GB 5009.213-2016 English PDFUS$519.00 · In stock
Delivery: <= 5 days. True-PDF full-copy in English will be manually translated and delivered via email. GB 5009.213-2016: Determination of paralytic shellfish poison in shellfish Status: Valid GB 5009.213: Historical versions
Basic dataStandard ID: GB 5009.213-2016 (GB5009.213-2016)Description (Translated English): Determination of paralytic shellfish poison in shellfish Sector / Industry: National Standard Classification of Chinese Standard: C53 Classification of International Standard: 67.040 Word Count Estimation: 26,226 Date of Issue: 2016-12-23 Date of Implementation: 2017-06-23 Older Standard (superseded by this standard): GB/T 23215-2008; GB/T 5009.213-2008; SC/T 3023-2004; SN 0352-1995; SN/T 1735-2006; SN/T 1773-2006 Regulation (derived from): National Health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 Summary: This standard specifies the mouse biology, enzyme-linked immunosorbent assay, liquid chromatography and liquid chromatography-tandem mass spectrometry for the determination of paralytic shellfish toxins in shellfish. This standard is applicable to the detection of paralytic shellfish toxins in shellfish and its products. GB 5009.213-2016: Determination of paralytic shellfish poison in shellfish---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 paralytic shellfish poison in shellfish National Standards of People's Republic of China National Food Safety Standard Determination of shellfish paralytic shellfish toxins Issued on. 2016-12-23 2017-06-23 implementation National Health and Family Planning Commission People's Republic of China China Food and Drug Administration released ForewordThis standard replaces GB/T 5009.213-2008 "shellfish paralytic shellfish toxins Determination", GB/T 23215-2008 "shellfish ", SC/T 3023-2004" Determination of paralytic shellfish toxins fluorescence detection - more paralytic shellfish poisoning Determination Liquid Chromatography Biological ", SN0352-1995" export shellfish paralytic shellfish toxin testing methods ", SN/T 1735-2006" Importers and shellfish The paralytic shellfish toxin testing method of high performance liquid chromatography "and SN/T 1773-2006" Importers shellfish paralytic shellfish toxins The method of enzyme-linked immunosorbent assay test method. " This standard compared with GB/T 5009.213-2008, the main changes are as follows. --- Standard name was changed to "national food safety standards in shellfish paralytic shellfish toxins determination"; --- Increased enzyme linked immunosorbent assay; --- Increased liquid chromatography - tandem mass spectrometry. National Food Safety Standard Determination of shellfish paralytic shellfish toxins1 ScopeThis standard specifies the shellfish paralytic shellfish toxin assay mouse bioassay, enzyme-linked immunosorbent assay, liquid chromatography and liquid chromatography Spectrum - tandem mass spectrometry. This standard applies to oysters, scallops and other shellfish and their products paralytic shellfish toxin testing. Mouse bioassay Principle 2 Extraction shellfish paralytic shellfish poisoning (PSP) with hydrochloric acid. Record mice injected liquid extraction after the time of death, according to the paralytic Shellfish toxins cause death time in mice and rats for relations table isolated mouse units (MU), and press the mouse body weight of rats correction unit Corrected mouse unit (CMU), calculated mouse units per 100g sample of the PSP. In saxitoxin as a standard, the rats unit In terms of the number of micrograms of toxin calculated per 100g shellfish meat PSP micrograms. The measurement results in the presence of representatives of a variety of chemical structures shellfish The total amount of PSP toxins.3 Reagents and materialsUnless 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 sodium hydroxide (NaOH). 3.1.2 hydrochloric acid (HCl). 3.1.3 absolute ethanol (CH3CH2OH). 3.2 reagent preparation 3.2.1 sodium hydroxide solution (0.1mol/L). the 4.0g of sodium hydroxide in 1L of water. 3.2.2 hydrochloric acid solution (0.18mol/L). The 15.5mL hydrochloric acid diluted to 1L with distilled water. 3.2.3 hydrochloric acid (5mol/L). 