GB 5009.206-2016 English PDFUS$399.00 · In stock
Delivery: <= 4 days. True-PDF full-copy in English will be manually translated and delivered via email. GB 5009.206-2016: Determination of tetrodotoxin in aquatic products -- HPLC-fluorescence detection method Status: Valid GB 5009.206: Historical versions
Basic dataStandard ID: GB 5009.206-2016 (GB5009.206-2016)Description (Translated English): Determination of tetrodotoxin in aquatic products -- HPLC-fluorescence detection method Sector / Industry: National Standard Classification of Chinese Standard: X04 Word Count Estimation: 20,241 Date of Issue: 2016-12-23 Date of Implementation: 2017-06-23 Older Standard (superseded by this standard): GB/T 23217-2008; GB/T 5009.206-2007; SN/T 1569.2-2013 Regulation (derived from): National Health and Family Planning Commission Notice No.17 of 2016 Issuing agency(ies): National Health and Family Planning Commission of the People's Republic of China, State Food and Drug Administration GB 5009.206-2016: Determination of tetrodotoxin in aquatic products -- HPLC-fluorescence detection 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 tetrodotoxin in aquatic products - HPLC-fluorescence detection method National Standards of People's Republic of China National Food Safety Standard Determination of aquatic tetrodotoxin Published 2016-12-23 2017-06-23 implementation National Health and Family Planning Commission People's Republic of China China Food and Drug Administration issued ForewordInstead of the standard GB/T 23217-2008 "aquatic tetrodotoxin liquid chromatography - fluorescence detection method", GB/T 5009.206- 2007 "Determination of tetrodotoxin in puffer fish fresh" and SN/T 1569.2-2013 "puffer fish tetrodotoxin outlet Detection Method 2 Point. mouse bioassay. " As compared with the present standard GB/T 5009.206-2007, major changes are as follows. --- standard name was changed to "National Food Safety Standard Determination of tetrodotoxin in aquatic products"; --- an increase of purification steps; --- modified enzyme-linked immunosorbent assay. National Food Safety Standard Determination of aquatic tetrodotoxin1 ScopeThis standard specifies the method for the determination of tetrodotoxin in aquatic products. The standard method for the determination of a first puffer fish muscle, liver, skin and gonad tissues tetrodotoxin. The second method is suitable for puffer fish Muscle, liver measured, tetrodotoxin skin and gonads. The third method is suitable for puffer fish, snails, shrimp, oysters, squid, and Clam Determination of tetrodotoxin. The fourth method is suitable for determination of puffer fish muscle, liver, skin and gonad tissues tetrodotoxin. The first mouse bioassay method Principle 2 The principle of tetrodotoxin soluble in an acidic solution, the sample was prepared acetic acid solution twice (0.5%) boiling extraction, centrifugation 20000g The supernatant was collected and the two supernatants were combined volume to 25mL, a mouse bioassay. The mice died after injection of the sample extract Time, isolated rat Units and weight correction unit mice rats, calculated to determine the content of tetrodotoxin.3 Reagents and materialsUnless otherwise indicated, the methods used were of analytical grade reagents, water was used three predetermined water GB 6682. 3.1 Reagent 3.1.1 glacial acetic acid (CH3COOH). 3.1.2 sodium hydroxide (NaOH). 3.2 Reagent preparation 3.2.1 acetic acid (0.5%). 5mL of glacial acetic acid diluted with water to 1L. 3.2.2 sodium hydroxide solution (1mol/L). 40g of sodium hydroxide dissolved in water and dilute to 1000mL. 3.3 Standard Tetrodotoxin (C11H17N3O8, CAS number. 4368-28-9), purity ≥98%, or certified by the state and granted a certificate of standard reference materials Reference material. Boiling water bath for 10min, with constant stirring to prevent clumping tissue. Cooled to room temperature, 20000g high-speed centrifugal 20min, supernatant was pipetted 25mL volumetric flask. Acetic acid solution was added to the precipitate (0.5%) 11mL, mix thoroughly, boiled in a boiling water bath for 5min, stirring constantly to prevent Only organization agglomeration. Cooled to room temperature, 20000g high-speed centrifugal 20min, the supernatant was pipetted. The combined supernatant was mixed with a solution of acetic acid (0.5%) volume to 25mL. This extract is equivalent to 0.4g 1mL sample. 