(整理)EP80细菌内毒素检查法中英文对照.

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EP8.0 2.6.14 细菌内毒素 （中英文 ）

2.6.14. BACTERIAL ENDOTOXINS 细菌内毒素

The test for bacterial endotoxins (BET) is used to detect or quantify

endotoxins from gram-negative bacteria using amoebocyte lysate from

the horseshoe crab (Limulus polyphenmus or Tachypleus tridentatus).

There are 3 techniques for this test: the gel-clot techniques, which is

based on gel formation; the turbidimetric technique, based on the

development of turbidity after cleavage of an endogenouse substrate;

and the chromogenic technique, based on the development of colour

after cleavage of a synthetic peptide-chromogen complex.

本法利用鲎试剂（从鲎——美洲鲎或中国鲎——变形细胞溶解物制备而来）检测

由革兰氏阴性菌产生的细菌内毒素或对内毒素进行定量。该检查包括三种方法：

一为凝胶法，系利用鲎试剂与内毒素产生凝集反应的原理；第二种为浊度法（基

于内源性底物断裂后，产生的浊度变化）；最后一种为显色法（得到的肽－呈色

基团复合物断裂后，检测反应混合物的色度）。

The following 6 methods are described in the present chapter:这一章阐述

了下面6种方法：

Method A. Gel-clot method: limit test

Method B. Gel-clot method: quantitative test

Method C. Turbidimetric kinetic method

Method D. Chromogenic kinetic method

Method E. Chromogenic end-point method

Method F. Turbidimetric end-point method

方法A：凝胶法：限度试验

方法B：凝胶法：定量试验

方法C：动态浊度法

方法D：动态显色法

方法E：终点显色法

方法F：终点浊度法

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Proceed by any of the 6 methods for the test. In the event of doubt or

dispute, the final decision is made based upon method A unless

otherwise indicated in the monograph.

检测时，可用6种方法的任一种进行试验。当测定结果可疑或有争议时，除非另

有规定，以专论中的方法A的测定结果为准。

The test is carried out in a manner that avoids endotoxin contamination.

试验操作过程应防止内毒素的污染。

1. APPARATUS仪器

Depyrogenate all glassware and other heat-stable apparatus in a hot-air

oven using a validated process. A commonly used minimum time and

temperature is 30 minutes at 25?C. If employing plastic apparatus, such

as microtitre plates and pipette tips for automatic pipetters, use

apparatus shown to be free of detectable endotoxin and which does not

interfere in the test.

所有的玻璃器皿及由其他耐热材料制成的器皿需用已验证的工艺在热烘箱内进

行去热原处理。去热原时，常用的最小时间和温度设置分别为30分钟和250℃。

若使用塑料器械，如微孔板和微量进样器配套的吸头等，它们必须标明无内毒素

并确对试验无干扰。

NOTE: in this chapter, the term ‘tube’ includes all types of receptacles,

for example microtitre plate wells.

注：这一章中，“管”的意思包括其他任何反应容器，如微孔板中的孔。

2. REAGENTS, TEST SOLUTIONS试剂、试液

(1) Amoeboyte lysate鲎试剂

Amoebocyte lysate is a lyophilized product obtained from amoebocyte

lysate from the horseshoe crab (Limulus polyphemus or Tachypleus

tridentatus). This reagent refers only to a product manufactured in

accordance with the regulations of the competent authority.

鲎试剂物是从鲎（美洲鲎或中国鲎）的变形细胞产生的低压冻干产物。该试剂仅

指在符合有关权威法规的生产条件下生产的鲎试剂产品。

NOTE: amoebocyte lysate reacts with some β-glucans in addition to

endotoxins. Amoebocyte lysate preparations which do not reacto with

glucans are available; they are prepared by removing from amoebocyte

lysate the G factor, which reacts with glucans, or by inhibiting the G

factor reactoing system of amoebocyte lysate. These preparations may

be used for endotoxin testing in the presence of glucans.

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注：除了内毒素外，鲎试剂和β-葡聚糖反应。也存在不与葡聚糖反应的鲎试剂；

它们通过从变形细胞溶解物中除去G因子来制备，因为G因子会和葡聚糖反应，

或者通过抑制变形细胞溶解物的G因子反应系统来制备。这些制剂可被用于葡

聚糖存在情况下的内毒素测试。

(2) Lysate solution鲎试液

Dissolve amoebocyte lysate in water for BET or in a buffer, as

recommended by the lysate manufacturer, by gentle stirring. Store the

reconstituted lysate, refrigerated or frozen, as indicated by the

manufacturer.

通过缓慢搅拌，将鲎试剂溶于水或溶于厂家推荐的缓冲液中来进行细菌内毒素试

验（BET）。按照厂商的指示，冷藏或冷冻储存重新鲎试液。

(3) Water for BET (water for bacterial endotoxins test) BET用水

Water for injections R or water produced by other procedures that shows

no reaction with the lysate employed at the detection limit of the

reagent.

