CPSC-CH-E1001-8.1
LCICPMS法对农残中代森锌检测实验方案
LC-ICP-MS法对农残中代森锌检测试验方案一、研究项目本试验针对农产品中重金属农药代森类化合物进行提取富集检测，检测措施采用LC-ICP-MS法。
弥补金属农药ICP-MS法检测旳空白，为重金属农药检测提供更低旳检测限和更精确旳检测措施。
二、研究旳背景；代森锌在我国是1956年首先由原华北农业科学研究所农药研究所试制成功，其后由山东农药厂上产。
代森锰锌是继代森锌和代森锰之后发展起来旳新品种。
这几种农药对农作物，尤其是果树和蔬菜徐福哦病害有效，因此一直在使用中。
其中代森锰锌旳发展最快。
代森类农药旳研制成为我国化工部“七五”攻关项目，由沈阳化工研究院承担“代森锰锌生产技术开发”。
由于发展过快，代森类农药旳生产已经处在超饱和状态，继而向多种农药配用旳方向发展。
有研究表明，代森锌络合物能减少乙撑硫脲旳量，并且药效也比代森锌有所提高。
伴随代森类农药在农作物旳使用旳普及，越来越多旳农民采用其作为田间杀虫剂，同步为保证更好旳防治效果，使用量也有所增大。
不过越来越多旳研究发现代森类物质及其代谢物质对人体有较强旳毒性。
有研究发现，代森锌在人和动物体内可分解为乙二胺、二硫化碳和氧化锌或分解成为二硫化碳、硫化氢和乙二胺。
二硫化碳和硫化氢一般由肺脏排出。
尚有学者提出，代森类农药在空气中、体内、食物处理等多种条件下不稳定，分解成旳乙撑硫脲（ETU）能对人体引起甲状腺肿瘤。
曾有学着通过小鼠试验发现，代森类农药对甲状腺体染色体有致突变作用。
英国和美国均提议减少EBDC类旳用药次数，减少用药量，延长安全间隔期等，以到达减少作物中旳残留量，而容许在小范围内食用EBDC，但未有主线性旳变化。
农产品旳药物残留问题已成为各级政府部门和消费者关注旳焦点。
目前农产品中药物残留问题旳严重性已拉响了我国食品安全旳警报，尤其是农药残留问题，已成为我国食品安全旳重要问题之一。
这一问题同步也是我国食品出口旳障碍之一。
由于重金属农药旳使用会导致药物旳滞留或积蓄，并以残留旳方式进入动物体内及生态系统，虽然在体内分解代谢，金属离子也会在体内蓄积，从而对人类生命健康和环境导致一定旳危害。
pocH-100i全自动三分类血液分析仪
pocH-100i主要优势
名牌进口产品与完善的售后服务支持 全自动三分类仪器提供准确 结果
– 试剂无需手工添加 – 鞘流电阻原理检测RBC/PLT – 三个直方图均有浮动界标功能 – 静脉血、末梢血独立校准模式
操作轻松方便
– 全中文、图形操作界面
安全、环保试剂
pocH-100i测试原理及流程
强大的联网功能
LAN接口：TCP/IP 连接
– 支持远程诊断功能 – 对HIS联网的急诊化验室有价值
pocH100i
实验室1
Sysmex XE-2100
pocH100i
实验室2
pocH100i
实验室3
中心实验室
pocH-100i / KX-21
小巧的外观，适合任何实验室
37 cm
19 cm
10 kg
检测模块
全血、预稀释标本 RBC/PLT HGB
WBC
检测原理 鞘流电阻测定 比色法 DC测定
检测通道 直方图 检测参数
RBC/PLT通道
RBC PLT 直方图 直方图 PLT RBC
HGB通道 WBC 通道
WBC 直方图
HGB
WBC、W-
SCR、W-MCR、
W-LCR
两种模式、结果可靠
15 uL
简单的维护保养
仪器预设保养程序，一按即可
1. 每天：执行“关机”程序 2. 每季 (或每1500标本)
– 废液室清洗 – 转换器清洗
– 无易耗配件
无氰化物试剂检测HGB
溶血素STROMATOLYSER-WH. 比色法检测HGB:
– 亚铁血红素中的Fe2+被季胺盐氧化形成季胺盐血红蛋白 （QAS-HGB）
CPSC-CH-E1001-08
美国·消费品安全委员会·实验室科学理事会·化学分部马里兰州Gaithersburg城Darnestown路10901号，邮编：20878测试方法：CPSC-CH-E1001-08[技术翻译：piery2006 校对：林雪霞]测定儿童金属产品(包括金属首饰)中总铅(Pb)含量的标准作业程序2008年12月4日本文件提供了美国消费品安全委员会（CPSC）测试实验室（LSC）分析儿童金属制品中的铅(Pb)含量所使用的测试方法的相关信息。
所述的方法为测定金属物品（例如：儿童金属首饰，但不局限于此）中的总铅含量。
CPSC工作人员认为此测试方法能够满足大多数测定消费品的金属和合金零部件中铅含量的要求。
对于某些特殊合金产品，需要以相关化学知识、材料科学和合金基质所适用的酸为基础，对方法进行调整。
所选择的测试方法应可以将被分析的样品材料完全消解。
如果与方法规定的适用范围不相符合，或是样品材料不能达到高效、高性能的完全消解，最好不要使用这些测试方法。
本文法适用于金属和金属合金零部件，CPSC工作人员并不建议使用到其它材料上。
因为在下述条件下，材料可能不会溶解，或是当样品与规定的酸混合时，造成与材料不适当的混合，与安全实验室操作不相符。
一般做法是将样品的可接触零部件磨成粉末。
然后在浓硝酸与浓盐酸的混合酸中完全溶解，最后采用电感受耦合等离子体原子发射光谱法（ICP-OES）分析。
其它分析方法如：电感耦合等离子体质谱法（ICP-MS）、火焰原子吸收光谱法（FLAA）、石墨炉原子吸收光谱法（GFAA）在合适的条件下，可以作为ICP-OES法的替代方法使用。
当使用替代分析方法时，应采用合适的、公认的分析技术（标准）。
定义：1. 样品―待测试的单个消费品或同一批次中一组相同的消费品。
2. 零部件―整个样品上的一个单独的次级单元。
例：一个手镯可以被分解成珠子、钩子和悬饰等数个零部件，每个零部件应单独分析。
CPSC-CH-E1002-08_2儿童非金属产品中总铅测试标准程序
U NITED S TATESC ONSUMER P RODUCT S AFETY C OMMISSIOND IRECTORATE FOR L ABORATORY S CIENCESD IVISION OF C HEMISTRY5R ESEARCH P LACER OCKVILLE,MD20850Test Method: CPSC-CH-E1002-08.2Standard Operating Procedure for Determining Total Lead (Pb) in NonmetalChildren’s Products, RevisionApril 10, 2012*This document provides detailed information on the test method that will be used by the U.S. Consumer Product Safety Commission’s testing laboratory (LSC) in the analysis of nonmetal children’s products for lead (Pb) content. This method is divided into three sections. The first section describes how to digest samples to determine the total lead content in crystal, ceramic, and other siliceous materials and contains a subsection on the use of X-ray fluorescence spectrometry (XRF) for determination of lead in such siliceous materials. The second section describes how to digest samples to determine the total lead content in polymeric (including natural and synthetic polymers), or plastic materials, and it contains a subsection on the use of X-ray fluorescence spectrometry (XRF) for determination of lead in such polymeric materials. The third section describes how to analyze the digested samples from the first two sections. This revision recognizes use of X-ray fluorescent spectroscopy measurement techniques in additional materials with certain limitations and acceptable ranges and replaces the previously issued Test Method CPSC-CH-E1002-08.1.The method applies to most nonmetal components other than paint, but it is not recommended by CPSC staff for materials, that when combined with the specified acid(s), results in an inappropriate combination of materials that would be inconsistent with safe laboratory practices. In such cases, the chemist should make a knowledge-based decision on the proper modifications of the method to maintain laboratory safety, while following the general approach described here.The general approach is to grind or cut any accessible component part of a sample into small pieces or a powder; digest an aliquot completely in nitric acid or for siliceous products in a combination of hot, concentrated nitric and hydrofluoric acids; and analyze by Inductively Coupled Plasma Optical Emission Spectroscopy (ICP-OES). Other analytical methods, such as Inductively Coupled Plasma – Mass Spectrometry (ICP-MS), Flame Atomic Absorption Spectroscopy (FLAA), and Graphite Furnace Atomic Absorption Spectroscopy (GFAA), may be used under appropriate conditions as an alternative to ICP-OES, using applicable,* This is a revision of Test Method CPSC-CH-E1002-08.1. This document was prepared by CPSC staff, has notbeen reviewed or approved by, and may not necessarily reflect the views of, the Commission.recognized analytical techniques for the alternative analytical method. Nonmetal materials may also be analyzed, using XRF, following the standard test method of ASTM F2617-081 or ASTM F2853-10e1,2 with limitations described below. The general approach in that case is to consider any XRF result to be indeterminate and in need of digestion and ICP analysis if that result falls within 30 percent of the Consumer Product Safety Improvement Act (CPSIA) limit.CPSC staff has concluded that the test methodologies provided in detail below are sufficient to determine lead content in most products. Knowledge-based adjustments on a case-by-case basis may be necessary for products made from certain materials.Definitions1.Sample–An individual consumer product or a group of identical consumer productsfrom a batch to be tested.ponent Part–An individual subunit within the total sample. An item, such as abracelet, may be broken into component parts, such as a bead, crystal, a hook, and apendant, with those component parts individually analyzed.3.Instrument Detection Limit (IDL)–3 times the standard deviation of 10 replicatemeasurements of reagent blank.4.Method Detection Limit (MDL)–Reagent blank fortified with 2–3 times the IDL.Seven replicate measurements are made. Calculate