细菌内毒素检查方法国际药典0424Word格式.docx

1、有以下三种方法： Method A. The gel-clot technique, which is based on gel formation;方法A：凝胶凝结技术，此方法基于凝胶的形成。 Method B. The turbidimetric technique, based on the development of turbidity after cleavage of an endogenous substrate;方法B：浊度技术，此方法基于内源性底物分裂后所产生的浊度。 Method C. The chromogenic technique, based on the dev

2、elopment of colour after cleavage of a synthetic peptide-chromogen complex.方法C：显色技术，此方法基于合成的肽显色复合物裂解后产生的颜色。Unless otherwise indicated in the individual monograph proceed by Method A.除在单独的检测专著中另有规定外，采用方法A。The test is carried out in a manner that avoids endotoxin contamination.试验过程中应防止内毒素污染。APPARATUS器

3、械Depyrogenate all glassware and other heat stable materials in a hot air oven using a validated process. A commonly used minimum time and temperature is 30 minutes at 250 C. If employing plastic apparatus such as microplates and pipet tips for automatic pipetters, use apparatus shown to be free of d

4、etectable endotoxin and which does not interfere in the test.用已验证过的方法在热风烘箱内对所有的玻璃器具和其他耐热材料进行去热原处理，通常此操作设定的最短时间和最低温度问30分钟和250 C。若使用到塑料器具，如微孔板和微量加样器配套的吸头等，应选用标明无内毒素并试验无干扰的器械。Note: In this chapter the term tube includes any other receptacle such as a micro-titre well.注意：此章节中的“管状容器”包括其他任何例如微孔板中的孔一类的容器，。

5、REAGENTS AND TEST SOLUTIONS试剂和检测用溶液 Amoebocyte lysate鲎试剂A lyophilized product obtained from the lysate of amoebocytes （white blood cells） from the horseshoe crab （Limulus polyphemus or Tachypleustridentatus）.由鲎（Limulus polyphemus或Tachypleustridentatus）的变形细胞（血液白细胞）的溶解产物得到的冻干产品。 Amoebocyte lysate reac

6、ts to some -glucans in addition to endotoxins. Amoebocyte lysate preparations which do not react to glucans are available: they are prepared by removing the G factor reacting to glucans from amoebocyte lysate or by inhibiting the G factor reacting system of amoebocyte lysate and may be used for the

7、endotoxin testing in the presence of glucans.注：鲎试剂除内毒素外还与一些-葡聚糖反应，但也有不与葡聚糖反应的鲎试剂。通过在制备时除去鲎试剂中能与葡聚糖反应的G因子或通过抑制鲎试剂中G因子的反应系统来得到此种鲎试剂，这种鲎试剂可用来检测含有葡聚糖的产品中的细菌内毒素。 Lysate solution 鲎试剂溶液Dissolve amoebocyte lysate in water BET or in a buffer recommended by the lysate manufacturer, by gentle stirring. Store t

8、he reconstituted lysate, refrigerated or frozen, according to the specifications of the manufacturer.用BET水或鲎试剂生产商推荐的缓冲液来溶解鲎试剂，缓缓搅拌。根据生产厂家的说明书对新形成的鲎试剂进行冷藏或冷冻保存。 Water BET （water for bacterial endotoxins test） BET水（细菌内毒素检测用水）Water for injections or water produced by other procedures that shows no reac

9、tion with the lysate employed, at the detection limit of the reagent.注射用水或用其他程序生产的检测限度内不与鲎试剂发生反应的水。Preparation of Standard Endotoxin Stock Solution标准内毒素贮备液的配制A Standard Endotoxin Stock Solution is prepared from the WHO International Standard for endotoxin （available from the National Institute for B

10、iological Standards and Control （NIBSC）, United Kingdom of Great Britain） or an endotoxin reference standard that has been calibrated against the WHO International Standard for endotoxin. Follow the specifications in the package leaflet and on the label for preparation and storage of the Standard En