BET用水指注射用水R或由其他方法制取的内毒素含量低于所用鲎试剂的检测

限的水。

3. PREPARATION OF THE STANDARD ENDOTOXIN STOCK SOLUTION

（内毒素储备标准溶液的制备）

The standard endotoxin stock solution is prepared from an endotoxin

reference standard that has been calibrated against the International

Standard, for example endotoxin standard BRP.

用内毒素标准品制备内毒素储备标准溶液；所用的内毒素标准品必须先用国际标

准品校准，如内毒素标准BRP。

Endotoxin is expressed in International Units (IU). The equivalence in IU

of the International Standard is stated by World Health Organisation.

内毒素以国际单位（IU）表示。IU的换算见国际卫生组织（WHO）公布的国际

标准。

NOTE: one International Unit (IU) of endotoxin is equal to one Endotoxin

Unit (E.U.).

注：一国际单位（IU）内毒素相当于一个内毒素单位（E.U.）。

Follow the specifications in the package leaflet and on the lable for

preparation and storage of the standard endotoxin stock solution.

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根据包装说明书上的标准和内毒素储备标准溶液的标签上关于制备和贮存的说

明。

4. PREPARATION OF THE STANDARD ENDOTOXIN SOLUTIONS（ 内毒素

标准溶液的制备）

After vigorously mixing the standard endotoxin stock solution, prepare

appropriate serial dilutions of this solution using water for BET.

充分混合内毒素储备标准溶液后，用细菌内毒素试验检查用水（BET用水）稀释，

制成适当的系列稀释液，即得BET用内毒素标准溶液。

Use the solution as soon as possible to avoid loss of activity by

adsorption.

得到的稀释液应尽快使用，以免因吸附而导致活性损失。

5. PREPARATION OF THE TEST SOLUTIONS供试品溶液的制备

Prepare the test solutions by dissolving or diluting active substances or

medicinal products using water for BET. Some substances or

preparations may be more appropriately dissolved or diluted in other

aqueous solutions. If necessary, adjust the pH of the test solution (or

dilution thereof) so that the pH of the mixture of the lysate and test

solution falls within the pH range specified by the lysate manufacturer,

usually 6.0 to 8.0. the pH may be adjusted by the use of acid, base or a

suitable buffer, as recommended by the lysate manufacturer. Acids and

bases may be prepared from concentrates or sol8ids with water for BET

in containers free of detectable endotoxin. Buffers must be validated to

be free of detectable endotoxin and interfering factors.

除非另有说明，以BET用水溶解或稀释活性成分或药品来制备供试品溶液。某

些成分或药品可能在其它的水溶液中溶解度更高。如有必要，调节待测溶液（或

它的稀释液）的pH值，使鲎试剂和供试品溶液的混合物的pH值在所选鲎试剂

生产商的指定pH范围内。一般要求供试品溶液的pH值范围为6.0——8.0。可

用鲎试剂生产商推荐的酸、碱或适当的缓冲溶液来调解pH值。酸或碱溶液须用

BET检查用水在去除内毒素的容器中配制。缓冲液必须经过验证不含内毒素和干

扰因子。

DETERMINATION OF THE MAXIMUM VALID DILUTION确定最大有

效稀释倍数

The Maximum Valid Dilution (MVD) is the maximum allowable dilution of

a sample at which the endotoxin limit can be determined. Determine the

MVD using the following formulae:

最大有效稀释倍数（MVD）指可检测出内毒素限值的供试品溶液的最大稀释倍

数。MVD按下列公式确定：

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Endotoxin limit × concentration of test solution

MVD=

λ

MVD = 内毒素限值×供试品溶液浓度/λ

Endotoxin limit: the endotoxin limit for active substances administered

parenterally, defined on the basis of dose, is equal to

K

M

内毒素限值：在剂量的基础上，注射药物的活性成分的内毒素限度为K/M

K = threshold pyrogenic dose of endotoxin per kilogram of body mass,

M = maximum recommended bolus does of product per kilogram of

body mass.

K=人每公斤体重每小时最大可接受的内毒素剂量（EU/kg）

M＝人用每公斤体重每小时的最大供试品剂量。

When the product is to e injected at frequent intervals or infused

continuously, M is the maximum total dose administered in a single hour

period.

The endotoxin limit for active substances administered parenterally is

specified in units such as: IU/mL, IU/mg, IU/Unit of biological activity, ect,

in monographs.

专论中，注射剂活性成分的内毒素限度的单位有IU/ml、IU/mg、IU/生物活性

单位等。

Concentration of test solution:

供试品溶液的浓度表示法：

— mg/mL if the endotoxin limit is specified by mass (IU/mg),

— 当内毒素限度以质量（IU/mg）表示时，用mg/ml表示浓度。

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— Units/mL if the endotoxin limit is specified by unit of biological

activity (IU/Unit),

— 当内毒素限度以（IU/ml）表示时，用ml/ml表示浓度。

— ml/mL if the endotoxin limit is specified by volume (IU/mL).