the MDL as follows: MDL = t X S, t= 3.14 (99 percent confidence level for 7 replicates), S= standard deviation.boratory Reagent Blank (LRB)–An aliquot of the digestion reagents that is treatedexactly as a sample, including exposure to glassware, digestion media, apparatus, and conditions used for a particular Pb test but with no added sample. LRB data are used to assess contamination from the laboratory environment.6.Calibration Blank–Deionized water acidified with nitric acid (3 ml concentrated nitricacid diluted to 100 ml with deionized water).7.Stock Standard Solution–1,000 ppm solution of Pb purchased from a reputablecommercial source, used to prepare calibration standards. Replace before expirationdate.8.Calibration Standards–Solutions containing 0 to 25 ppm of Pb in 3 percent nitric acidmatrix are used. A minimum of 4 calibration standards are used. Calibrationstandards should be prepared on a biweekly basis at minimum.9.Quality Control Sample (QCS)–A solution containing Pb that is used to evaluate theperformance of the instrument system. QCS is obtained from a source external to the laboratory and Stock Standard Solution.10.Certified Reference Material (CRM)–CRMs are materials with a similar matrix as testsamples with known lead levels. CRMs are used to verify digestion and analysismethods. For example, standard reference materials (SRMs) are CRMs that are1 Standard Test Method for Identification of Chromium, Bromine, Cadmium, Mercury, and Lead in Polymeric Material Using Energy Dispersive X-ray Spectrometry.2Standard Test Method for Determination of Lead in Paint Layers and Similar Coatings or in Substrates and Homogenous Materials by Energy Dispersive X-Ray Fluorescence Spectrometry Using Multiple Monochromatic Excitation Beams.available from the National Institute of Standards and Technology (NIST), such asthose listed in the Equipment and Supplies section below. Appropriate CRMs fromother sources are also acceptable.Equipment and Supplies: The materials used for sampling and analyses are as follows:1.Nitric Acid, Trace Metal Grade2.Hydrofluoric Acid, Trace Metal Grade3.Distilled Water4.Microwave Digestion Apparatus5.Cryogenic Mill6.Liquid Nitrogen7.CRMs such as ERM®-EC680k3 and EC681k, low-density polyethylene materials thatcontain lead and NIST SRM 89 and 610, leaded glass.8.Internal Standard (such as yttrium, from a stock standard solution of that elementappropriate to the instrument parameters of the ICP used for the analysis)I.Total Lead in Ceramics, Glass and Crystal, and other Siliceous MaterialsA.Acid DigestionWhen preparing a sample, the laboratory should make every effort to ensure that thealiquot removed from a component part of a sample is representative of the component to be tested and is free of contamination. Each unique component type from a subsample is analyzed for total Pb content. CPSC staff uses a method based on EPA 30524(/epawaste/hazard/testmethods/sw846/pdfs/3052.pdf) for determining lead content in ceramic or crystal materials. Certified reference materials, such as NIST SRM 89 and 610, which closely match the material of the tested product, should be used to verify accuracy of digestion and analysis methods. After digesting the sampleaccording to this procedure, it should be tested by ICP, as described below in Section III.1.Weigh out a 30–100 mg piece of crystal, glass, or ceramic item into an appropriatemicrowave vessel equipped with a controlled pressure-relief mechanism. Ceramicitems generally weigh several grams or more, and consist of the base ceramic with aglaze and decoration fired on. The lead in ceramics is generally in the glaze ordecoration. When analyzing ceramics or glass, the entire item, including the glaze,decoration, and ceramic base material should be ground in a cryogenic mill and 30–100 mg of the ground ceramic/glass powder weighed in an appropriate microwavevessel. If used, the grinding apparatus must be cleaned thoroughly to prevent cross-contamination. Record actual weight to the nearest 0.1 mg.2.At room temperature, add 3 ml of concentrated nitric acid and 1 ml of concentratedhydrofluoric acid to each vessel. Wait for completion of the initial reaction of theacid and the sample before sealing vessels. Seal vessels in accordance with themanufacturer’s directions.3 European Reference Material, produced and certified under Institute for Reference Material and Measurements (IRMM).4 Microwave Assisted Acid Digestion of Siliceous and Organically Based Matrices.3.The microwave method should involve increasing the temperature of each sample toat least 180°C in approximately 5.5 minutes, and holding at 180°C for 9.5 minutes.4.Allow the samples to cool for a minimum of 5 minutes before removal frommicrowave. Vent the microwave vessels in fume hood before uncapping.5.Add 30 ml of 4 percent (w/w) boric acid to each vessel to permit the complexation offluoride to protect the ICP quartz plasma torch. Quantitatively transfer the sample toa 50 ml plastic volumetric flask or disposable volumetric digestion cup. Dilute to 50ml with deionized water.Caution: The analyst should wear protective gloves and face protection and must n ot at any time permit solution containing hydrofluoric acid to come in contact with skin orlungs. This document does not address all safety concerns; additional safety precautions are necessary for all steps, particularly when using hydrofluoric acid. This method is not to be used except by qualified, properly trained workers.B.Identification and Quantification of Pb in Siliceous Materials Using EnergyDispersive XRF Spectrometry Using Multiple Monochromatic Excitation Beams Alternately, Energy Dispersive XRF Spectrometry Using Multiple MonochromaticExcitation Beams (HDXRF) can be used with limitations to determine quantitatively the amount of Pb in siliceous materials by following ASTM F 2853-10e1. This standard isapplicable only for homogeneous siliceous materials and for XRF instruments meetingthe requirements given in the ASTM method. The following limitations apply:1.Applicable only for analysis of homogeneous materials. It is not suitable for testingglazed ceramics.2.Multiple measurements on different locations of the sample component part should beperformed to ensure some degree of spatial homogeneity. If the relative standarddeviation on 3 or more XRF measurements of a sample componen t part exceeds 30percent, analysis using wet chemical procedures (after preparing a homogenized aliquot