5.2.2 liver tissue samples Accurately weighed 10g (to the nearest 0.1g) in 50mL centrifuge sample tube, was added acetic acid (0.5%) 8mL, mix well, at room temperature High speed centrifugation at 20000g 20min, supernatant was pipetted 25mL volumetric flask. 8mL extracted repeatedly with acetic acid (0.5%) 1 time. The combined supernatant was mixed with a solution of acetic acid (0.5%) volume to 25mL. This extract is equivalent to 0.4g 1mL sample. 5.2.3 skin tissue sample Accurately weighed 10g (to the nearest 0.1g) in 50mL centrifuge sample tube, was added acetic acid (0.5%) 11mL, mix well, chamber High speed centrifugation at 20000g temperature 20min, supernatant was pipetted 25mL volumetric flask. The extraction was repeated with 11mL acetic acid (0.5%) 1 time. The supernatant was combined with a solution of acetic acid (0.5%) volume to 25mL. This extract is equivalent to 0.4g 1mL sample. Note 1. The samples were prepared, frozen samples need to be operated in a semi-thawed state. If not timely test sample, should be stored at 4 ℃, tested within 24h. Note 2. If the case where the puffer fish organs or tissue sample of less than 10g, is extracted according to the above method of operation, need to reduce the amount of acetic acid (0.5%) added. Note 3. extract should contain not be separated from the liquid surface of the oil was centrifuged, gum or lipids. 5.3 mouse assay Select 19.0g ~ 21.0g of specific pathogen free (SPF) male Kunming 6 healthy mice were weighed and body weight was recorded. Randomly divided Of the experimental group and the control group two, three per group. Extract or acetic acid solution to each test mouse by intraperitoneal injection of 1mL sample (0.5%) (control solution). If overflow injection during injection, the mice should be discarded and a re-injected mice. Record Note Shooting start and finish time, carefully observe and record the time of death when the mice stopped breathing (to the last breath is only a mouse). If the sample extract after injection, mice were less than the time of death 7min, press Appendix A Table A.1 calculated 1mL sample extract Virulence, as a reference, formulated ambassador mice died 7min ~ 13min required dilution, use solution of acetic acid (0.5%) in dilute Release, the diluent re-injected mice at least 3 to determine the virulence of the sample. Specific examples in Appendix B. If the sample solution injection, mice Time of death is greater than or equal to 7min, virulence is determined directly from the sample median lethal time. Symptoms of poisoning death in mice. Mice were injected with tetrodotoxin quiet early, followed by difficulty in breathing, shortness of breath, abdominal contraction accelerated, Unresponsive, then sudden Rush, sprint jerks, flee, moved, flipped jumping operation like struggling, after several tens of seconds, mice Leg twitch violently, after 2 or 3 times to climb lying immobile, weak abdominal breathing gradually slowed down, and finally death. To stop breathing as a judgment of death standard.6 expression analysis results6.1 calculate virulence The time to death of mice is obtained in the test, the median lethal time is calculated, and the calculated Table A.1 virulence (if the bit When the time is right in the middle of the number of lethal two times given in Table A.1, whichever is greater of the two values of the time period; if given in Table A.1 of between When the time out of the two, but not exactly in the middle, then take a time value closer to the median time to death). If the mouse body weight is not exactly 20.0 g of, as shown in Table A.2 murine unit (MU) for correcting the extraction is shown by the mouse virulence was corrected unit. Is defined such that 1MU weighing 20.0g of specific pathogen free (SPF) male Kunming mice virulence 30min death phase When to 0.18μg tetrodotoxin. Tetrodotoxin content in the samples according to the formula (1). X = M × E × f × W (1) Where. X-tetrodotoxin