— 当内毒素限度以生物单位（IU/Unit）表示时，用Units/ml表示浓度。

λ = the lablelled lysate sensitivity in the gel-clot techniques (IU/mL) or

the lowest concentration used in the standard curve of the turbidimetric

or chromogenic techniques.

λ=指凝胶法中鲎试剂的标示灵敏度，或指浊度法或显色法使用的标准回归曲线

上最低点（IU/ml)的对应值。

7. GEL-CLOT TECHNIQUE (METHODS A AND B) 凝胶法（方法A和B）

The gel-clot techniques allow detection or quantification of endotoxins

and is based on clotting of the lysate in the presence of endotoxins. The

minimum concentration of endotoxins required to cause the lysate to

clot under standard conditions is the labeled lysate sensitivity. To ensure

both the precision and validity of the test, confirm the labeled lysate

sensitivity and perform the test for interfering factors as described under

1. Preparatory testing.

凝胶法系通过鲎试剂与内毒素产生凝集反应的原理来检测或定量内毒素的方法。

在标准条件下，可引起鲎试液凝集的内毒素的最低浓度即为鲎试剂的标示灵敏

度。为确保试验结果精确、有效，应根据预备试验中的说明开展试验，复核鲎试

剂的标示灵敏度和试验的干扰因素。

1. PREPARATORY TESTING预备试验

(i) Confirmation of the labeled lysate sensitivity鲎试剂的标示灵敏度复核试

验

Confirm in 4 replicates the labeled sensitivity λ, expressed in IU/mL, of

the lysate solution prior to use in the test. Confirmation of the lysate

sensitivity is 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.

灵敏度用IU/ml表示。应在鲎试剂用于检查内毒素之前对灵敏度进行复核；复

核试验需要制备标示灵敏度λ的4支平行管。当使用新批号的鲎试剂或试验条件

发生了任何可能影响检验结果的改变时，应进行鲎试剂灵敏度复核试验。

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Prepare standard solutions of at least 4 concentrations equivalent to 2λ,

λ, 0.5λ and 0.25λ by diluting the standard endotoxin stock solution

with water for BET.

用BET用水稀释内毒素储备标准溶液，制成2λ、λ、0.5λ和0.25λ四种浓度

的标准溶液。

Mix a volume of the lysate solution with an equal volume of 1 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 the

recommendations of the lysate manufacturer (usually at 37±1?C for

60±2 min), avoiding vibration. Test the integrity of the gel: for tubes,

take each tube in turn directly from the incubator and invert it through

approximately 180? in one smooth motion. If a firm gel has formed that

remains in place upon inversion, record the result as positive. A result is

negative if an intact gel is not formed.

在每支试管中，分别混合等体积（通常为0.1mL)的鲎试液和标准溶液。如使用

的是装有鲎试剂冻干粉末的西林瓶或安瓿，可向其中直接加入溶液。按照鲎试剂

生产商的建议，将反应混合物孵育一段时间(通常在37±1℃下保温60±2min)，

在此过程中，不能振动试管。凝胶的完整性测试：如使用试管作为容器，先将试

管从保温箱中取出，再缓缓将试管倒转180o。如果形成了坚实的凝胶，倒转后

凝胶不移位，此时将该项检查的结果记录为阳性。如未能形成坚实且不变形的凝

胶，该项检查的结果即为阴性。

The test is considered valid when the lowest concentration of the

standard solutions shows a negative result in all replicate tests. The

end-point is the lowest concentration in the series of decreasing

concentrations of standard endotoxin that clots the lystae. Determine

the geometric mean end-point concentration by calculating the mean of

the logarithms of the end-point concentrations of the 4 dilution series,

take the antilogarithm of this value, as indicated by the following

expression:

仅在最低浓度的标准溶液的所有平行管的检查结果均为阴性的情况下，试验方为

有效。反应终点浓度指系列递减的内毒素浓度中最后一个呈阳性结果的浓度。将

终点浓度取对数，计算它们的平均值，再将平均值的结果取反对数，最后的表达

式如下：

Σe

Geometric mean end-point concentration = antilog

f

Σe = sum of the log end-point concentrations of the dilution series

used,

f = number of replicates.

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终点浓度的几何平均值＝log-1(Σe/f)

Σe＝所用系列溶液的终点浓度的对数值的和

f＝平行管的数量

The geometric mean end-point concentration is the measured sensitivity

of the lysate solution (IU/mL). If this is not less than 0.5λ and not more

than 2λ, the labeled sensitivity is confirmed and is used in the test

performed with this lysate.