by grinding sufficient sample) should be done before determining that the items meetCPSIA requirements for lead.3.Any XRF measurement of lead concentration, where the interval comprised of thereported result, plus or minus the instrument’s reported 95 percent uncertainty, includes the range with 30 percent above or below the CPSIA limit, shall be considered“inconclusive.” An average of at least 3 measurements, none of which is“inconclusive,” as defined in this paragraph, should be obtained in order to have a“conclusive” result.55 For example, if the XRF instrument reports a result of 65 ppm lead with an uncertainty of 10 ppm lead for a material subject to the CPSIA limit of 100 ppm lead content, this measurement would be consideredinconclusive because 65 ppm +10 ppm = 75 ppm, which is less than 30 percent below the applicable limit of 100 ppm. A reported result of 60 ppm with a reported uncertainty of 8 ppm would be a conclusive measurement of a material subject to the CPSIA limit of 100 ppm as 68 ppm is more than 30 percent below the applicable limit of 100 ppm.4.For “inconclusive” results, additional testing is necessary in order to make adetermination, such as by digestion and ICP analysis, per sections IA and III.C. Identification and Quantification of Pb in Siliceous Materials Using Other Formsof XRF SpectrometryOther types of XRF spectrometers that do not meet the requirements of ASTM F2853-10 can be used to determine quantitatively the amount of Pb in siliceous materials, withlimitations. The following limitations, in addition to those outlined in Section I-B forHDXRF, apply:1.Follow sampling, testing, calibration, quality control guidelines described in section 6of International Electrotechnical Commission (IEC) Method 62321 ED 1.0 B.2. A set of at least 4 glass calibration standards should be used to validate that theinstrument is suitable for testing for Pb in siliceous materials. The calibrationstandards should cover the applicable range to certify that the sample meets CPSIAlead content requirements (0-2000 mg/kg). At least 1 standard in each calibration setshould have lead concentration less than 100 mg/kg.3.Verify the instrument performance daily, by analyzing one or more reference materialsof the same matrix or metal type as the materials on which analyses will be performed.The lead concentration of the reference material should be in the range of 50–300mg/kg, and the determined concentration from the measurement must be in agreementwith the known or certified value. The measured result with the given uncertainty (at95 percent confidence) should overlap with the reported certified values and givenuncertainty of the reference materials.4.The limit of detection (LOD) for lead in glass should be determined followingguidelines in section 6 of IEC 62321. The lead LOD shall be equal to or less than 30mg/kg for the specific material or metal type tested. Some types of XRFspectrometers may not have sensitivity to obtain sufficient LOD for certifying to leadrequirements.II. Total Lead in Plastics, Polymers, and Other Non-Siliceous MaterialsA.Acid DigestionWhen preparing a sample, the laboratory should make every effort to ensure that thealiquot removed from a component part of a sample is representative of the component to be tested and is free of contamination. Each unique component type from a subsample is analyzed for total Pb content. CPSC staff uses a method based on methodology found in Canada Product Safety Bureau Method C-02.36 (http://www.hc-sc.gc.ca/cps-spc/prod-test-essai/_method-chem-chim/c-02_3-eng.php) for determining lead content in plasticmaterials, such as polyethylene and polyvinyl chloride (PVC). EPA Method 3051A7(/epawaste/hazard/testmethods/sw846/pdfs/3051a.pdf), with6 Determination of Total Lead in Polyvinyl Chloride Products by Closed Vessel Microwave Digestion.7 Microwave Assisted Acid Digestion of Sediments, Sludges, Soil, and Oils.modifications in sample weight, temperature, time, and acid volumes to match those given below, is also acceptable. Certified reference materials that closely match the material of the tested product, such as ERM®-EC680k and EC681k (described above), should be used to verify the accuracy of digestion and analysis methods. After digesting the sample according to this procedure, it should be tested by ICP, as described below in Section III.1.Cut the test specimen into small pieces. Hard to digest plasti cs may need to becryomilled to get finer powder. Weigh out 150 mg of the milled or cut plastic into an appropriate microwave vessel equipped with a controlled-pressure relief mechanism.Ensure that the milling apparatus is thoroughly clean between test specimens to avoid cross-contamination. Record actual weight to the nearest 0.1 mg.2.At room temperature, add 5 ml of concentrated nitric acid to each vessel. Wait forcompletion of the initial reaction of the acid and the sample before sealing vessels.Seal vessels in accordance with manufacturer’s directions.3.The microwave method should involve increasing temperature of each sample to atleast 200°C in approximately 20 minutes, and holding for 10 minutes.4.Allow the samples to cool for a minimum of 5 minutes before removal frommicrowave. Vent the microwave vessels in a fume hood before uncapping.5.Quantitatively transfer the sample to a 50 ml volumetric flask or disposable volumetricdigestion cup. Dilute to 50 ml with deionized water.B.Identification and Quantification of Pb in Polymeric and Other NonmetalMaterials Using XRFAlternately, Energy Dispersive XRF can be used with limitations to determine quantitatively the amount of Pb in polymeric materials by following ASTM F 2617-08 or ASTM F2853-10e1. These standards are applicable only for homogeneous polymeric materials and for XRF instruments meeting the requirements given in the ASTM methods. Components could be analyzed intact, without any modification, if they have suitable surface characteristics, geometry, and homogeneity. Destructive sample preparation techniques may be required for certain components to create a uniform sample for testing. Excessive curvature, rough surface texture, or specimen thickness less than 2 millimeters, may require sample preparation techniques, such as compression molding, as outlined in ASTM F 2617-08. Based on the interlaboratory study of reference materials reported in this standard and the fact that actual consumer products to be tested are likely to be less homogeneous