content --- sample, in units of units per gram rats (MU/g); Rat number M --- 1mL injection unit, in units of murine units (a MU); E --- extraction coefficient, the method for the extraction coefficient 3.11; f --- Sample dilution liquid extract; --- W is the weight correction coefficient mice. 6.2 report the results In the control group of normal mice, the puffer fish tetrodotoxin content in various tissues is determined and expressed as follows. If the time of death in mice is greater than 30min, results are reported as < 3.11MU/g muscle/liver/skin. If the time of death of mice is smaller than 30min, the results according to equation (1) is calculated to obtain the actual results reported, the sample content of tetrodotoxin ××× MU/g muscle/liver/skin. When 3 mice were dead time is not less than or not greater than 30min 30min, re-injection of 3 mice need to ensure small 3 Time of death mice are greater than or less than 30min 30min, whereby the determination result; if repeated injection three mice still occurs the first time to death Mice mortality times, need to lethal time determination result according to the first injection median of 3 mice.7 OtherThe detection limit of this method is 3.11MU/g. The second method of liquid chromatography - tandem mass spectrometry Principle 8 Sample with 1% acetic acid - methanol solution was extracted, purified immunoaffinity column, liquid chromatography - tandem mass spectrometry, external standard.9 Reagents and materialsUnless otherwise indicated, the reagents used were of analytical grade method, a test water to a predetermined water GB 6682. 9.1 Reagent 9.1.1 methanol (CH3OH). chromatography. 9.1.2 acetonitrile (CH3CN). HPLC grade. 9.1.3 formic acid (HCOOH). chromatography. 9.1.4 glacial acetic acid (CH3COOH). excellent pure. 9.1.5 ammonium acetate (CH3COONH4). chromatography. 9.1.6 dodecahydrate disodium hydrogen phosphate (Na2HPO4 · 12H2O). 9.1.7 sodium phosphate dihydrate (NaH2PO4 · 2H2O) dihydro. 9.1.8 Sodium chloride (NaCl). 9.1.9 sodium hydroxide (NaOH). 9.2 Reagent preparation 9.2.1 acetate - methanol (199). The glacial acetic acid and methanol at a volume ratio of 199 were mixed to homogeneity. 9.2.2 acetate - methanol (298). The glacial acetic acid and methanol at a volume ratio of 98 2 were mixed to homogeneity. 9.2.3 formic acid (1999). 1mL of formic acid was added to the 999mL of water, and mix. 9.2.4 0.1% formic acid solution containing 5mmol/L ammonium acetate. Weigh 0.19g of ammonium acetate, 0.1% formic acid solution was brought to 500mL. 9.2.5 formic acid (0.1%) - acetonitrile (11). formic acid (0.1%) mixed with an equal volume of acetonitrile. 9.2.6 phosphate buffer solution (PBS solution). Weigh-hydrogen phosphate dodecahydrate, disodium 6.45g, sodium dihydrogen phosphate dihydrate 1.09g, chloride Sodium 4.25g, dissolved in water and dilute to 500mL. 9.2.7 sodium hydroxide solution (1mol/L). 20.0g of sodium hydroxide was weighed accurately, dissolved in water and dilute to 500mL. 9.3 Standard Tetrodotoxin (C11H17N3O8, CAS number. 4368-28-9), purity ≥98%, or certified by the state and granted a certificate of standard reference materials Reference material. 9.4 Standard Preparation 9.4.1 tetrodotoxin Standard stock solution (100μg/mL). Weigh accurately 10 mg tetrodotoxin standard (accurate to 0.1mg), with a small amount of methyl Acid solution (0.1%) was dissolved to methanol to 100mL, -18 ℃ below stored. 9.4.2 tetrodotoxin intermediate standard solution (1μg/mL). exact amount of tetrodotoxin standard stock solution (100μg/mL) amount to formic acid (0.1%) - was diluted in acetonitrile (1 1) and set the volume to prepare a standard intermediate liquid 1μg/mL, here -18 ℃ stored. 9.4.3 tetrodotoxin series of standard working solution. accurately draw tetrodotoxin intermediate standard solution (1μg/mL) amount to formic acid (0.1%) - Acetonitrile (11) and set the volume was diluted to give a concentration of 1μg/L ~ 1000μg/L of the working standard solution series, using now. 9.5 Material 9.5.1 tetrodotoxin immunoaffinity column. column dimensions 3mL, the maximum capacity of the column 1000ng, or equivalent column. The organic phase 9.5.2 0.22μm microporous membrane. 