反应终点的浓度的几何平均值即为鲎试剂灵敏度的测量值（IU/ml）。当终点浓

度的几何平均值在0.5λ至2.0λ之间时，可判定受试鲎试剂的标示灵敏度为λ，

可用于内毒素的检查。

(ii) Test for interfering factors干扰因素试验

Prepare solutions A, B, C and D as shown in Table 2.6.14-1, and use the

test solutions at a dilution less than the MVD, not containing any

detectable endotoxins, operating as described under 1. preparatory

testing, (i) Confirmation of the labeled lysate sensitivity.

按表2.6.14.-1制备溶液A、B、C、D。供试品的稀释度不得超过MVD，且供试

品溶液不能检查出内毒素，具体操作见（1）预备试验，（i）鲎试剂标示灵敏度

的复核试验项。

The geometric mean end-point concentrations of solutions B and C are

determined using the expression described in 1. Preparatory testing, (i)

Confirmation of the labeled lysate sensitivity.

根据（1）预备试验下的（i）鲎试剂标示灵敏度的复核试验项中列出的公式，计

算B和C溶液的终点浓度的几何平均值。

Table 2.6.14.-1

Solutio

n

Endotoxin

concentration/solut

ion to which

endotoxin is added

None/Test solution

2λ/Test solution

Diluen

t

DilutioEndotoxin

n factor

concentrati

on

Numbe

r of

replicat

es

4

4

4

A

B

-

Test

solutio

n

-

1

2

-

2λ

1λ

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4

8

C 2λ/Water for BET Water 1

for BET

2

4

8

D None/Water for BET - -

0.5λ

0.25λ

2λ

1λ

0.5λ

0.25λ

-

4

4

2

2

2

2

2

Solution A = solution of the preparation being examined that is free of

detectable endotoxins.

Solution B = test for interference.

Solution C = control of the labeled lysate sensitivity.

Solution D = negative control (water for BET).

表2.6.14-1

溶内毒素浓度/

液

配制内毒素的

溶液

A 无/供试品溶

液

B 2λ/供试品溶

液

稀释剂 稀释倍数 稀释后内毒素

的浓度

平行管数

－ － － 4

供试品溶液 1

2

4

8

2λ

1λ

0.5λ

0.25λ

4

4

4

4

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C 2λ/BET用水 BET用水 1

2

4

8

2λ

1λ

0.5λ

0.25λ

－

2

2

2

2

2 D 0/BET用水 － －

A=经检查无内毒素的溶液

B=干扰实验用

C=鲎试剂标示灵敏度的对照品

D=阴性对照品（BET检查用水）

The test for interfering factors must be repeated when any changes are

made to the experimental conditions that are likely to influence the

result of the test.

如试验条件发生了任何可能影响到试验结果的变化，须重新进行干扰因素试验。

The test is considered valid when all replicates of solutions A and D show

no reaction and the result of solution C confirms the labeled lysate

sensitivity.

只有当溶液A和D的所有平行管中无反应、且溶液C的结果在复核的鲎试剂灵

敏度范围内时，试验方有效。

If the sensitivity of the lysate determined with solution B is not less than

0.5λ and not greater than 2λ, the test solution does not contain

interfering factors under the experimental conditions used. Otherwise,

the test solution interferes with the test.

经测定，如果溶液B的灵敏度在[0.5λ，2.0λ]间，可判定供试品溶液在该实验

条件下，对实验无干扰；反之则判定供试品溶液对试验能形成干扰。

If the preparation being examined interferes with the test at a dilution

less than the MVD, repeat the test for interfering factors using a greater

dilution, not exceeding the MVD. The use of a more sensitive lysate

permits a greater dilution of the preparation being examined and this

may contribute to the elimination of interference.

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若供试品溶液在小于MVD的稀释倍数下对试验有干扰，应将供试品溶液进行不

超过MVD的进一步稀释，再重复干扰试验。使用灵敏度更高的鲎试剂进行内毒

素的检查时，因为更高灵敏度鲎试剂能排除实验的干扰因素，供试品的稀释倍数

也可适当放宽。

Interference may be overcome by suitable validated treatment, such as

filtration, neutralization, dialysis or heat treatment. To establish that the

treatment chose effectively eliminates interference without loss of

endotoxins, repeat the test for interfering factors using the preparation

being examined to which the standard endotoxin has been added and

which has then been submitted to the chosen treatment.

可通过其他适宜的方法（如过滤、中和、透析或加热处理等）排除干扰。为确保

所选择的处理方法能有效地排除干扰且不会使内毒素失去活性，要使用预先添加

了标准内毒素再经过处理的供试品溶液进行干扰实验。

2. LIMIT TEST (METHOD A) 限度试验（方法A）

(i) Procedure方法

Prepare solutions A, B, C and D as shown in Table 2.6.14.-2, and perform

the test on these solutions following the procedure described under 1.

Preparatory testing, (i) Confirmation of the labeled lysate sensitivity.

