中/美/歐/日四大藥典澄清度檢查規(guī)范-中英雙譯

中國(guó)藥典2020

0902 澄清度檢查法

澄清度檢查法系將藥品溶液與規(guī)定的濁度標(biāo)準(zhǔn)液相比較，用以檢查溶液的澄清度。除另有規(guī)定外，應(yīng)采用第一法進(jìn)行檢測(cè)。

品種項(xiàng)下規(guī)定的“澄清”，系指供試品溶液的澄清度與所用溶劑相同，或不超過(guò)0.5號(hào)濁度標(biāo)準(zhǔn)。“幾乎澄清”，系指供試品溶液的濁度介于0.5號(hào)至1號(hào)濁度標(biāo)準(zhǔn)液的濁度之間。

第一法（目視法）

除另有規(guī)定外，按各品種項(xiàng)下規(guī)定的濃度要求，在室溫條件下將用水稀釋至一定濃度的供試品溶液與等量的濁度標(biāo)準(zhǔn)液分別置于配對(duì)的比濁用玻璃管（內(nèi)徑15-16 mm,平底，具塞，以無(wú)色、透明、中性硬質(zhì)玻璃制成）中，在濁度標(biāo)準(zhǔn)液制備5 分鐘后，在暗室內(nèi)垂直置于傘棚燈下，照度為1000 lx，從水平方向觀察、比較。除另有規(guī)定外外，供試品溶解后應(yīng)立即檢視。

第一法無(wú)法準(zhǔn)確判定兩者的澄清度差異時(shí)，改用第二法進(jìn)行測(cè)定，并以其測(cè)定結(jié)果進(jìn)行判定。

濁度標(biāo)準(zhǔn)存貯液的制備 稱(chēng)取于105℃干燥至恒重的硫酸肼1.00 g，置于100 ml量瓶中，加水適量使溶解，必要時(shí)可在40℃的水浴中溫?zé)崛芙?，并用水稀釋至刻度，搖勻，放置4-6小時(shí)；取此溶液于等容量的10%烏洛托品溶液混合，搖勻，于25℃避光靜置24小時(shí)，即得。該溶液置冷處避光保存，可在2個(gè)月內(nèi)使用，用前搖勻。

濁度標(biāo)準(zhǔn)原液的制備 取濁度標(biāo)準(zhǔn)貯備液15.0 ml，置1000 ml量瓶中，加水稀釋至刻度，搖勻，取適量，置1 cm吸收池中，照紫外-可見(jiàn)分光光度法（通則0401），在550 nm的波長(zhǎng)處測(cè)定，其吸光度在0.12-0.15范圍內(nèi)，該溶液應(yīng)在48小時(shí)內(nèi)使用，用前搖勻。

濁度標(biāo)準(zhǔn)液制備 取濁度標(biāo)準(zhǔn)原液與水，按照下表配置，即得。濁度標(biāo)準(zhǔn)液應(yīng)臨用時(shí)制備，使用前充分搖勻。

第二法（濁度儀法）

供試品的濁度可采用濁度儀測(cè)定。溶液中不同大小、不同特性的微粒物質(zhì)包括有色物質(zhì)均可使入射光產(chǎn)生散射，通過(guò)測(cè)定透射光或者散射光的強(qiáng)度，可以檢查供試品的濁度。儀器測(cè)定模式通常有三種類(lèi)型，透射光式、散射光式和透射光-散射光比較測(cè)量模式（比率濁度模式）。

1.儀器的一般要求

采用散射光式濁度儀時(shí)，光源峰值波長(zhǎng)為860 nm；測(cè)量范圍應(yīng)包含0.01-100NTU。在0-10NTU范圍內(nèi)分辨率應(yīng)為0.01NTU；在10-100NTU范圍內(nèi)分辨率應(yīng)為0.1NTU.

2.適用范圍及檢測(cè)原理

本法采用散射光式濁度儀，適用于低、中濁度無(wú)色供試品溶液的濁度測(cè)定（濁度值為100NTU以下的供試品。）因?yàn)楦邼岫鹊墓┰嚻窌?huì)造成多次散射現(xiàn)象，時(shí)散射光強(qiáng)度迅速下降，導(dǎo)致散射光強(qiáng)度不能正確反映供試品的濁度值。0.5-4號(hào)濁度標(biāo)準(zhǔn)液的濁度值范圍約為0-40NTU。

采用散射光式濁度儀測(cè)定時(shí)，入射光和測(cè)定的散射光呈90℃夾角，入射光強(qiáng)度和散射光強(qiáng)度關(guān)系式如下。

I=K’T I₀

式中 I為散射光強(qiáng)度，單位為cd；

     I₀ 為入射光強(qiáng)度，單位為cd；

     K’為散射系數(shù)；

     T為供試品溶液的濁度值，單位為NTU（NTU是基于福爾馬肼濁度標(biāo)準(zhǔn)液液測(cè)定的散射濁度單位，福爾馬肼濁度標(biāo)準(zhǔn)液即為第一法中的濁度標(biāo)準(zhǔn)貯備液）。