45mL of hydrochloric acid diluted with water to 100mL. 3.2.4 acidic ethanol solution. the amount of anhydrous ethanol 200mL, diluted with water to 1000mL, and mix, using hydrochloric acid solution (5mol/L) to pH 2.0 to 4.0. 3.3 Standard Standard saxitoxin (STX, C10H17N7O4 · 2HCl, CAS No. 35554-08-6). purity ≥98.0%. 3.4 preparation of standard solution 3.4.1 saxitoxin standard stock solution (100μg/mL). accurately weighed amount STX standard, with an acidic ethanol solution was dissolved and set the volume, STX is formulated as a concentration of 100μg/mL standard stock solution. 3.4.2 saxitoxin standard working solution (1μg/mL). Imbibe 1mL saxitoxin standard stock solution (100μg/mL), water Diluted, adjusted with hydrochloric acid solution (5mol/L) pH to 2.0 to 4.0, and water volume to 100mL. The standard working solution at 0 ℃ ~ 4 ℃ under Save 30d. 3.5 Material 3.5.1 Mouse. weight healthy ICR male mice of 19g ~ 21g. 3.5.2 pH meter or pH paper.4 instruments and equipment4.1 homogenizer. 4.2 Analysis of balance. a sense of the amount of 0.1g and 0.0001g. 4.3 centrifuge. speed ≥2000r/min. Step 5 Analysis Note. In order to avoid endangering toxins should wear gloves to operate. Pipette and pipette class used equipment, etc. should be discarded extract in sodium hypochlorite solution Solution (5%) or more in order to make immersed 1h toxin. For the sewage generated in the process of animal waste and animal carcass disposal, reference should be GB 14925 execution. 5.1 Sample Collection Take adequate number of shellfish samples from more than 2kg, shelled shellfish make up to 200g or more. Not timely submission of fresh shellfish, open The separated shell shellfish meat, 200g of shellfish meat after draining into 200mL of hydrochloric acid solution (0.18mol/L), and at 4 ℃, equipment seized. 5.2 Sample Preparation 5.2.1 oysters, clams and mussels The appearance of the sample with water shellfish thoroughly washed, cut off the adductor muscle, open the shell, rinsed with distilled water to remove sediment and other foreign matter inside. will Adductor muscle and tissue glue connecting the unit separately, carefully remove the shellfish meat, do not cut the body shell. Non-heating or anesthetic open the shell. Collect about 200g shellfish meat dispersed into the sieve draining 5min (Do not allow the accumulation of meat), fish out the broken shells and other debris, will be homogenized shellfish meat alternate. 5.2.2 scallops Take edible part used as a detector, press 5.2.1 homogeneous spare. 5.2.3 frozen shellfish At room temperature, the sample was frozen (shelled or husked) natural thawing, according to 5.2.1 open the shell, rinse, take the meat, homogenization, spare. 5.2.4 Canned shellfish The entire contents of the tank (meat and liquid) sufficiently homogeneous. If the pot is large, the draining and collecting shellfish liquid asphalt were weighed and under Storage of solids and soup, the original solids and soup cans proportions homogenized spare. 5.2.5 with an acid preserved shellfish Lek to the acid, acid meat and shellfish were stored and set aside. 5.2.6 shellfish meat products Volume of the hydrochloric acid solution (0.18mol/L) and other dry products can be soaked in 24h ~ 48h (4 ℃ refrigeration), drain to the acid, were stored shellfish meat And acid backup. 5.3 PSP standards controlled trials 5.3.1 PSP formulated standard working solution With 10mL, 15mL, 20mL, 25mL and 30mL water are diluted with 10mL saxitoxin standard working solution to prepare a series Concentration of standard dilution. PSP standard median time to death of the working fluid selection 5.3.2 Take a series of concentrations 5.3.1 formulated standard dilutions of each 1mL, the number of mice by intraperitoneal injection only, select the median time to death Concentration dose of 5min ~ 7min. If a dilution solution has to meet the requirements, the need to increase or decrease the amount of water 1mL supplement dilution test. Each weighed before the test mice to a group of 10 mice, two concentrations median time of death within 5min ~ 7min range Standard dilution injected mice were measured and recorded for each mouse by intraperitoneal injection is completed to the time required to stop breathing death. 