按表2.6.14-2制备溶液A、B、C、D。实验方法见（1）预备试验的（i）鲎试剂

标示灵敏度的复核试验项。

Table 2.6.14.-2

Solution Endotoxin concentration/Solution to which Number of

endotoxin is added replicates

A

B

C

D

表2.6.14-2

None/Diluted test solution

2λ/Diluted test solution

2λ/Water for BET

None/Water for BET

2

2

2

2

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溶液 内毒素浓度/配制内平行管数

毒素的溶液

0/供试品溶液

2λ/供试品溶液

2λ/BET用水

0/BET用水

2

2

2

2

A

B

C

D

Prepare solution A and solution B (positive product control) using a

dilution not greater than the MVD and treatments as described in 1.

Preparatory testing, (ii) Test for interfering factors. Solutions B and C

(positive controls) contain the standard endotoxin at a concentration

corresponding to twice the labeled lysate sensitivity. Solution D (negative

control) consists of water for BET.

制备溶液A、B（供试品阳性对照），稀释倍数不得超过MVD。按照（1）预备

实验，（ii）干扰因素实验项下的说明开展实验。溶液B、C（阳性对照）所含标

准内毒素的浓度为鲎试剂标示灵敏度的两倍。溶液D（阴性对照）为BET用水。

(ii) Interpretation结果判断

The test is considered valid when both replicates of solution B and C are

positive and those of solution D are negative.

只有溶液B和C的平行管的试验结果均为阳性、溶液D的平行管的试验结果均

为阴性时，试验方有效。

When a negative result is found for both replicates of solution A, the

preparation being examined complies with the test.

溶液A的平行管的试验结果均为阴性时，可判定供试品符合试验的内毒素限度

要求。

When a positive result is found for both replicates of solution A, the

preparation being examined does not comply with the test.

溶液A的平行管的检查结果均为阳性时，可判定供试品不符合试验的内毒素限

度要求。

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When a positie result is found for one replicate of solution A and a

negative result is found for the others, repeat the test.

当溶液A的平行管其中一个检查结果为阳性，另一个为阴性时，重复试验。

In the repeat test, the preparation being examined complies with the test

if a negative result is found for both replicates of solution A. The

preparation does not comply with the test if a positive result is found for

one or both replicates of solution A.

在重复试验中，溶液A的平行管的试验结果均为阴性时，可判定供试品符合试

验的内毒素限度要求。溶液A的平行管的检查结果有一个或均为阳性时，可判

定供试品不符合试验的内毒素限度要求。

However, if the preparation does not comply with the test at a dilution

less than the MVD, the test may be repeated using a greater dilution, not

exceeding the MVD.

但是，如果供试品不符合规定是在供试品的稀释倍数小于MVD的情况下，应将

供试品溶液稀释至较大但不超过MVD的倍数，再次开展试验。

3. QUANTITATIVE TEST (METHOD B)

(i) Procedure

The test quantifies bacterial endotoxins in the test solution by titration to

an end-point. Prepare solutions A, B, C and D as shown in Table 2.6.14.-3,

and test these solutions according to the procedure described under 1.

Preparatory testing, (i) Confirmation of the labelled lysate sensitivity.

通过将供试品滴定至反应终点，对供试品所含的细菌内毒素定量。按表2.6.14-3

制备溶液A、B、C、D。具体实验操作见（1）预备试验的（i）鲎试剂标示灵敏

度的复核试验项。

Table2.6.14-3

Endotoxin

Concentration/

Solution to

which Initial Number

Endotoxin is Dilution Endotoxin of

Solution Added Diluent Factor Concentration Replicates

A

a

none/sample

solution

Water

for BET

1

2

4

8

—

—

—

—

2

2

2

2

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Endotoxin

Concentration/

Solution to

which Initial Number

Endotoxin is Dilution Endotoxin of

Solution Added Diluent Factor Concentration Replicates

B

b

2/sample

solution

C

c

2/ Water for

BET

Water

for BET

1

2

4

8

2

1

0.5

0.25

D

d

none/ Water for

BET

Solution A = test solution at the dilution, not exceeding the MVD, with

which the test for interfering factors was carried out. Subsequent dilution

of

the test solution must not exceed the MVD. Use water for BET to make a

dilution series of 4 tubes containing the test solution at concentrations

of 1,

1/2, 1/4 and 1/8, relative to the dilution used in the test for interfering

factors. Other dilutions up to the MVD may be used as appropriate.

Solution B = solution A containing standard endotoxin at a

concentration of 2λ (positive product control).

Solution C = a dilution series of 4 tubes of water for BET containing the

standard endotoxin at concentrations of 2λ, λ, 0.5λ and 0.25λ.

Solution D = water for BET (negative control).