than the reference materials, CSPC staff has concluded that analysis using wet chemical procedures outlined in Sections II-A and III should be done on any samples with Pb results determined using XRF to be greater than 70 percent of the Pb requirements of the Consumer Product Safety Improvement Act (CPSIA) before certifying that the item meets the CPSIA.Other homogeneous nonmetal materials such as wood and fabric can be analyzed by XRF following ASTM F2853-10e1 subject to the same limitations given in Section I-B aboveor using other types of XRF spectrometers that do not meet the requirements of ASTMF2853-10e1 subject to same limitions given in Section I-C above.III.Total Pb in Acid Digests of Polymeric or Siliceous Materials - Analysis of Sample Using ICP MethodAnalyze diluted samples for Pb concentration using an ICP spectrometer (or AtomicAbsorption spectrometer). Analysis procedures for ICP-OES and FLAA and GFAA are based on the methodology in ASTM E1613-04.8 ICP-MS may also be employed with appropriate procedures, such as EPA 6020A.9 Calculate total lead concentration in the component part from that of the diluted sample, accounting for all dilution. Report as percent by weight of the component part itself.ICP Operating Procedures and Quality Control MeasuresAnalysis1.Ignite plasma. Perform wavelength calibration or torch alignments per instrumentmanufacturer recommendations.2.Allow the instrument to become thermally stable before beginning.3.Ensure the following element and wavelength are selected in analytical method:a.Pb 220.353.One other Pb line, such as Pb 217.00, should be used to ensure spectral interferences are not occurring during analysis.4.An internal standard, such as 2 µg/ml yttrium, is used.5.Perform calibration using calibration blank and at least 3 standards. Calibrationshould be performed a minimum of once a day when used for analysis, or each timethe instrument is set up. Results for each standard should be within 5 percent of thetrue value, and the calibration blank should be < 5 times MDL. If the values do notfall within this range, recalibration is necessary.6.Analyze the QCS after the calibration and before any samples. The analyzed value ofPb should be within ±10 percent of the expected value. If Pb value is outside the ±10 percent limit, recalibration is required.a.At least one LRB must be analyzed with each sample set. If the Pb valueexceeds 10 times the MDL, laboratory or reagent contamination should beexpected. The source of the contamination should be identified and resolvedbefore continuing analyses. The LRBs should be the same acid concentrationsas added to the sample and should be taken through the same digestionprocedure.7.At least one certified reference material (CRM) should be analyzed with each batch ofsamples. The CRM should be a similar material as the test specimen with a knownamount of Pb. Analyte recoveries should be within ±20 percent of expected values. If recoveries are outside this limit, the source of the problem should be identified andresolved before continuing analyses.8.Dilute any samples that have Pb values exceeding 1.5 times the high calibrationstandard, and reanalyze.8 Standard Test Method for Determination of Lead by Inductively Coupled Plasma Atomic Emission Spectrometry (ICP-AES), Flame Atomic Absorption Spectrometry (FAAS), or Graphite Furnace Atomic Absorption Spectrometry (GFAAS) Techniques.9 Inductively Coupled Plasma-Mass Spectrometry.Calculations and Results ReportedResults for the Pb test methods are calculated and reported as follows:1.Total Pb - % Pb (wt/wt) = 0.10cd/wa.c= concentration of Pb detected (in units of ppm)b.d= dilution factor (in ml units)c.w= weight of aliquot digested (in mg units)Examples:Table 1: Total Pb Analysis(c) (d) (w)Item ppmPb DilutionfactorTotalPb(µg)Samplewt(mg)% PbCrystal 20 1,000 20,000 50 40Summary of changes in Revision CPSC-CH-E1002-08.11.Page 1, revised test method # and date.2.Page 1, last paragraph first sentence, allowed for polymeric materials to be cut intosmall pieces.3.Page 2, removed IDL and MDL CPSC lab values; not relevant to method and newinstruments will have different values.4.Page 2, definition 10, last sentence revised to include other sources for CRMs.5.Page 3, removed reference to NIST SRM 1412 and added 610. 1412 no longeravailable.6.Page 3, step 3, changed temperature requirements from “180±5°C” to “at least180°C”7.Page 4, step 1, removed requirement for cryomilling.8.Page 4, step 3, changed temperature requirements from “210±5°C” to “at least200°C.”9.Page 5, first paragraph, changed “>200mg/kg” to “greater than 70% of CPSIA Pblimit” to generalize for future changes to limit.Summary of Changes in Revision CPSC-CH-E-1002-8.21.Page 1, revised test method # and date, added statement that SOP contains asubsection on the use of X-ray fluorescence spectrometry (XRF) for determination oflead in such siliceous materials.2.Page 2, change to biweekly from weekly minimum time between calibration standardpreparation.3. Page 7, Analysis step 5, added statement that calibration blank should be < 5 timesMDL.4. Page 7, Analysis step 6, removed “immediately” after QCS and added” and beforeany sample” after calibration.5. Page 7, Analysis step 6a, changed “the LRB shall not exceed 3 times MDL” to“the LRB shall not exceed 10 times MDL.” This change reflects lower MDLsachieved on some instruments in particular ICP-MS and difficulty in LRB meeting3 times MDL.6. Page 4, added subsections B and C to section I that outline XRF testing proceduresfor determining Pb in glass or siliceous materials.。
中文CPSC-CH-C1001-09.1测试邻苯二甲酸酯的标准作业程序
相关离子（m/z）
91.1, 105, 194, 212 149, 167, 205, 223
91.1, 149, 206 149, 167, 279
149, 167, 261, 279 149, 167, 293 149, 167, 307
分析 1． 六种目标邻苯二甲酸酯每种至少配制浓度不同（浓度范围为 0.5~10μg/ml）的四个校正标准溶液和一个校正空 白（环己烷）。每个校正标准溶液含 1 μg/ml 的内标。 2． 用 GC-MS 分析标准溶液和空白。定量分析出结果，确保合适的保留时间和不受到污染。 3． 对每种标准溶液在两个波谷之间（时间范围见表 2）进行积分。在第一通道和第二通道扫描的化合物可以通过 总离子流图（TIC）或离子色谱图（字体加粗的为建议定量离子）进行积分。SIM 第三通道扫描的邻苯二甲酸酯会 重叠且必须使用它们的定量离子（仍然为字体加粗）积分。 4． 根椐规一化的邻苯二甲酸酯信号制作校正曲线。规一化是以邻苯二甲酸酯信号积分面积除以内标信号积分面积 来实现的。 5． 分析一个认证参考材料（CRM），确保校正是正确无误的。分析结果与标称值相差不超过±10%。如果不是， 请配制新的标准溶液重新进行校准。 6． 分析实验室方法空白和所有的样品。每隔一段时间分析一个认证参考材料（CRM）进行校准查核。 7． 量化结果。如果结果处于校正范围外，重新回到邻苯二甲酸酯萃取第 5 步（进行另一次稀释使结果处于校正范 围内）。 0
测定邻苯二甲酸酯的浓度 这些方法需要使用到有危险性的材料。接触所有危险性材料均应在通风橱内进行，并作好充分的个人防护，这是最 重要的。
邻苯二甲酸酯是一种常见的污染物。即便是低浓度的污染都可能会影响定量分析结果。应避免与塑胶材料接触，并 且只使用仔细清洗过的玻璃器皿和仪器设备。所有试剂均应测试邻苯二甲酸酯含量。定期使用 GC-MS 分析试剂空 白监控可能的污染。当可行时，建议使用一次性玻璃器皿。
ASTM F963-11 测试方法中文