5.3.3 toxin conversion factor (CF) is calculated Select 5.3.3.1 mice the median time to death 5.3.2 Calculation of the concentration of the selected median standard dilutions of the test group of death. Discard the median time to death less than 5min to or greater than the test group 7min; selecting the median time to death in 5min ~ 7min of the test group, the test group are allowed, a Do the time of death in mice may be less than or greater than 5min 7min. 5.3.3.2 Calibration mouse unit (CMU) Calculation For the selected median time to death for the test group 5min ~ 7min, according to the Appendix A Richard deaths each mouse in the group when Mouse between units corresponding to (MU), then in accordance with Appendix B Richard groups per mouse body weight corresponding to the weight correction coefficient, the same mice Body weight correction coefficient obtained by multiplying the unit with the mouse mouse correction unit (CMU) tested only in mice. 5.3.3.3 toxin conversion factor (CF) is calculated Toxin conversion factor (CF) single mice according to formula (1). Where. CF3 --- toxin conversion factor, in micrograms per milliliter (μg/mL); c --- endotoxin per milliliter STX standard dilution liquid, in micrograms per milliliter (μg/mL); CMU --- correction mouse units. Get a single mice toxin conversion factor (CF), and then calculate the average value of CF n = 10 mice in the group is the conversion factor toxin Number (CF1). Calculation toxin conversion factor (CF2) between groups 5.3.3.4 The average conversion factor of endotoxin in the group taking a different test group, the group is among the toxin conversion factor (CF2). In between the two groups toxin conversion factor Calculate the number of samples virulence. 5.3.3.5 CF values checked regularly As PSP detection interval is longer, use the appropriate standard dilution was injected 5 mice at each measurement remeasurement CF values. Such as If detected several times a week, with the median time to death 5min ~ 7min standard dilution checked once a week, the measured value should be in CF Original determination within ± 20% CF value range. If the results do not match, then the same standard dilutions additional injection of 5 mice prior to injection integrated The results of five mice, the calculated value of CF. And with the same standard dilution was injected a second group of 10 mice, the second group obtained CF CF values and the first set of values are averaged, that is, a new CF value. Repeat CF value check is usually within ± 20% of the original results, if often found large deviation should be investigated before making routine testing Are variable factors influence the process. 5.4 Sample Extraction 100g samples taken in a beaker, add hydrochloric acid solution (0.18mol/L) 100mL sufficiently stirred, homogenized, adjusted to pH 2.0 to 4.0, will When to be added dropwise a solution of hydrochloric acid (5mol/L) or sodium hydroxide solution (0.1mol/L) adjusting the pH, when the alkali slowly, while not required Off stirring to prevent local damage alkalized toxins. The mixture was heated to boiling 5min, cooled to room temperature, move the cylinder and diluted to 200mL, Adjusting the pH to 2.0 to 4.0. The mixture was poured back to the beaker, stir, to natural sedimentation supernatant was translucent, not to clog the needle, mixing, if necessary Or supernatant 3000r/min centrifugal 5min, or filtered with a filter paper. The supernatant was collected for use. 5.5 Mice Experiment Take 19.0g ~ 21.0g healthy ICR male mice 6, weighed and the weight recorded. Randomly divided into experimental group and control group, two groups of three. Each experimental group of mice by intraperitoneal injection of 1mL sample extract, for each control group injected 1mL hydrochloric acid solution (0.18mol/L). If more than one drop of the injection process extracts overflow, the mice should be discarded and re-injection of a mouse. recording Time after injection, careful observation and record the time of death of the mice stopped breathing (to the last breath mouse only). If the