表

2.6.14-3

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— 1

2

2

2

2

2

2

— — — 2

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溶

液

A

内毒素浓度

/配制内毒

素的溶液

无/供试品

溶液

稀释

剂

BET

用水

稀释倍

数

1

2

4

8

稀释后平行

内毒素管数

的浓度

－

-

-

-

2λ

2λ

1λ

0.5λ

0.25λ

－

2

2

2

2

2

B

C

2λ/供试品

溶液

2λ/BET用

水

－

BET

用水

1

1

2

4

8

－

2

2

2

2

2

D

无/BET用

水

－

溶液A=不超过MVD并且通过干扰试验的供试品溶液。从通过干扰试验的稀释倍数开始，用BET用水稀

释至1倍、2倍、4倍和8倍，每一浓度准备两份稀释液，最后的稀释倍数不得超过MVD，并且确定稀释

液通过了干扰试验。也可以准备其它合适倍数的稀释液。

溶液B=2λ浓度标准内毒素的溶液A（供试品阳性对照）

溶液C=含有2λ、λ、0.5λ和0.25λ浓度标准内毒素的BET用水系列，每一浓度准备两份。

溶液D=BET用水（阴性对照）

(ii) Calculation and interpretation

The test is considered valid when the following 3 conditions

are met:

(a) both replicates of solution D (negative control) are negative,

(b) both replicates of solution B (positive product control)

are positive,

(c) the geometric mean end-point concentration of solution C

is in the range of 0.5λ to 2λ.

(ii)计算和结果判断

符合下列三个条件时，可将试验判为有效：

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（a） 溶液D（阴性对照）的两组平行管均显阴性，

（b）

（b）溶液B（供试品阳性对照）的两个平行管均显阳性，

（c）溶液C的终点浓度的几何平均值在0.5λ—2λ之间。

To determine the endotoxin concentration of solution A, calculate the

end-point concentration for each replicate, by multiplying each

end-point dilution factor by λ.

The endotoxin concentration in the test solution is the end-point

concentration of the replicates. If the test is conducted with a diluted test

solution, calculate the concentration of endotoxin in the original solution

by multiplying the result by the dilution factor.

If none of the dilutions of the test solution is positive in a valid test,

report the endotoxin concentration as less than λ (or, if a diluted sample

was tested, report as less than the lowest dilution factor of the sample ×

λ). If all dilutions are positive, the endotoxin concentration is reported

as equal to or greater than the largest dilution factor multiplied by λ (e.g.

in Table 2.6.14.-3, the initial dilution factor × 8 × λ).

The preparation being examined meets the requirements of the test if

the endotoxin concentration in both replicates is less than that specified

in the monograph.

测定溶液A的内毒素浓度，计算溶液A的系列稀释液的终点浓度时，应将内毒素浓度

记为终点稀释倍数乘以λ。

供试品的内毒素浓度指这些平行管的反应终点浓度的几何平均值。如试验使用的是供试

品的稀释液，计算时，将检查结果乘以稀释倍数，即得到原始溶液的内毒素浓度。

如在有效的试验中，供试品的检查结果均显阴性，应记为内毒素浓度小于λ（如实验使

用的是供试品的稀释液，检查结果应记为内毒素浓度小于λ乘以供试品的最小稀释倍数）。

如供试品的所有结果均为阳性，应记为内毒素浓度大于或等于最大的稀释倍数乘以λ（如，

在表2.6.14-3中，初始稀释倍数×8×λ）。

如供试品内毒素的浓度小于其专论中的规定浓度，可判定供试品符合检查要求。

QUANTITATIVE TECHNIQUES(METHODS C, D, E AND F)

8.

光度测定法（方法C、D、E和F）

（1）. TURBIDIMETRIC TECHNIQUE (METHODS C AND F)

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（1）浊度法（方法C和F）

This technique is a photometric test to measure the increase in turbidity.

Based on the test principle employed, this technique may be classified as

being either the end-point-turbidimetric test or the kinetic-turbidimetric

test.

本法是利用光学试验来测定浊度增加的方法。根据检测原理，这种方法又可分为终点浊

度法和动态浊度法。

The end-point-turbidimetric test (Method F) is based on the quantitative

relationship between the endotoxin concentration and the turbidity

(absorbance or transmission) of the reaction mixture at the end of an

incubation period.

终点浊度法（方法F）系依据反应混合物在孵育终止时，反应混合物的浊度（吸光度或

透光率）与内毒素浓度之间的量化关系来测定内毒素含量的方法。

The kinetic-turbidimetric test (Method C) is a method to measure either

the time (onset time) needed for the reaction mixture to reach a

predetermined absorbance or transmission, or the rate of turbidity

development.

动态浊度法（方法C）是检测反应混合物的浊度达到某一预先设定的吸光度或透光率需

要的反应时间（起效时间），或是检测浊度变化率。

The test is carried out at the incubation temperature recommended by

the lysate manufacturer (usually 37 ± 1 °C).

根据鲎试剂生产商的建议，光度测定试验应在孵育温度下进行（通常为37±1℃）。

(2)CHROMOGENIC TECHNIQUE (METHODS D AND E)

(2)

显色法（方法

D

和

E

）

This technique is used to measure the chromophore released from a

suitable chromogenic peptide by the reaction of endotoxins with the

lysate. Depending on the test principle employed, this technique may be

classified as being either the end-point-chromogenic test or the

kinetic-chromogenic test.