10 instruments and equipment 10.1 liquid chromatography - tandem mass spectrometry. ion source distribution. 10.2 Analytical Balance. a sense of 0.1mg and 0.01g. 10.3 homogenizer. ≥12000r/min. 10.4 a vortex. 10.5 High Speed Refrigerated Centrifuge. speed ≥8000r/min. 10.6 SPE. 10.7 Organomation. 11 analysis steps Note. To avoid hazardous toxins, should wear gloves to operate. Extraction pipette top in the equipment used, the waste liquid or the like to be in a sodium hydroxide solution (1mol/L) Soak for more than 1h to allow toxin. 11.1 Preparation of samples After washing with water the surface of the fish waste, fish surface moisture absorbed by filter paper with scissors decomposed into the fish muscle, liver, skin, and of Glands (testis or ovaries) and other parts, each part of the tissue with blood wash with water, dry paper tissue after water on the surface of each cut into pieces, were sufficiently Mass, into a clean container, and labeled tag. 11.2 sample collection 5g weighed (accurate to 0.01g) sample was homogenized 50mL stoppered centrifuge tube, was added 11mL acetic acid - methanol solution (199), vortex Rotary oscillation 2min, 50 ℃ water bath ultrasonic extraction 15min, at 8000r/min centrifugal 5min, supernatant was transferred to a 25mL volumetric flask. The residue was added 11mL acetic acid - methanol solution (199), the extraction was repeated once, the supernatant was combined with acetic acid - methanol (199) fixed Volume to 25mL. The extract was pipetted to the other about 10mL 50mL stoppered centrifuge tube, -20 ℃ frozen 30min, then at 8000r/min from Heart 5min, 5mL accurate pipetting the supernatant was 20mLPBS solution is diluted with sodium hydroxide solution (1mol/L) adjusted at pH Range of 7 to 8, to be purified. Purification Sample 11.3 The immunoaffinity column in PBS natural sequestration discharge flow rate, liquid to be purified into a flow rate of 1 drop/s and passed through the column, then 10mL water rinsed, drained, with 5mL of acetic acid - methanol solution (298), eluent dry nitrogen at 45 ℃, formic acid was added 1.0mL Solution (0.1%) - acetonitrile (11) was dissolved the residue, ultrasonic 1min, after the organic phase through a microporous membrane 0.22μm for liquid chromatography - Cascade With mass spectrometry. 11.4 instrument reference conditions HPLC conditions refer to 11.4.1 HPLC conditions are listed below with reference to. a) Column. GelAmide-80 column, column length 150mm, inner diameter 2mm, particle size 5 m, column or equivalent. b) flow rate. 0.3mL/min. c) Column temperature. 40 ℃. d) Injection volume. 10μL. e) The mobile phase and gradient conditions are shown in Table 1. Mobile phase Solution A. 0.1% formic acid solution containing 5mmol/L ammonium acetate; mobile phase Solution B. acetonitrile. Table 1 Mobile phase and gradient conditions Time/min A /% B /% 0.0 1090 2.0 10 90 2.1 90 10 6.0 90 10 6.1 1090 8.0 1090 11.4.2 Mass Reference Conditions Mass spectrum Reference conditions are listed below. a) ion source. electrospray ionization source (ESI); b) scan mode. positive ion scan; The standard series tetrodotoxin working solution were injected into the liquid chromatography - tandem mass spectrometer, the measured peak area corresponding to the standard series of work Concentration of the solution as the abscissa tetrodotoxin, TTX peak area of peaks of the vertical axis, the standard curve. 11.6 Determination of the sample solution The sample solution is injected into the liquid chromatography - tandem mass spectrometer, the peak area obtained in response, the test solution to give a standard curve tetrodotoxin The concentration of pigment. Tetrodotoxin extracted ion chromatograms of standard solutions See Appendix C in FIG C.1. 