11、dotoxin Stock Solution.标准内毒素贮备液是用WHO内毒素国际标准（英国国家生物标准和控制学会（NIBSC）制定的标准）或由根据WHO内毒素国际标准校准后的内毒素标准品而制备得到的。根据说明书和标签上的要求制备及储存标准内毒素贮备液。Endotoxin is expressed in International Units （IU） of endotoxin.内毒素用国际单位（IU）来表示。 One International Unit （IU） of endotoxin is equal to one Endotoxin Unit （EU）.1个国际单位（IU）等于1个内

12、毒素单位（EU）。Preparation of Standard Endotoxin Solution标准内毒素溶液的制备After mixing the Standard Endotoxin Stock Solution vigorously, prepare appropriate serial dilutions of Standard Endotoxin Solution, using water BET.在剧烈的混匀标准内毒素贮备液后，用BET 水对其进行稀释以制备适当的系列稀释液，即标准内毒素溶液。Use dilutions as soon as possible to avoid

13、 loss of activity by adsorption.稀释液应尽快使用，以避免因吸附而导致活性损失。Preparation of sample solutions供试品溶液的制备Prepare sample solutions by dissolving or diluting the pharmaceutical substance or the finished preparation using water BET. Some substances or preparations may be more appropriately dissolved or diluted in

14、 other aqueous solutions. If necessary, adjust the pH of the solution to be examined （or dilution thereof） so that the pH of the mixture of the Lysate solution and sample solution falls within the pH range specified by the lysate manufacturer, usually 6.08.0. The pH may be adjusted by the use of aci

15、d, base or suitable buffer as recommended by the lysate manufacturer. Acids and bases may be prepared from concentrates or solids with water BET in containers free of detectable endotoxin. Buffers must be validated to be free of detectable endotoxin and interfering factors.通过BET水溶解或稀释药用物质或成品制剂来制备供试品

16、溶液。一些药用物质或成品制剂更适于用其他的水溶液来进行溶解或稀释。如果需要的话，调节供试品溶液（或其稀释液）的PH值以使鲎试剂溶液与供试品溶液的混合液的PH值符合鲎试剂生产商规定的PH值范围，此范围通常为6.8-8.0。可使用鲎试剂生产商推荐的酸、碱或适当的缓冲液来调节PH值。所使用的酸和碱溶液可以用其浓缩物或固体物在没有内毒素的容器中，用BET水进行配制。所使用的缓冲液必须进行验证以证明其没有内毒素和干扰因子。DETERMINATION OF MAXIMUM VALID DILUTION确定最大有效稀释倍数The maximum valid dilution （MVD） is the max

17、imum allowable dilution of a sample at which the endotoxin limit can be determined.最大有效稀释倍数（MVD）指在能够确定内毒素限度时供试品允许被稀释的最大倍数。Determine the MVD from the following equation根据下面的公式计算MVD:MVD = Endotoxin limit内毒素限度The endotoxin limit for parenteral preparations, defined on the basis of dose, equals K/M, whe

18、re K is a threshold pyrogenic dose of endotoxin per kg of body weight and M is equal to the maximum recommended bolus dose of product per kg of body weight. When the product is to be injected at frequent intervals or infused continuously, M is the maximum total dose administered in a single hour per

19、iod.非胃肠道给药制剂内毒素限度根据其剂量来确定，等于K/M。K指每千克体重可接受的内毒素的致热剂量阈值，M指每千克体重建议的最大注射剂量。如果一种药品以较短的时间间隔频繁注入或连续输入体内，那么M就是指在一小时内使用的最大总剂量。 The endotoxin limit depends on the product and its route of administration and is stated in the individual monograph. Suggested values for K are:内毒素标准取决于产品和其使用途径，并且在单独的专论中有规定。建议的K值如下

20、：- intravenous route: K = 5 IU endotoxin per kg body weight;静脉注射给药：K= 5 IU内毒素/千克体重- intravenous route for radiopharmaceuticals: K = 2.5 IU endotoxin per kg body weight;放射性药物静脉注射给药：K= 2.5 IU 内毒素/千克体重- intrathecal route: K = 0.2 IU endotoxin per kg body weight.鞘内注射给药：K = 0.2 IU内毒素/千克体重For other routes