该法系利用检测鲎试剂与内毒素反应使特定底物释放出呈色团的多少而测定内毒素的

含量的方法。根据检测量之间存在的量化关系来测定内毒素含量的方法。

The end-point-chromogenic test (Method E) is based on the quantitative

relationship between the endotoxin concentration and the quantity of

chromophore released at the end of an incubation period.

终点显色法（方法E）是依据反应混合物中内毒素浓度和其在孵育终止时释放出的生色

团的的量之间存在的量化关系来测定内毒素含量的方法。

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The kinetic-chromogenic test (Method D) measures either the time

(onset time) needed for the reaction mixture to reach a predetermined

absorbance, or the rate of colour development.

The test is carried out at the incubation temperature recommended by

the lysate manufacturer (usually 37 ± 1 °C).

动态显色法（方法D）是检测反应混合物的色度达到某一预定吸光度（或透光率）所需

要的反应时间（起效时间），或检测色度增长速度的方法。

根据鲎试剂生产商的建议，光度测定试验应在孵育温度下进行（通常为37±1℃）。

3. PREPARATORY TESTING

3

预备试验

To assure the precision or validity of the turbidimetric and chromogenic

techniques, preparatory tests are conducted to show that the criteria for

the standard curve are satisfied and that the test solution does not

interfere with the test.

Validation of the test method is required when any changes are made to

the experimental conditions that are likely to influence the result of the

test.

为确保浊度法和显色法试验结果精确、有效，应根据下面的说明，预先进行标准曲线

的可靠性和干扰因素试验。

当实验条件发生了任何可能会影响检验结果的改变时，需要验证试验方法。

(i) Assurance of criteria for the standard curve

The test must be carried out for each lot of lysate reagent.

Using the standard endotoxin solution, prepare at least 3 endotoxin

concentrations within the range indicated by the lysate manufacturer to

generate the standard curve. Perform the test using at least 3 replicates

of each standard endotoxin solution as recommended by the lysate

manufacturer (volume ratios, incubation time, temperature, pH, etc.).

If the desired range is greater than 2 log10 in the kinetic methods,

additional standards must be included to bracket each log10 increase in

the range of the standard curve.

The absolute value of the correlation coefficient, | r |, must be greater

than or equal to 0.980, for the range of endotoxin concentrations set up.

（i） 标准曲线的可靠性试验

每批鲎试剂都必须进行此项试验。

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用内毒素标准溶液制成至少三个浓度的稀释液，以获取标准曲线。按照鲎试剂生产商的

建议（体积比、孵育时间、温度、pH等），每一内毒素标准浓度的溶液至少做三支平行管。

使用动态法检测时，如预期范围超过2 log，为使标准曲线反映出每个对数增长，应相

应增加标准品溶液。

在鲎试剂生产商规定的内毒素浓度范围内，相关系数的绝对值︱r︱必须大于等于0.980。

(ii)Test for interfering factors

Select an endotoxin concentration at or near the middle of the endotoxin

standard curve.

Prepare solutions A, B, C and D as shown in Table 2.6.14.-4. Perform the

test on at least 2 replicates of these solutions as recommended by the

lysate manufacturer (volume of test solution and lysate solution, volume

ratio oftest solution to lysate solution, incubation time, etc.)

(ii)干扰因素试验

选择标准曲线中点或一个靠近中点的内毒素浓度。

按表2.6.14-4制备溶液A、B、C、D。在鲎试剂生产商的建议测试条件（供试品和鲎试液

体积、供试品于鲎试液的体积比、孵育时间等）下，对这些溶液开展试验，每种溶液至少做

两组平行。

Table2.6.14-4

Solution to which

Endotoxin is Number of

Solution Endotoxin Concentration Added Replicates

A

a

none sample solution not less than

2

B

b

middle concentration of sample solution not less than

the standard curve 2

C

c

at least 3 concentrations

Water for BET

each

(lowest concentration is

concentration

designated )

not less than

2

D

d

none

Water for BET not less than

2

Solution A = test solution, that may be diluted not to exceed the MVD.

Solution B = preparation to be examined at the same dilution as

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solution A, containing added endotoxin at a concentration equal to or

near the middle of the standard curve.

Solution C = standard endotoxin solution at the concentrations used in

the validation of the method as described under 3. Preparatory testing,

(i) Assurance of criteria for the standard curve (positive controls).

Solution D = water for BET (negative control).