在入射光I₀不變的情況下，散射光強(qiáng)度I與濁度值成正比。因此，可以將濁度測(cè)量轉(zhuǎn)化為散射光強(qiáng)度的測(cè)量。

3.系統(tǒng)的適用性試驗(yàn)

儀器應(yīng)定期（一般每月一次）對(duì)濁度標(biāo)準(zhǔn)液的線(xiàn)性和重復(fù)性進(jìn)行考察，采用0.5號(hào)至4號(hào)濁度標(biāo)準(zhǔn)液進(jìn)行濁度值測(cè)定，濁度標(biāo)準(zhǔn)液的測(cè)定解果（單位NTU）與濃度間應(yīng)呈線(xiàn)性關(guān)系，線(xiàn)性方程的相關(guān)系數(shù)應(yīng)不低于0.999；取0.5號(hào)至4號(hào)濁度標(biāo)準(zhǔn)液，重復(fù)測(cè)定5次，0.5號(hào)和1號(hào)濁度標(biāo)準(zhǔn)液測(cè)量濁度值的相對(duì)標(biāo)準(zhǔn)偏差應(yīng)不大于5%，2-4號(hào)濁度標(biāo)準(zhǔn)液測(cè)量濁度值的相對(duì)標(biāo)準(zhǔn)偏差不大于2%。

4.測(cè)定法

按照儀器說(shuō)明書(shū)要求并采用規(guī)定的濁度液進(jìn)行儀器校正。溶液劑直接取樣測(cè)定；原料藥或者其它劑型按照個(gè)論項(xiàng)下的標(biāo)準(zhǔn)規(guī)定制備供試品溶液，臨用時(shí)制備。分別取供試品溶液和相應(yīng)濁度標(biāo)準(zhǔn)液進(jìn)行測(cè)定，測(cè)定前應(yīng)搖勻，并避免產(chǎn)生氣泡，讀取濁度值。供試品溶液濁度值不得大于相應(yīng)濁度標(biāo)準(zhǔn)液的濁度值。

美國(guó)藥典USP44

<630> VISUAL COMPARISON 視覺(jué)比較

The purpose of this test is to provide the details for the visual comparison of the color and/or turbidance of sample solutions of certain concentration to a standard solution or a series of standard solutions of known concentration. Where a color or turbidity comparison is directed, follow the procedures and conditions outlined below for performing these tests.

本試驗(yàn)的目的是提供特定濃度的樣品溶液與已知濃度的標(biāo)準(zhǔn)溶液或一系列標(biāo)準(zhǔn)溶液的顏色和/或濁度的視覺(jué)比較細(xì)節(jié)。如果需要進(jìn)行顏色或濁度比較，請(qǐng)遵循以下程序和條件進(jìn)行這些測(cè)試

Comparison vessels: Color-comparison tubes matched as closely as possible in internal diameter, in depth of sample solution, and in all other respects should be used.

對(duì)比容器：應(yīng)使用內(nèi)徑、樣品溶液深度和所有其他方面盡可能匹配的顏色對(duì)比管。

Viewing conditions for turbidity comparison: Tubes should be viewed horizontally against a dark background with the aid of a light source directed from the sides of the tubes.

濁度比較的觀察條件：應(yīng)在黑暗背景下，借助從管子側(cè)面發(fā)出的光源水平觀察管子。

Viewing conditions for color comparison: Tubes should be viewed downward against a white background. Most of the time, common room lighting is sufficient to perform the assessment. A light source directed from beneath the bottoms of the tubes may be used if needed and if the practice is consistent between the materials under comparison.

顏色比較的觀察條件：管子應(yīng)在白色背景下向下觀察。大多數(shù)情況下，公共空間照明足以進(jìn)行評(píng)估。如果需要，并且對(duì)比材料之間的實(shí)踐一致，可以使用從管底部下方引導(dǎo)的光源

<855> NEPHELOMETRY AND TURBIDIMETRY      散射光濁度法和透射光比濁法

1. INTRODUCTION 介紹

Nephelometry and turbidimetry are analytical techniques that are based on the principles of light-scattering phenomena. Light scattering is the physical phenomenon in which a beam of light changes its direction of propagation (known as deflection) as a result of interaction with sufficiently small matter particles. It has been established from the Maxwell electromagnetic theory that a prerequisite for scattering to occur is that the refractive indexes of the suspended particles must be different from those of the suspending liquid. The larger the difference, the more intense the scattering becomes. There are two types of light scattering: 1) elastic scattering, in which the wavelength of the scattered light and incident light are the same; and 2) inelastic light scattering, in which the wavelength of the scattered light and incident light are different. Only the first type of light scattering (elastic) is relevant to nephelometry and turbidimetry.