8.测试方法8．1概述——此章节中的检验和测试程序用于确定产品是否符合本规范要求。
本规范涵盖的所有类型玩具必须按照8.5到8.10进行正常使用和误用情况测试。
其他测试适用于第4节中规定的玩具类型。
除非有特别规定，一种类型玩具上适用的相关测试可以按任何次序进行。
除非有特别规定，每一类测试可在不同的玩具上进行。
这些测试可以认为是质量鉴定试验，而不一定是质量控制试验。
当玩具的设计和材料有变化时也要进行这些测试。
每一家将其产品作为符合本规范的合格产品来推销的制造商或分销商可使用基于统计规律的、合适的抽样方案，但要保留那些可有力证明其产品符合本规范所有要求的声明所必需的记录。
由CPSC制定的任何强制性测试方法要代替本章节中相应的适用测试方法。
8.2危险物质含量测试——FHSA颁布、用于测定有毒物质存在的16CFR中的适用章节见表4。
表4的参考条例参见1973年9月27日发布的法规。
注6——确定玩具是否符合本规范中要求时要采用最新发布的法规。
根椐法律规定，FHSA的要求为强制性要求。
8.3玩具﹑玩具部件和材料中重金属含量的测试方法8.3.1元素总含量筛选8.3.1.1受验玩具材料的消解通过如下适当的CPSC方法:(1)CPSC-CH-E1001-08.1(金属材料)(2)CPSC-CH-E1002-08.1(非金属材料)(3)CPSC-CH-E1003-09(油漆和类似表面涂层材料)8.3.1.2采用如下改进:浓硝酸消解液采用王水替代(3份浓硝酸:1份浓盐酸).玻璃和陶瓷材料可以使用3份HF:1份浓HNO3进行消解.某些聚合物如PVC和CPVC需要使用3份浓硝酸:1份30%双氧水完全消解.在任何情况下,对于某些材料制成的产品所用的上述消解混液可视情况做一些调整,同时也是可行的,只要完全消解过程完成并且要考虑到尽可能的避免不溶金属盐的形成.在任何情况下,为了减少不溶金属盐形成的可能性,请尽量避免使用浓硫酸.8.3.1.3将消解后的材料过滤,根据实际条件稀释,然后用原子光谱或其他合适已验证的方法对表1和表2所列的所有8种元素的总含量进行分析.如果测试结果都小于对应表中所规定的每种元素可的溶性限值,那此材料可被认为是符合4.3.5和4.3.5.2节中要求的,并且不需要作进一步测试.相反,如果超过了对应表中可溶性限值,则需要按照8.3.2(对油漆和类似表面涂层材料)和8.3.5(对基材)方法做进一步测试来证明符合性.另外,如果玩具或玩具组件是金属小物体,需要根据8.3.5.5(5)进行测试.像很多材料(如三种不同颜色的聚苯乙烯塑料)在总元素筛选应用方面,最多三个混测样品是可以接受的,但在可溶性元素测试方面是不合适的.注7:除了要求的总铅含量的测试之外,可以选择不进行总元素筛选测试,只根据8.3.2-8.3.6进行可溶性元素测试.8.3.2溶解可溶性物质的方法(关于表面涂层的)---模拟材料吞咽后在消化道停留4小时的条件,在此条件下,从玩具中提取可溶性元素,测定提取物中可溶性元素的含量.8.3.2.1仪器-常规实验室仪器和如下仪器(1)金属筛,额定孔径为0.5mm的平纹不锈钢金属筛,规格如下:(a)额定金属丝直径为0.315mm(b)每个筛孔尺寸的最大偏差:±0.090mm(c)平均孔径公差:±0.018mm已及(d)中等偏差(6%以下的孔径应大于额定孔径与下列数值之和)＋0.054mm(2)p H值测试仪,最小精度为0.2p H单位(3)滤膜过滤器,孔径为0.45μm(4)离心机:分离能力达(5000士500)g(g=9.80665m/s2).(5)恒温搅拌工具搅拌时温度恒定为(37士2)0C,(6)系列化学容器总容量为盐酸溶液提取剂体积的1.6倍~5.0倍。
QPCR及QRT-PCR系列产品
Invitrogen的ICFC系列产品促销1．QPCR及QRT-PCR系列产品Invitrogen公司专门为中国客户提供的定量PCR试剂盒，结合了 UDG 防止残余污染技术和SYBR® Green I 荧光染料（存在于SYBR® Green I荧光定量PCR试剂盒中），在美国接受了严格的质量监控，可提供极高灵敏度的目的序列定量检测，线性剂量低，反应浓度范围很大。
qPCR Supermix-- 即用型反应剂，专为高特异性、实时定量DNA扩增设计UDG-- 防止携带污染物，减少克隆片段假阳性结果ROX参考染料-- 适用ABI仪器的校正染料产品信息活动时间：即日起至2009年4月30日2．Gibco南美胎牛血清即日起凡优惠价¥1780购买Gibco胎牛血清500ml（目录号:C2027050）即可获赠送价值¥250现金抵用券。
您可以凭现金抵用券在英韦创津公司购买任何商品，此券有效期至2009年5月31日。
产品信息活动时间：即日起至2009年4月30日独特的采集方式：GIBCO采用无菌心脏穿刺的方式采血原装直送，避免污染：原产地采集、加工、检测、包装。
完善的质控：采集、处理、检测、运输等环节都有文件和证书。
3．Invitrogen TA Cloning克隆产品专门用于克隆Taq聚合酶扩增的PCR产物。
采用pCR载体，能产生80%以上的重组产物，90%以上重组产物都包含插入片段。
产品信息活动时间：即日起至2009年5月31日附：pCR载体优点及图谱：3’-T突出端可直接连接Taq扩增的PCR产物可选择T7或T7和Sp6启动子进行体外RNA转录和测序侧向EcoRⅠ位点的通用多接头位点方便了插入片段的切离可以选择卡那霉素或氨苄青霉素进行筛选非常简便的蓝/白克隆筛选具有M13正向和反向引物位点，方便测序4．GIBCO液体培养基系列产品创立近50年的历史，品质优秀，产品种类丰富；为了中国用户利益，特建立国内生产线；所有产品，从原材料到生产全部按照GIBCO质量标准进行，每批均送抵美国公司总部质检合格后，才在国内销售。
E.coli Electro-Cells HB101 说明书
E.coli Electro-CellsHB101使用说明书Takara Code : D9021包装量Electro-Cells HB101 50 μl × 10 支Control DNA（pBR322，0.01 ng/μl）10 μl × 1 支保存: -80℃制品说明用高电压脉冲电流瞬间击穿大肠杆菌，从而使外源DNA转入大肠杆菌内的转化方法称为电穿孔法。
电穿孔法是各种转化方法中效率最佳的方法之一，比经Ca2+处理而获得的感受态细胞的转化效率高。
在制作基因文库、进行亚克隆时，尤其在转化少量DNA时，特别能发挥其威力。
GenotypesupE44，Δ(mcrC-mrr)，recA13，ara-14，proA2，lacY1，galK2，rpsL20，xyl-5，mtl-1，leuB6，thi-1细胞种类高效常用宿主菌E.coli HB101E.coli HB101在基因重组实验起始时便十分常用。
遗传性能稳定，使用十分方便，适合于各种基因重组实验。
细胞浓度: 1～2×1010 Bacteria/ml质量标准1. 使用10 pg的质粒DNA进行转化时：50 μl E.coli Electro-Cells HB101/10 pg pBR322 Plasmid＞5×108 transformants/μg pBR322 Plasmid。
2. 50 μl的E.coli Electro-Cells HB101在含有100 μg/ml的Ampicillin L-琼脂平板培养基上过夜培养40 hr不产生菌落。
使用方法质粒DNA的转化方法1. Electro-Cell（50 μl）使用前在冰中融化。
2. 在融化的Electro-Cell中加入1～2 μl DNA溶液（当DNA溶液中有盐存在时用乙醇沉淀脱盐）。
3. 将Electro-Cell及DNA混合液注入到冰中预冷的0.1 cm冲击槽内（Cuvette）。
邻苯二甲酸酯测量不确定度评定报告(CPSC 09.4)
邻苯二甲酸酯测量不确定度评定报告1.0 目的:本实验室采用气相色谱-质谱联用仪（GC-MS），对PVC样品中邻苯二甲酸酯含量进行分析检测。
基于JJF1059-1999《测量不确定度评定与表示》的一般要求，分析测试过程中不确定度的主要来源，评估标准不确定度、合成不确定度和扩展不确定度的数值，通过不确定度的分析结果来评定该方法的适用性。
本文主要以测试PVC样品中的邻苯二甲酸二(2-乙基己基)酯(DEHP)为例子，其他邻苯二甲酸酯分析测试过程相同。
2.0 实验原理及步骤2.1 实验原理本实验室依据CPSC-CH-C1001-09.4的标准方法，进行萃取、鉴别和量化 PVC 样品中含有的单体邻苯二甲酸酯。
试样经冷冻粉碎成粉末，在四氢呋喃中将它完全溶解，用正己烷将PVC 聚合物沉淀析出，过滤，然后用正己烷将溶液稀释定容，采用气相色谱-质谱联用仪（GC-MS），鉴别并量化各种邻苯二甲酸酯。
2.2试剂及设备2.2.1 标准物质：邻苯二甲酸二（2-乙基）己酯(DEHP)2.2.2 (BSS224S) 电子天平2.2.3 QP2010Plus气相色谱-质谱联用仪2.2.4 移液管：1mL、5mL2.2.5 容量瓶：50mL 、100mL2.3实验步骤准确称取0.1g（精确至0.1mg）试样。
将试样置于刻度试管中，在试管中加入10mL四氢呋喃超声溶解，之后再加入20ml正己烷沉淀。
实验步骤可见下页图1。
第 1 页共6 页图13.0数学模型Cx =(C-C0)×V/W 式中：Cx = 样品中相应邻苯二甲酸酯计算浓度，mg/kg；C = 样品中相应邻苯二甲酸酯测试浓度，mg/L；C0 = 空白相应溶液浓度，mg/L；V = 样品定容体积，ml；W = 样品质量，g.第 2 页共6 页第 3 页 共 6 页4.0识别各不确定度分量的来源4.1称重引入的不确定度 4.2容积引入的不确定度4.3配制标准溶液引入的不确定度 4.4测量重复性引入的不确定度4.5气相色谱-质谱联用仪引入的不确定度 4.6空白分析引入的不确定度空白分析显示未检出邻苯二甲酸酯，故在使用公式计算时C 0为零，所以不考虑空白分析引入的不确定度。
安捷伦产品目录
15
Real-Time PCR
16
Mx3000P QPCR System
17
Brilliant III Ultra-Fast SYBR Green QPCR and QRT-PCR Reagents
18
Brilliant III Ultra-Fast QPCR and QRT-PCR Reagents
Agilent / STRATAGENE
Agilent website: /genomics
Welgene | Agilent Stratagene
威健股份有限公司 | Stratagene 總代理
Table of Content
Table of Contents
/ XL1-Red Competent Cells SoloPack Gold Supercompetent Cells
/ TK Competent Cells Specialty Cells
/ Classic Cells / Fine Chemicals For Competent Cells
適用於 UNG 去汙染或 bisulphite
sequencing
適用於 TA Cloning
最高敏感性
取代傳統 Taq 的好選擇
-
2
威健股份有限公司 | Stratagene 總代理
PCR Enzyme & Instrument
Agilent SureCycler 8800
市場上領先的 cycling 速度和 sample 體積 10 ～ 100 μL 簡易快速可以選擇 96 well 和 384 well 操作盤 優秀的溫控設備讓各個 well 都能保持溫度的穩定 七吋的高解析度觸控螢幕讓操作上更為簡便 可以透過網路遠端操控儀器及監控儀器 Agilent 專業的技術支援可以幫助您應對各種 PCR 的問題
《2024年丝胶靶向Akt1调控糖酵解及氧化应激保护STZ致损伤INS-1细胞》范文
《丝胶靶向Akt1调控糖酵解及氧化应激保护STZ致损伤INS-1细胞》篇一一、引言随着糖尿病发病率的逐年攀升，研究胰岛素敏感度调节和β细胞损伤的机制对于防治糖尿病具有重要意义。
STZ（链脲佐菌素）诱导的INS-1细胞损伤模型是研究糖尿病发病机制及药物筛选的常用模型。
丝胶作为一种天然生物活性物质，具有多种生物功能，包括抗氧化、抗炎和促进细胞修复等。
本文旨在探讨丝胶靶向Akt1（蛋白激酶B）对糖酵解及氧化应激的影响，并分析其在STZ致损伤的INS-1细胞中的保护作用。
二、材料与方法2.1 材料实验所需材料包括：丝胶、STZ、INS-1细胞、相关试剂及仪器等。
2.2 方法（1）INS-1细胞培养及STZ处理：培养INS-1细胞，并给予不同浓度的STZ处理，建立细胞损伤模型。
（2）丝胶处理：将丝胶加入到STZ处理的INS-1细胞中，观察其对细胞的保护作用。
（3）Akt1活性检测：采用相关方法检测Akt1的活性变化。
（4）糖酵解及氧化应激指标检测：检测相关指标包括乳酸脱氢酶、丙二醛等。
（5）统计分析：采用SPSS软件进行数据分析，P<0.05为差异有统计学意义。
三、结果3.1 丝胶对STZ致损伤的INS-1细胞的保护作用实验结果显示，丝胶能够显著降低STZ对INS-1细胞的损伤程度，提高细胞存活率。
3.2 丝胶对Akt1活性的影响丝胶处理后，Akt1活性显著提高，表明丝胶可能通过激活Akt1发挥其保护作用。
3.3 丝胶对糖酵解及氧化应激的影响丝胶处理后，糖酵解相关指标如乳酸脱氢酶活性降低，氧化应激相关指标如丙二醛含量降低，表明丝胶能够调节糖酵解及氧化应激水平。
四、讨论4.1 丝胶保护INS-1细胞的机制丝胶通过激活Akt1，进而调控糖酵解及氧化应激，从而对STZ致损伤的INS-1细胞产生保护作用。
Akt1作为一种重要的信号分子，在细胞生存、增殖和凋亡等方面发挥重要作用。
丝胶可能通过与Akt1相互作用，激活其下游信号通路，从而发挥保护作用。
一种新型咔唑类菁染料黏度荧光探针
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CP1H操作手册中文
关于在国外的使用
当出口（或提供给非居住者）本产品中属于外汇及外国贸易管理法所规定的出口许可、 承认对象货物（或技术）范围的产品时，必须有以相关法律为基准的出口许可、承认（或 官方交易许可）。
4
关于 CP 系列的「单元版本」
关于 CP 系列的「单元版本」
单元版本是指
在 SYSMAC CP 系列中，为了管理由于版本升级等引起的 CPU 单元配置功能的差异，引 入了「单元版本」这个概念。
W451
CP1H-X40D□-□ CP1H-XA40D□-□ CP1H-Y20DT-D CS1G/H-CPU□□H CS1G/H-CPU□□-V1 CS1D-CPU□□H CS1D-CPU□□S CS1W-SCU21 CS1W-SCB21-V1/41-V1 CJ1G/H-CPU□□H CJ1G-CPU□□P CP1H-CPU□□ CJ1G-CPU□□ CJ1W-SCU21-V1/41-V1 WS02-CXPC1-EV6 WS02-CXPC1-EV6
6
关于 CP 系列的「单元版本」
3）通过单元版本标签进行识别
单元版本标签（下图）附带在产品中。
Ver. Ver.