21、 of administration the acceptance criterion for bacterial endotoxins is generally determined on the basis of results obtained during development of the preparation.采用其他给药途径时，细菌内毒素的可接受标准一般是根据研发过程中得到的结果来确定的。The endotoxin limit for parenteral preparations is specified in units such as IU/ml, IU/mg, IU/

22、Unit of biological activity, etc., in the individual monograph.非胃肠道给药制剂的内毒素限度以IU/ml、IU/mg,、IU/生物活性单位等等来表示，在单独的专论中有规定。Concentration of sample solution:供试品溶液的浓度- mg/ml in the case of endotoxin limit specified by weight （IU/mg）;如果内毒素限度以质量（IU/mg）表示时，用mg/ml表示供试品浓度；- units/ml in the case of endotoxin limi

23、t specified by unit of biological activity （IU/Unit）;如果内毒素限度以生物活性单位（IU/Unit（单位）表示时，用units（单位）/ml表示供试品浓度；- ml/ml when the endotoxin limit is specified by volume （IU/ml）.如果内毒素限度以体积（IU/ml）表示时，用ml/ml表示供试品浓度；: the labelled lysate sensitivity in the gel-clot technique （IU/ml） or the lowest concentration u

24、sed in the standard curve for the turbidimetric or chromogenic techniques.：指用凝胶法检测时鲎试剂标示灵敏度（IU/ml）或指用浊度或显色法检测时运用于标准曲线上的最低浓度。METHOD A: GEL-CLOT TECHNIQUE方法A：凝胶法The gel-clot technique is for detecting or quantifying endotoxins based on clotting of the Lysate solution in the presence of endotoxin. The

25、minimum concentration of endotoxin required to cause the lysate to clot under standard conditions is the labelled sensitivity of the Lysate solution. To ensure both the precision and validity of the test, perform the tests for confirming the labelled lysate sensitivity and for interfering factors as

26、 described below under Preparatory testing.凝胶法指基于溶液中的内毒素与鲎试剂产生凝聚反应而检测或定量内毒素的方法。在标准条件下要求的使鲎试剂凝结的内毒素浓度即为鲎试剂的标示灵敏度。为了保证检测的精确性和有效性，按照准备试验下规定的方法来复核鲎试剂的标示灵敏度和干扰因子。Preparatory testing准备试验Test for confirmation of labelled lysate sensitivity鲎试剂标示灵敏度确认试验Confirm in four replicates the labelled sensitivity, , e

27、xpressed in IU/ml of the lysate prior to use in the test. The test for confirmation of the lysate sensitivity is to be carried out when a new lot of lysate is used or when there is any change in the test conditions which may affect the outcome of the test.用4个平行样来确认鲎试剂的标示灵敏度，在开始试验前将其转换为用IU/ml的形式来表示。当

28、使用新批号的鲎试剂或当试验条件发生了任何可能影响试验结果的改变时，应进行鲎试剂灵敏度确认试验。Prepare standard solutions having at least four concentrations equivalent to 2 , , 0.5 and 0.25 by diluting the Standard Endotoxin Stock Solution with water BET.用BET水将标准内毒素贮备液稀释，至少制备相当于2 , , 0.5 和 0.25四个浓度的标准溶液。Mix a volume of the Lysate solution with a

29、n equal volume of one of the standard solutions （such as 0.1 ml aliquots） in each tube. When single test vials or ampoules, containing lyophilized lysate are employed, add solutions of standards directly to the vial or ampoule. Incubate the reaction mixture for a constant period according to directi

30、ons of the lysate manufacturer （usually at 371C for 60 2 minutes）, avoiding vibration. Test the integrity of the gel for tests carried out in tubes, take each tube in turn directly from the incubator and invert it through approximately 180 degrees in one smooth motion. If a firm gel has formed that remains in place upon inversio