sample extract injection after the time of death one or two mice greater than 7min, should be subject to at least three mice were injected. If after the time of death in mice less than 5min, diluted sample extracts were then injected with three mice until the mice within 5min ~ 7min Death; diluted sample extract, hydrochloric acid solution was added dropwise required (0.18mol/L) adjusted to pH 2.0 to 4.0.6 expression analysisTo 6.1 mass fraction of PSP virulence calculations and results are expressed 6.1.1 Calculation PSP virulence to mass fraction of PSP content per 100g sample according to the formula (2). Where. X --- content per 100g sample PSP, the unit is microgram per hundred grams (μg/100g); The median correction mouse units CMU1 --- sample test group of mice; Between CF2 --- group toxins conversion factor, in micrograms per milliliter (μg/mL); DF --- dilution; --- Volume of the sample extract 200 milliliters (mL). Note. Depending on the time of death in mice test sample of the test group, found in Appendix A corresponding murine unit; isolated mouse weight corresponding weight correction according to Appendix B Positive coefficient, obtained by multiplying the CMU both the mice. Select the test group, three mice CMU median, is CMU1. 6.1.2 to mass fraction of PSP virulence Expression of results If the control group of normal mice, is reported in the test sample PSP toxin content. ××× μg/100g. 6.2 In terms of the PSP MU virulence calculations and results are expressed Calculation PSP virulence 6.2.1 MU meter Each 100g sample in terms of PSP MU virulence according to formula (3) Calculated. Where. Y --- MU value per 100g sample, in units of mouse units per hundred grams (MU/100g); The median CMU1 --- murine experimental group of mice the virulence correction unit; DF --- dilution; --- Volume of the sample extract 200 milliliters (mL). Note. For PSP standards made difficult laboratories, according to equation (3) to calculate the terms of the PSP MU virulence. MU 6.2.2 to determine the count of the PSP and the results are expressed virulence The following judgments and statements in the control group of mice a normal situation. If the experimental group, the median time to death less than 5min, then extract the sample should be diluted, then select three mice tested, straight The median time to death to get to 5min ~ 7min far, the sample is calculated based on the last dilution experiments mice virulence units, reported The sample unit reported murine virulence is. ××× MU/100g. If the experimental group, the median time to death more than 7min, directly calculate and determine the sample unit of mouse virulence report murine virulence of the sample units It is. ××× MU/100g. If the experimental group was observed in all mice died within 15min not, can report the sample unit mice virulence less than 400MU/100g. Enzyme-linked immunosorbent assay Principle 7 Free paralytic shellfish poisoning their competitive enzyme markers of paralytic shellfish toxin antibodies, and paralytic shellfish toxin antibody and anti-capture Body connection. Is not bound enzyme label is removed in the washing step. Bound enzyme markers will be colorless chromogenic agent into a blue product. After adding the stop solution the color from blue to yellow. Pore solution using a microplate reader measuring the absorbance at 450nm wavelength of Value, sample content and paralytic shellfish poisoning absorbance is inversely proportional to the standard curve drawn quantitative calculations.8 Reagents and materialsUnless otherwise indicated, the methods used were of analytical grade reagents and water as a water GB/T 6682 regulations. 8.1 Reagents 8.1.1 hydrochloric acid (HCl). 8.1.2 dodecahydrate disodium hydrogen phosphate (Na2HPO4 · 12H2O). 8.1.3 Sodium chloride (NaCl). 8.1.4 Potassium chloride (KCl). 8.1.5 potassium dihydrogen phosphate (KH2PO4). 8.1.6 Tween -20 (C58H114O26). 8.1.7 bovine serum albumin (BSA). 8.1.8 enzyme labels. 8.1.9 paralytic shellfish toxin antibodies. 8.1.10 hydrogen peroxide (H2O2). 8.1.11 3,3,5,5-tetramethylbenzidine (TMB, C16H20N2). 8.1.12 sulfuric acid (H2SO4). 