1.0 1.0
Ver. Ver.
为了管理由于版本升级等引起的 CPU 单元配置功能的差异的标 签。
请根据需要贴在产品的正面。 These Labels can be used to manage differences in the available functions among the Units. Place the appropriate label on the front of the Unit to show what Unit version is actually being used.
人类C-C CKR-8 ELISA试验套件说明书
Human C-C CKR-8(C-C chemokine receptor type 8) ELISA KitCatalogue No.: EKF58052Size: 48T/96TReactivity: HumanRange: 0.313-20ng/mlSensitivity: 0.188ng/mlApplication: For quantitative detection of C-C CKR-8 in serum, plasma, tissue homogenates and other biological fluids.Storage: 2-8°C for 6 monthsExpiry Date: see kit labelPrinciple: SandwichNOTE: FOR RESEARCH USE ONLY.Kit ComponentsItem Specifications(48T/96T) StorageELISA Microplate(Dismountable) 8×6/8×12 2-8°C/-20°CLyophilized Standard 1vial/2vial 2-8°C/-20°CSample Dilution Buffer 10ml/20ml 2-8°CBiotin-labeled Antibody(Concentrated) 60ul/120ul 2-8°C(Avoid Direct Light)Antibody Dilution Buffer 5ml/10ml 2-8°CHRP-Streptavidin Conjugate(SABC) 60ul/120ul 2-8°C(Avoid Direct Light)SABC Dilution Buffer 5ml/10ml 2-8°CTMB Substrate 5ml/10ml 2-8°C(Avoid Direct Light)Stop Solution 5ml/10ml 2-8°CWash Buffer(25X) 15ml/30ml 2-8°CPlate Sealer 3/5piecesProduct Description 1copyTypical Data & Standard CurveResults of a typical standard operation of a C-C CKR-8 ELISA Kit are listed below. This standard curve was generated at our lab for demonstration purpose only. Users shall obtain standard curve as per experiment by themselves. (N/A=not applicable)STD.(ng/ml) OD-1 OD-2 Average Corrected0 0.127 0.131 0.129 00.312 0.3 0.308 0.304 0.1750.625 0.432 0.444 0.438 0.3091.25 0.781 0.803 0.792 0.6632.5 1.064 1.094 1.079 0.955 1.639 1.687 1.663 1.53410 2.007 2.065 2.036 1.90720 2.539 2.613 2.576 2.447SpecificityThis assay has high sensitivity and excellent specificity for detection of C-C CKR-8. No significant cross-reactivity or interference between C-C CKR-8 and analogues was observed.Note: Limited by current skills and knowledge, it is difficult for us to complete the cross-reactivity detection between C-C CKR-8 and all the analogues, therefore, cross reaction may still exist.RecoveryMatrices listed below were spiked with certain level of C-C CKR-8 and the recovery rates were calculated by comparing the measured value to the expected amount of C-C CKR-8 in samples.Matrix Recovery Range (%) Average (%)Serum(n=5) 95-101 98EDTA Plasma(n=5) 91-102 94Heparin Plasma(n=5) 95-103 95LinearityThe linearity of the kit was assayed by testing samples spiked with appropriate concentration of C-C CKR-8 and their serial dilutions. The results were demonstrated by percentage of calculated concentration to the expectation.Sample 1:2 1:4 1:8Serum(n=5) 97-103% 92-99% 92-100%EDTA Plasma(n=5) 89-105% 94-98% 90-99%Heparin Plasma(n=5) 89-105% 95-100% 90-95%PrecisionIntra-Assay: CV<8%Inter-Assay: CV<10%StabilityThe stability of ELISA kit is determined by the loss rate of activity. The loss rate of this kit is less than 10% within the expiration date under appropriate storage condition.Standard(n=5) 37°C for 1 month 2-8°C for 6 monthsAverage (%) 80 95-100To minimize extra influence on performance, operation procedures and lab conditions, especially room temperature, air humidity, incubator temperature should be strictly controlled. It is strongly suggested that the same operator performs the whole assay from the beginning to the end.Operation ProcedurePrinciple of the AssayThis kit was based on sandwich enzyme-linked immune-sorbent assay technology. Antibody was pre-coated onto 96-well plates. And the biotin conjugated Antibody was used as detection Antibody. The standards, test samples and biotin conjugated detectio n Antibody were added to the wells subsequently, and washed with wash buffer. HRP-Streptavidin was added and unbound conjugates were washed away with wash buffer. TMB substrates were used to visualize HRP enzymatic reaction. TMB was catalyzed by HRP to produce a blue color product that changed into yellow after adding acidic stop solution. The dens ity of yellow is proportional to the target amount of sample captured in plate. Read the O.D. absorbance at 450nm in a microplate reader, a nd then the concentration of target can be calculated.Precautions1.To inspect the validity of experiment operation and the appropriateness of sample dilution proportion, pilot experimentusing standards and a small number of samples is recommended.2.After opening and before using, keep plate dry.3.Before using the kit, spin tubes and bring down all components to the bottom of tubes.4.Storage TMB reagents avoid light.5.Washing process is very important, not fully wash easily cause a false positive and high background.6.Duplicate well assay is recommended for both standard and sample testing.7.Don’t let microplate dry at the as say, for dry plate will inactivate active components on plate.8.Don’t reuse tips and tubes to avoid cross contamination.9.Please do not mix the reagents in different kits of our company. Do not mix reagents from other manufacturers.10.To ensure accurate results, proper adhesion of plate sealers during incubation steps is necessary.Material Required but Not Supplied1.Microplate reader (wavelength:450nm)2.37°C incubator3.Automated plate washer4.Precision single and multi-channel pipette and disposable tips5.Clean tubes and Eppendorf tubes6.Deionized or distilled waterWashingManual: Discard the solution in the plate without touching the side walls. Clap the plate on absorbent filter papers or other absorbent material. Fill each well completely with 350ul wash buffer and soak for 1 to 2 minutes, then aspirate contents from the plate, and clap the plate on absorbent filter papers or other absorbent material.Automatic: Aspirate all wells, and then wash plate with 350ul wash buffer. After the final wash, invert plate, and clap the plate on absorbent filter papers or other absorbent material. It is recommended that the washer shall be set for soaking 1 minute. (Note: set the height of the needles; be sure the fluid can be sipped up completely)Sample Collection and Storage (universal)●Serum: Place whole blood sample at room temperature for 2 hours or put it at 2-8°C overnight and centrifugation for 20minutes at approximately 1000×g, Collect the supernatant and carry out the assay immediately. Blood collection tubes should be disposable, non-pyrogenic, and non-endotoxin.●Plasma: Collect plasma using EDTA-Na2 or heparin as an anticoagulant. Centrifuge samples for 15 minutes at 1000×g at 2-8°Cwithin 30 minutes of collection. Collect the supernatant and carry out the assay immediately. Avoid hemolysis, highcholesterol samples.