11.7 Qualitative Under the same test conditions, the sample solution is compared to the retention time of the standard solution of tetrodotoxin, a deviation within ± 2.5%, and the subject Measured the relative abundance of ions, it should be consistent with the concentration of the standard working solution similar relative abundances of ions, which should be consistent with the abundance ratio deviation Table 3 Claim. Table 3 Maximum allowable deviation relative ion abundances The relative ion abundances > 50% > 20% ~ ≤50% > 10% ~ ≤20% ≤10% Permissible relative deviation of ± 20% ± 25% ± 30% ± 50% 11.8 blank test Except that no sample addition are by the measuring step. 12 expression analysis results Tetrodotoxin content in the samples according to the formula (2). X = c × V × f (2) Where. 16.1.3 acetonitrile (CH3CN). HPLC grade. 16.1.4 glacial acetic acid (CH3COOH). chromatography. 16.1.5 ammonium acetate (CH3COONH4). chromatography. 16.1.6 dodecahydrate disodium hydrogen phosphate (Na2HPO4 · 12H2O). 16.1.7 Sodium phosphate dihydrate (NaH2PO4 · 2H2O) dihydro. 16.1.8 Sodium chloride (NaCl). 16.1.9 Sodium hydroxide (NaOH). 16.1.10 heptane sulfonate (C7H15NaO3S). 16.1.11 sodium carbonate (Na2CO3). 16.2 reagent preparation 16.2.1 Sodium hydroxide solution (1mol/L). Weigh 40g of sodium hydroxide, dissolved in water and diluted to 1000mL. 16.2.2 phosphate buffered saline (PBS solution). Weigh 6.45g of disodium hydrogen phosphate dodecahydrate, 1.09 g of sodium dihydrogen phosphate dihydrate, 4.25g of sodium chloride, dissolved in water and dilute to 500mL. 16.2.3 acetate - methanol (199). The glacial acetic acid and methanol at a volume ratio of 199 were mixed to homogeneity. 16.2.4 acetate - methanol (298). The glacial acetic acid and methanol at a volume ratio of 98 2 were mixed to homogeneity. 16.2.5 ammonium acetate buffer solution. Weigh 4.6g of ammonium acetate and 2.02g of heptane sulfonate, was dissolved by adding about 700mL of water, adjusted with glacial acetic acid pH 5.0, diluted with water to 1L. 16.2.6 sodium hydroxide solution (4mol/L). Weigh 160g of sodium hydroxide, dissolved in and diluted to 1L with water. 16.2.7 acetic acid solution (1%). glacial acetic acid and water at a volume ratio of 199 were mixed to homogeneity. 16.3 Standards Tetrodotoxin (C11H17N3O8, CAS number. 4368-28-9), purity ≥98%, or certified by the state and granted a certificate of standard reference materials Reference material. 16.4 Standard Preparation 16.4.1 standard stock solution (100μg/mL). Weigh accurately 10 mg tetrodotoxin standard (accurate to 0.1 mg), was dissolved with a small amount of water. In methanol to 100mL. It can be stored at 4 ℃ month. 16.4.2 standard working solution. stock standard solution appropriate amount according to need, standard working solution diluted to an appropriate concentration in a solution of acetic acid (1%). Pro with the existing service. 16.4.3 matrix-matched series of standard working solution. Prepare a standard solution in a series of blank substrate working solution of appropriate concentration. Using now. 16.5 Materials 16.5.1 tetrodotoxin immunoaffinity column. column dimensions 3mL, the maximum capacity of the column 1000ng, or equivalent column. 16.5.2 0.22μm aqueous phase microporous membrane. 17 instruments and equipment 17.1 liquid chromatograph. with the column derivatization and fluorescence detection means. 17.2 Electronic balance. a sense of the amount of 0.1mg and 0.01g. 17.3 organization stamp mill. 17.4 oscillator. 17.5 ultrasonic generator. 17.6 SPE. 17.7 Centrifuge. ≥8000r/min. 17.8 Organomation. 18 analysis steps Note. To avoid hazardous toxins, should wear gloves to operate. Extraction pipette top in the equipment used, the waste liquid or the like to be in 1mol/L sodium hydroxide solution 1h soaking solution above, so toxin. 18.1 Preparation of samples After washing with water the surface of the fish waste, fish surface moisture absorbed by filter paper with scissors decomposed into the fish muscle, liver, skin, and of Glands (testis or ovaries) a......Tips & Frequently Asked Questions:Question 1: How long will the true-PDF of GB 5009.206-2016_English be delivered?Answer: Upon your order, we will start to translate GB 5009.206-2016_English as soon as possible, and keep you informed of the progress. The lead time is typically 2 ~ 4 working days. The lengthier the document the longer the lead time.Question 2: Can I share the purchased PDF of GB 5009.206-2016_English with my colleagues?Answer: Yes. 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