表

2.6.14-4

溶液

A

B

C

D

内毒素浓度

无

标准曲线的

中点

至少3个浓

度（最低浓度

规定为

λ

）

无

配制内毒素

的溶液

供试品溶液

供试品溶液

BET检查

平行管或微孔数

不少于2个

不少于2个

每一浓度不少于2个

BET检查用

不少于2个

水

- 溶液A=稀释倍数不超过MVD的供试品溶液。

- 溶液B=加入了标准曲线终点或靠近中点的一个已知浓度内毒素的、且与溶液A有

相同稀释倍数的供试品溶液。

- 溶液C=如“3.预备实验（i）标准曲线的可靠性试验”项下描述的、用于制备标准

曲线的标准内毒素溶液。

-

溶液D=BET用水（阴性对照）

The test is considered valid when the following conditions

are met:

– the absolute value of the correlation coefficient of the standard curve

generated using solution C is greater than or equal to 0.980;

– the result with solution D does not exceed the limit of the blank value

required in the description of the lysate reagent employed, or it is less

than the endotoxin detection limit of the lysate reagent employed.

试验必须符合以下条件方有效：

- 溶液C标准曲线的相关系数绝对值大于或等于0.980；

- 溶液D（阴性对照）的试验结果不得大于所用鲎试剂的说明书中规定的空白值的限

度，或小于鲎试剂的内毒素检测限。

Calculate the mean recovery of the added endotoxin by subtracting the

mean endotoxin concentration in the solution (if any) (solution A, Table

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2.6.14.-4) from that in the solution containing the added endotoxin

(solution B, Table 2.6.14.-4).

用含标准内毒素的供试品溶液（溶液B，表2.6.14.-4）的平均内毒素含量减去供试品

溶液（溶液A，表2.6.14.-4）的平均内毒素含量，计算内毒素的平均回收率。

The test solution is considered free of interfering factors if under the

conditions of the test, the measured concentration of the endotoxin

added to the test solution is within 50-200 per cent of the known added

endotoxin concentration, after subtraction of any endotoxin detected in

the solution without added endotoxin.

当内毒素的回收率在50％－200％之间时，可认为在此试验条件下供试品溶液中不存

在干扰因素。在溶液中不添加任何内毒素的条件下，计算任何所测内毒素的减少。

When the endotoxin recovery is out of the specified range,the test

solution is considered to contain interfering factors. Repeat the test

using a greater dilution, not exceeding the MVD. Furthermore,

interference of the test solution or diluted test solution not to exceed the

MVD may be eliminated by suitable validated treatment, such as

filtration, neutralisation, dialysis or heat treatment. To establish that the

treatment chosen effectively eliminates interference without loss of

endotoxins, repeat the test for interfering factors using the preparation

being examined to which the standard endotoxin has been added and

which has then been submitted to the chosen treatment.

当内毒素的回收率不在指定范围内，应根据凝胶法章节的“1.预备实验（ii）干扰因素实验”

项下的说明排除干扰因素。重复干扰因素实验以验证处理的有效性。此外，可通过其他适宜

的方法（如过滤、中和、透析或加热处理等）排除供试液或未超过MVD的稀释供试液的干

扰。为确保所选择的处理方法能有效地排除干扰且不会使内毒素失去活性，要使用预先添加

了标准内毒素再经过处理的供试品溶液进行干扰实验。

4. TEST

检查法

(i) Procedure

Follow the procedure described in 3. Preparatory testing,(ii) Test for

interfering factors.

按 “（3）预备试验下的（ii）干扰因素试验”项下操作步骤。

(ii) Calculation

Calculate the endotoxin concentration of each replicate of solution A

using the standard curve generated by the positive control solution

test is considered valid when the following 3 requirements are met:

(1) the results obtained with solution C comply with the requirements for

validation defined under 3. Preparatory testing, (i) Assurance of criteria

for the standard curve,

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(2) the endotoxin recovery, calculated from the endotoxin concentration

found in solution B after subtracting the endotoxin concentration found

in solution A, is within the range of 50-200 per cent,

(3) the result obtained with solution D (negative control) does not

exceed the limit of the blank value required in the description of the

lysate employed, or it is less than the endotoxin detection limit of the

lysate reagent employed.

(ii)计算

用系列溶液C生成的标准曲线计算溶液A的每一个平行管的内毒素浓度。

试验必须符合以下三个条件方有效：

阳性对照系列溶液C的检查结果要符合“3.预备

(1)实验（i）标准曲线的可靠性试验”中的要求。

(2)用溶液B中的内毒素浓度减去溶液A中的内毒素浓度后，计算出的回收率在50％－

200％的范围内。

(3)溶液D（阴性对照）的试验结果不得大于所用鲎试剂的说明书中规定的空白值的限度或

小于鲎试剂的内毒素检测限。

(iii) Interpretation

The preparation being examined complies with the test if the mean

endotoxin concentration of the replicates of solution A,after correction

for dilution and concentration, is less than the endotoxin limit for the

ines on the test for bacterial endotoxins are given in

general chapter 5.1.10.

(iii)结果判断

若校正了稀释倍数和浓度后，由溶液A的内毒素浓度计算得到的供试品内毒素平均浓

度小于供试品的内毒素限度，可判断供试品符合细菌内毒素检查的要求。

细菌内毒素试验指导见通则5.1.10.

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