●Tissue Homogenates: As hemolysis blood has relation to assay result, it is necessary to remove residual blood by washingtissue with pre-cooling PBS buffer (0.01M, pH=7.4). Mince tissue after weighing it and get it homogenized in PBS (thevolume depends on the weight of the tissue. Normal, 9mL PBS would be appropriate to 1 gram tissue pieces. Some protease inhibitors are recommended to add into the PBS) with a glass homogenizer on ice. To further break the cells, you cansonicate the suspension with an ultrasonic cell disrupter or subject it to freeze-thaw cycles. The homogenates are then centrifuged for 5 minutes at 5000×g to get the supernatant. The total protein concentration was determined by BCA kit and the total protein concentration of each pore sample should not exceed 0.3mg.●Adherent and Suspension Cell Culture: Use three T25 flasks or one T75 flask for cell culture, the number of cells (1x107);1. Suspension cell: centrifuge at 2500 rpm at 2-8℃ for 5 minutes; collect clarified cell culture supernatant;2. Adherent cell: collect supernatant directly; centrifuge at 2500 rpm at 2-8℃ for 5 minutes; collect clarified cell culturesupernatant for immediate detection or store it separately at -80℃.●Cell Lysate Preparation: Two types of cell lysates are specified below.1. Suspension Cell Lysate：Centrifuge at 2500 rpm at 2-8℃ for 5 minutes; Then add pre-cooling PBS into collected cell and gentlymix. Recollect cell by repeating centrifugation. Add 0.5-1ml RIPA lysis buffer (NP-40 lysis buffer or Triton X-100 surfactant is not recommended due to the interfering with antigen-antibody reaction). Add suitable protease inhibitor (e.g. PMSF, workingconcentration: 1mmol/L). Lyse the cell on ice for 30min-1h. During lysate process, use the tip for pipetting or intermittently shake the centrifugal tube to completely lyse the protein. Alternatively, cells are subject to fragmentation by ultrasonic cell disruptor (300W, 3~5 s/time, 30s intervals, four-five times) or ultrasonic generator (14μm for 30s ). At the end of lysate or ultrasonicdisruption, centrifuge at 10000rpm at 2-8℃ for 10 minutes. Then, the supernatant is added into EP tube and stored at -80℃.2. Adherent Cell Lysate: Absorb supernatant and add pre-cooling PBS once. Then, add 0.5-1ml RIPA lysis buffer (NP-40 lysis bufferor Triton X-100 surfactant is not recommended due to the interfering with antigen-antibody reaction). Add the suitable protease inhibitor (e.g. PMSF, working concentration: 1mmol/L). Scrape adherent cell gently with a cell scraper. Add the cell suspension into centrifugal tube. Lyse the cell on ice for 30min-1h. During lysate process, use the tip for pipetting or intermittently shake thecentrifugal tube to completely lyse the protein. Alternatively, cells are subject to fragmentation by ultrasonic generator (14μm for 30s ) or ultrasonic cell disruptor (300W, 3~5 s/time, 30s intervals, four-five times). At the end of lysate/ultrasonic disruption,centrifuge at 10000rpm at 2-8℃ for 10 minutes. Then, the supernatant is added into EP tube and stored at -80℃.●Other Biological Fluids: Centrifuge samples for 20 minutes at 1000×g at 2-8°C. Collect supernatant and carry out the assayimmediately.Note: Samples used within 5 days can be stored at 2-8°C; otherwise, they must be stored at -20°C or -80°C or liquid nitrogen to avoid loss of biological activity and contamination. Avoid multiple freeze-thaw cycles. Hemolytic samples are not suitable for this test. Sample DilutionThe user should estimate the concentration of target protein in the test sample, and select a proper dilution factor to make the diluted target protein concentration fall in the optimal detection range of the kit. Dilute the sample with the provided dilu tion buffer, and several trials may be necessary. The test sample must be well mixed with the dilution buffer. And also standard curves and sample should be making in pre-experiment. If samples with very high concentrations, dilute samples with PBS first and then dilute the samples with Sample Dilution.The matrix components in the sample will affect the test results, which it need to be diluted at least 1/2 with Sample Dilution Buffer before testing!Reagent Preparation and StorageBring all reagents and samples to room temperature for 20 minutes before use.1, Wash Buffer:If crystals have formed in the concentrate, you can warm it with 40°C water bath (Heating temperature should not exceed 50°C) and mix it gently until the crystals have completely been dissolved. The solution should be cooled to room temperature before use. Dilute 30ml (15ml for 48T) Concentrated Wash Buffer to 750ml (375ml for 48T) Wash Buffer with deionized or distilled water(The recommended resistivity of deionized or distilled water is 18MΩ). Put unused solution back at 2-8°C.2, Standards:1). Add 1 ml Sample Dilution Buffer into one Standard tube (labeled as zero tube), keep the tube at room temperature for 10 minutes and mix them thoroughly.Note: If the standard tube concentration higher than the range of the kit，please dilute it and labeled as zero tube.2). Label 7 EP tubes with 1/2, 1/4, 1/8, 1/16, 1/32, 1/64 and blank respectively. Add 0.3ml of the Sample Dilution Buffer int o each tube. Add 0.3ml of the above Standard solution (from zero tube) into 1st tube and mix them thoroughly. Transfer 0.3ml from 1st tube to 2nd tube and mix them thoroughly. Transfer 0.3ml from 2nd tube to 3rd tube and mix them thoroughly, and so on. Sample Dilution Buffer was used for the blank control.Note: It is best to use Standard Solutions within 2 hours.3, Preparation of Biotin-labeled Antibody Working Solution:Prepare it within 1 hour before experiment.1）Calculate required total volume of the working solution: 0.1ml/well × quantity of wells. (Allow 0.1-0.2ml more than the total volume.)2）Dilute the Biotin-detection Antibody with Antibody Dilution Buffer at 1:100 and mix them thoroughly. (i.e. Add 1ul Biotin-labeled Antibody into 99ul Antibody Dilution Buffer.)4, Preparation of HRP-Streptavidin Conjugate (SABC) Working Solution:Prepare it within 30 minutes before experiment.1）Calculate required total volume of the working solution: 0.1ml/well × quantity of wells. (Allow 0.1-0.2ml more than the total volume.)2）Dilute the SABC with SABC Dilution Buffer at 1:100 and mix them thoroughly. (i.e. Add 1ul of SABC into 99ul of SABC Dilution Buffer.)Assay ProcedureWhen diluting samples and reagents, they must be mixed completely and evenly. Before adding TMB into wells, equilibrate TMB Substrate for 30 minutes at 37°C. It is recommended to plot a standard curve for each test.1.Set standard, test samples (diluted at least1/2 with Sample Dilution Buffer), control (blank) wells on the pre-coated platerespectively, and then, records their positions. It is recommended