8.2 reagent preparation 8.2.1 hydrochloric acid solution (0.1mol/L). Measure 9mL hydrochloric acid, diluted with water to 1000mL. 8.2.2 hydrochloric acid (5mol/L). Measure 450mL of hydrochloric acid, diluted with water to 1000mL. 8.2.3 in phosphate buffered saline (PBS solution, pH7.4). potassium dihydrogen phosphate were weighed 0.20g, disodium hydrogen phosphate dodecahydrate 2.90g, chloro Sodium 8.00g, potassium chloride 0.20g, dissolved in water and dilute to 1000mL. 8.2.4 antibody dilution. Weigh 1.0gBSA, plus PBS solution to dissolve and dilute to 1000mL. 8.2.5 enzyme label reagent. The antibody dilution with enzyme conjugate diluted to working concentration. 8.2.6 paralytic shellfish toxin antibody solution. paralytic shellfish toxin antibodies diluted antibody dilution to the working concentration. 8.2.7 eluent. lessons 0.5mL Tween-20, diluted to 1000mL with PBS solution. 8.2.8 sulfuric acid solution (1mol/L). lessons 53.2mL sulfuric acid, slowly added to the water containing 500mL beaker, and water to the 1000mL, and mix. 8.3 Standard Saxitoxin (STX, C10H17N7O4 · 2HCl, CAS No. 35554-08-6) standard solution. 8.4 Standard Solution Standard series working solution preparation. Imbibe saxitoxin standard solution was diluted with an appropriate volume of PBS solution (pH7.4), preparation A mass concentration of 0μg/L, 2.5μg/L, 5μg/L, 10μg/L, 20μg/L, 40μg/L in series of standard working solution. Using now. 8.5 Material Coated with paralytic shellfish toxin capture antibody microplate. NOTE. Commercial kit if the evaluation of technical parameters to achieve the requirements of this standard are also suitable for this standard, see Appendix C.9 instruments and equipment9.1 microplate reader. 9.2 homogenizer. 9.3 centrifuge. speed ≥6000r/min. 10 analysis steps 10.1 Sample Collection With 5.1. 10.2 Sample Preparation With 5.2. 10.3 Sample Extraction Weigh 10g (accurate to 0.1g) sample was added 70mL hydrochloric acid solution (0.1mol/L) [such as drier shellfish meat, added 140mL Hydrochloric acid solution 5min, at 4 ℃ 6000r/min centrifugal 10min, supernatant solution (0.1mol/L)], boiled and stirred hydrochloric acid (5mol/L) adjusted to pH 4.0 or less. Take 100μL extract was added 900μLPBS solution (pH7.4), mix, take into 50μL Determination row. 10.4 Determination It will be coated with capture antibodies paralytic shellfish poisoning wells shall be inserted microporous frame and mark, including the blank well, standard solution Bore holes and sample solution were doing parallel holes. To blank control wells added 50μL phosphate buffer, standard solution well add 50μL Paralytic Shellfish Endotoxin standard series working solution, the sample was well add 50μL sample solution. Added 50μL paralytic shellfish toxin enzyme conjugate to each well, gently Mix; then add 50μL paralytic shellfish toxin antibody to each well and thoroughly mixed with glue to seal porous paper to prevent solution evaporation, 20 ℃ ~ 25 ℃ dark dark place incubate 15min. After incubation, the liquid decanted holes, each micro-injected 250μL wash eluent, Flip the plate, pour squirt bottle, then repeat the washer 5 times, pat dry on absorbent paper. Each well 100μL hydrogen peroxide and TMB, mixed, 20 ℃ ~ 25 ℃ dark dark place incubate 15min. Sulfuric acid solution was added to each well 100μL (1mol/L) rapid mixing Even terminate the reaction, measure and record the absorbance at 450nm wavelength within 10min. If the measured concentration exceeds the standard sample solution After re-measured linear range of the curve, you can expand the dilution factor. 10.5 standard curve Logarithm to paralytic shellfish toxins series of standard working solution concentration to the base 10 as the horizontal, the percentage of the formula (4) Calculation The absorbance value of the vertical axis, the standard curve. Paralytic shellfish toxin stan......Tips & Frequently Asked Questions:Question 1: How long will the true-PDF of GB 5009.213-2016_English be delivered?Answer: Upon your order, we will start to translate GB 5009.213-2016_English as soon as possible, and keep you informed of the progress. The lead time is typically 3 ~ 5 working days. 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