tube added with 0.1 g of anhydrous sodium sulfate was added 2 mL of ethyl acetate and extracted repeatedly, The combined organic phases; stand for at least 0.5 h, transferred to another tube, in a stream of nitrogen blow to nearly dry, add 0.5 mL residue was dissolved in ethyl acetate (Note Italy. according to the sensitivity of the instrument, adjust the volume of the residue was dissolved in ethyl acetate, under normal circumstances, the use of tandem mass spectrometer, the amount used To 0.5 mL, single-stage mass spectrometer, used in an amount of 0.1 mL. ),spare. Derived from the standard series solution. Take the amount of the standard series solution of 1.0 mL, synchronous operation in accordance with the sample derived methods. 13 instrument reference conditions 13.1 Chromatographic conditions Column. DB-5 ms column (30 m × 0.25 mm ID × 0.25 μm) or equivalent columns. Inlet temperature. 120 ℃ to keep 2 min, to 40 ℃/min rate was raised to 240 ℃, and holding 5 min. Column temperature program. 65 ℃ held 1 min, to 15 ℃/min rate was raised to 200 ℃, then to 40 ℃/min ramped 240 ℃, And holding 5 min. Carrier gas. Helium (purity > 99.999%), pre-column pressure of 69 mPa, equivalent to 10 psi. Splitless injection volume 1 μL. 13.2 MS parameters Detection mode. selected ion monitoring (SIM) acquisition. Ionization mode. electron bombardment source (EI), energy of 70 eV. Transfer line temperature. 250 ℃. Ion source temperature.200 ℃. Solvent delay. 6 min; MS Acquisition time. 6 min ~ 12 min. Acrylamide monitoring ions m/z 106,133,150 and 152, quantitative ion m/z 150. C3- acrylamide internal standard monitoring ions m/z 108,136,153 and 155, quantitative ion m/z 155. 13.3 standard curve The standard series working solution derived respectively injected into the gas chromatography - mass spectrometry system to measure the corresponding acrylamide and the internal standard peak area, In each series of standard working solution of acrylamide sample concentration (μg/L) as the abscissa, acrylamide and quantitative internal standard 13C3 acrylamide from Sub-measured mass chromatogram peak area ratio of the vertical axis, linear curve. 13.4 Determination of the sample solution The derivatized sample solution injected into the gas chromatography - mass spectrometry system to get the peak a......Tips & Frequently Asked Questions:Question 1: How long will the true-PDF of GB 5009.204-2014_English be delivered?Answer: Upon your order, we will start to translate GB 5009.204-2014_English as soon as possible, and keep you informed of the progress. 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