to measure each standard and sample in duplicate.2.Prepare Standards: Aliquot 100ul of zero tube, 1st tube, 2nd tube, 3rd tube, 4th tube, 5th tube, 6th tube and Sample DilutionBuffer (blank) into the standard wells.3.Add Samples: Add 100ul of properly diluted sample into test sample wells.4.Incubate: Seal the plate with a cover and incubate at 37°C for 90 minutes.5.Wash: Remove the cover and discard the plate content, and wash plate 2 times with Wash Buffer. Do NOT let the wells drycompletely at any time.6.Biotin-labeled Antibody: Add 100ul Biotin-labeled Antibody working solution into above wells (standard, test sample andblank wells). Add the solution at the bottom of each well without touching the sidewall, cover the plate an d incubate at 37°C for 60 minutes.7.Wash: Remove the cover, and wash plate 3 times with Wash Buffer, and let the Wash Buffer stay in the wells for 1-2minutes each time.8.HRP-Streptavidin Conjugate (SABC): Add 100ul of SABC Working Solution into each well, cover the plate and incubate at37°C for 30 minutes.9.Wash: Remove the cover and wash plate 5 times with Wash Buffer, and let the wash buffer stay in the wells for 1-2 minuteseach time.10.TMB Substrate: Add 90ul TMB Substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 minutes.(Note: The reaction time can be shortened or extended according to the actual color change, but not more than 30 minutes.You can terminate the reaction when apparent gradient appeared in standard wells.)11.Stop: Add 50ul Stop Solution into each well. The color will turn yellow immediately. The adding order of Stop Solutionshould be as the same as the TMB Substrate Solution.12.OD Measurement: Read the O.D. absorbance at 450nm in Microplate Reader immediately after adding the stop solution. Regarding calculation, (the relative O.D.450) = (the O.D.450 of each well) – (the O.D.450 of blank well). The standard curve can be plotted as the relative O.D.450 of each standard solution (Y) vs. the respective concentration of the standard solution (X). The target concentration of the samples can be interpolated from the standard curve. It is recommended to use some professional software to do this calculation, such as Curve Expert 1.3 or 1.4.Note: If the samples measured were diluted, multiply the dilution factor to the concentrations from interpolation to obtain the concentration before dilution.SummaryStep1: Set standard, test samples, control (blank) wells on the pre-coated plate respectively, and then, records their positions.Step2: Add 100ul standard or sample to each well and incubate for 90 minutes at 37°C.Wash step: Aspirate and wash plates 2 times.Step3: Add 100ul Biotin-labeled Antibody working solution to each well and incubate for 60 minutes at 37°C.Wash step: Aspirate and wash plates 3 times.Step4: Add 100ul SABC Working Solution into each well and incubate for 30 minutes at 37°C.Wash step: Aspirate and wash plates 5 times.Step5: Add 90ul TMB Substrate Solution. Incubate 10-20 minutes at 37°C.Step6: Add 50ul Stop Solution. Read at 450nm immediately and calculation.。
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测试方法：CPSC-CH-E1001-8.1
测定金属材料中总铅的标准操作程序
儿童产品（包括儿童的金属手饰）
2010年6月21日修定
该文件提供了关于测试方法的详细信息，而这些方法被美国消费品安全委员会的实验室用来测试儿童金属产品中总铅的含量。
所描述的方法用来测定金属中总铅的含量，而不限于儿童的金属手饰。
该版本替代了先前发布的测试方法CPSC-CH-E1001-8，主要是在编辑上有一些变化。
美国消费品安全委员会得出结论，这些测试方法足以测试大多数消费品里的总铅含量。
对一些合金产品可能需要一些调整，但应基于足够的化学知识和针对基选择合适的酸.所选择的方法应是那些能很好证明它们能完全消解样品。
如果与申请不一致，或不能证明有很好的效果或不熟悉方法，那么这些方法不能被使用。
该方法适用于金属和金属部件，美国消费品安全委员会不建议用于其它一些材料，这些材料在具体的条件下不溶解或会与酸反应，生成一些不符合实验室安全的物质。
一般方法是将样品磨成粉末，采用HCL和HNO3的组合来完全消解样品，然后采用ICP进行测试。
其它的一些方法，比如ICP-MS，FLAA和GFAA在合适的条件下可作为一种对ICP-OES的替代。
定义：
1.样品-一个单一的消费产品或一组来自于同一批的完全一样的产品。
2.部件-样品里的单个分部件。
诸如手镯的一些物件可破解成一些部件，比如珠子，挂钩，
吊坠。
每一部分都要分析。
3.仪器侦测极限（IDL）-对试剂空白测量十次的3倍标准偏差。
4.方法侦测极限（MDL）-试剂空白，2～3倍的仪器侦测极限。
重覆测试该空白7次。
计
算方法侦测极限如下：MDL=t×S,t=3.14(99%的置信区间)，S=标准偏差。
5.实验室试剂空白（LRB）-铅测试时使用的萃取或消解介质。
LRB数据用来评估实验室
环境的污染。
6.校正空白-用硝酸酸化的去离子水（用去离子水将3ml浓硝酸稀释成100ml）
7.标准储备溶液-从声誉好的供应商购买铅标准溶液1000ppm，用来制备校正溶液。
8.校正标准品-3%的硝酸基质里含 0～25ppm的铅溶液，最少采用4个校正标准品，应每
周配一次。
9.质量控质样品（QCS）-一个含铅的溶液用来评估仪器系统的性能。
QCS从外部获得或
从储备液配置。
10.获认可的参考物质（CRM）-CRMs已知铅浓度，和样品含同一种基质。
用来验正消解和
分析方法。
CRMs的样品列在设备和消耗品部分，但不限于这些。
设备和消耗品：下面的材料用来取样和分析：
1.硝酸,痕量金属级
2.盐酸,痕量金属级
3.消解罐,50ml
4.金属切割工具
5.蒸馏水
6.微波消解设备
7.研磨机
8.振荡器
9.已知浓度的铅含量且用于金属方面的CRMs，例如NIST SAM 54d,1728
10.内标（比如钇，来自于该元素的标准储备液，适用于ICP分析的一些分析参数）
Ⅰ.金属消解的总铅
当制备一个样品时，实验室应确保来自于样品的部分是有代表性的，没有污染的。
每一个单一的，可接触的部分都要分析总铅含量，CPSC实验室用两个方法中的一个来测定金属中的总铅。
CPSC采用的一个方法热块消解，是基于加拿大产品安全局方法C-02.4.里的方法论,这个方法等同于CPSC测定总铅和其在儿童金属手饰的适用性的标准操作程序Ⅰ.总铅分析的筛选测试程序2/3/2005.注意到该参考程序第二部分是用来测定酸的萃取率而不适合用于测定总铅含量。
另一个方法是微波消解方法，是基于EPA3051A作了部分修改。
考虑到要合金需要合适的酸，该方法作一些修改是必要的。
CRMs应高度匹配测试产品的合金，应被用来验证消解和分析方法的精确性。
样品制备方法
LSC职员发现下面概述的两种方法进行元素分析时，在文件的范围内都适合于金属样品的制备。
A．热块消解方法
1.如果物件上有油漆或类似的涂层，（可能含铅），涂层需要单独地从基材上移走并被
测试和分析，按照CPSC里的标准操作程序来测试油漆的总铅。
尽可能的不移走基材。
2.称取30～100mg的样品加入到50ml消解罐里。
儿童产品的一些部件包括金属手饰，
一般重几克以上，只取一部分（没有油漆和类似的涂层，但是包含电镀层，电镀层被认为是基材的一部分）。
样品应被剪切成小片以增加溶解率。
如果用研磨设备，应该彻底清理，以阻止交叉污染。
记录实际重量，精确到0.1mg。
3.在化学通风橱里，加8ml的浓硝酸至烧杯里,通过105±3℃的热段消解蒸发至大约
3ml。
4.冷却后，加入2ml的浓HCL搅拌。
5.用蒸馏水冲洗烧杯壁，稀释至20ml.
6.将溶液放在60℃的振荡器里，慢慢地搅动，或用搅拌器搅动，或搅拌水浴至少4Hr。
7.定量地转移至50ml容量瓶里或一次性测量消解杯里，用去离子水定容至50ml。
8.稀释样品使铅的结果在仪器的校正范围内，一般50倍就足够了。
B．微波方法
1.如果物件上有油漆或类似的涂层，（可能含铅），涂层需要单独地从基材上移走并被
测试和分析，按照CPSC里的标准操作程序来测试油漆的总铅。
尽可能的不移走基材。
2.称取30～100mg的样品加入到合适的消解罐里,该消解罐应有卸压装置。
童产品的
一些部件包括金属手饰，一般重几克以上，只取一部分（没有油漆和类似的涂层）。
样品应被剪切成小片以增加溶解率。
如果用研磨设备，应该彻底清理，以阻止交叉污染。
记录实际重量，精确到0.1mg。
3.加入
4.5ml浓硝酸和1.5ml的浓盐酸至每一个消解罐里，在室温下，等候酸和样品
的反应完全（没有明显的烟气和气泡），然后密封消解罐。
密封消解罐按照厂商的说明进行。
4.微波方法应逐步在大约
5.5Mins内升温到至少175℃，在175℃时保持4.5Mins。
5.在移出微波前，让样品至少冷却5Mins，打开盖子前，在通风橱要先卸压。
6.定量地将样品转移到50ml容量瓶里，或一次性测量消解杯里。
用去离子水定容至
50ml。
Ⅱ.金属材料里的总铅的分析
采用ICP光谱仪分析稀释样品的铅浓度。
ICP-OES,FLAA和GFAA的分析过程都是基于ASTM E1613-04里的方法。
ICP-MS也可以和其它合适的方法结合起来使用，如EPA6020A。
（注:方法C-02.4描述了AAS光谱分析的替代程序）,从稀释的溶液计算出部件的总铅含量，并说明稀释，采用重量百分比报告浓度。
ICP操作程序和质量控制
分析
1.点燃等离子体。
按照制造商建议执行波长校正或矩管准直。
2.仪器在开始时，要先预热。
3.在分析方法里，确保元素和波长被选择：
a.Pb 220.353
b.Pb的另一条线217.00是确保光谱干扰不会发生。
4.采用内标，比如2µg/ml的钇。
5.采用至少3个标液和一个空白执行校正。
分析时，校正应每天至少一次，或仪
器每次使用时。
每个标液的结果应在5%的真值范围内。
如果值不在这个范围内，重新校正。
6.校正后立即分析QCS。
Pb的分析值应在期望值的10%范围内。
如果Pb的值在10%
的范围外，重新校正。
a.每次测试时，至少有一个LRB被分析。
如果Pb的值超过了3倍的MDL，则实
验室或试剂有可能被污染。
在继续分析前，应确认和解决污染源。
LRBs应是
和样品里的酸是同一种浓度，LRBs应引入到整个过程消解过程中。
7.在每批样品分析中，至少要有一个参考物质被分析。
作为已知浓度的样品，参
考物质应具有类似的材料。
分析物的回收率应在期望值的15%范围内。
如果回收率超出了这个范围，那么在确认和解决问题源之前，不能分析。
8.Pb的浓度超过校正曲线的1.5倍时，稀释样品，重新分析。
计算和结果报告
Pb测试结果的计算和报告如下所示：
总Pb的百分比：%Pb(wt./wt.)=C×D/W×100%/1000µg/mg
a.C=Pb的浓度（µg/ml）
b.D=稀释因子（ml）
c.W=样品质量（mg）
例如：
表格1：总铅分析
部件取50mg进行消解，稀释到1000ml来用ICP分析。
结果发现在溶液中有20µg/ml
CPSC-CH-E1001-8.1修定概述
1.第11页，修定测试方法#和日期
2.第2页，去掉了IDL和MDL的CPSC实验室值；与方法无关，新仪器有不同的值
3.第2页，定义10，修定了最后一句，允许CRMs有其它来源
4.第3页，改变了推介的CRMs，去掉了NIST 53e 和 1129(没有保证Pb),加入了1728
（新的NIST SRM 锡基质里的铅浓度545ppm）
5.第4页，改变了微波的温度要求，从“175±5℃”至“至少175℃”。