散射光濁度法和透射光比濁法是基于光散射現(xiàn)象原理的分析技術(shù)。光散射是一種物理現(xiàn)象，其中光束由于與足夠小的物質(zhì)粒子相互作用而改變其傳播方向（稱(chēng)為偏轉(zhuǎn)）。根據(jù)麥克斯韋電磁理論，散射發(fā)生的先決條件是懸浮顆粒的折射率必須不同于懸浮液體的折射率。差異越大，散射越強(qiáng)烈。光散射有兩種類(lèi)型：1）彈性散射，其中散射光和入射光的波長(zhǎng)相同；2）非彈性光散射，其中散射光和入射光的波長(zhǎng)不同。只有前一種光散射（彈性）與散射光濁度法和透射光比濁法有關(guān)。

In turbidimetry, the intensity of the transmitted light is measured and the attenuation of the intensity of incident light as a result of scattering is measured at the direction of incident light (i.e., 0°) and compared to the intensity of incident light (blank measurement). The measured property is an indirect measurement of the scattering effect of the suspended particles and is referred to as turbidance. Any absorbance of light by the suspended sample will result in additional attenuation of light intensity (see Ultraviolet-Visible Spectroscopy <857> and Ultraviolet-Visible Spectroscopy—Theory and Practice <1857>). Hence, it is important to ensure that the material being measured does not absorb light at the measurement wavelength. Indeed the equations governing absorption and turbidimetry are the same (albeit with different values for the attenuation constants). In nephelometric techniques, the intensity of the scattered light at a 90° angle from the propagation direction of the incident light is measured. Therefore, a nephelometric measurement is a direct measurement of the scattering effect of suspended matter.

在透射光比濁法中，測(cè)量透射光的強(qiáng)度，并在入射光方向（即0°）測(cè)量散射導(dǎo)致的入射光強(qiáng)度的衰減，并與入射光強(qiáng)度進(jìn)行比較（空白測(cè)量）。被測(cè)特性是懸浮顆粒散射效應(yīng)的間接測(cè)量，稱(chēng)為濁度。懸浮樣品對(duì)光的任何吸收都會(huì)導(dǎo)致光強(qiáng)度的額外衰減（參見(jiàn)<857> Ultraviolet-Visible Spectroscopy和<1857> Ultraviolet-Visible Spectroscopy—Theory and Practice）。因此，確保被測(cè)材料不會(huì)吸收測(cè)量波長(zhǎng)處的光非常重要。實(shí)際上，控制吸收和濁度測(cè)定的方程式是相同的（盡管衰減常數(shù)的值不同）。在散射光濁度法中，測(cè)量與入射光傳播方向成90°角的散射光強(qiáng)度。因此，散射光濁度法濁度測(cè)量是對(duì)懸浮物散射效應(yīng)的直接測(cè)量。

2. TERMS AND DEFINITIONS 術(shù)語(yǔ)和定義

Terms commonly used in describing turbidimetric and nephelometric techniques are:

• Turbidance (symbol, S): A measure of the decrease of the transmitted incident light beam intensity as a result of the light-scattering effect of suspended particles. The amount of suspended matter may be measured by observation of either the transmitted light (turbidimetry) or the scattered light (nephelometry).

log I₀/I_t = kbc = T

I₀ = intensity of incident light

I_t = intensity of transmitted light

k = molar turbidity coefficient

b = cell path length

c = concentration

T = turbidance

• Turbidity (symbol, τ): In turbidimetric measurements, the turbidity is the measure of the decrease in incident beam intensity/unit length of a given suspension. The International Organization for Standardization defines turbidity as “the reduction of transparency of a liquid caused by the presence of undissolved matter”.

• Turbidity Measurement Units: The turbidity units are stated using a descriptor which indicates the method of measurement.

• Nephelometric Turbidity Units (NTUs): When the turbidity is measured using a nephelometer, which measures the scattered light at a 90° angle from the direction of propagation of incident light, the units of turbidity are called nephelometric turbidity units (NTUs). The magnitude of NTU is defined based on the turbidity generated by primary formazin standard (a suspension made by mixing solutions of hydrazine sulfate and hexamethylenetetramine in water). Safer polymer-bead suspensions are now commercially available and are recognized as an acceptable alternative. However, all those standards are traced to formazin. The primary formazin standard solution has been assigned a turbidity of 4000 NTUs.

Other recognized units for turbidity include the formazin turbidity unit (FTU) and the formazin nephelometric unit (FNU). These units are equivalent to NTU for the range from 0–40 NTUs.

描述濁度法和濁度法的常用術(shù)語(yǔ)包括：

•濁度（符號(hào)S）：由于懸浮顆粒的光散射效應(yīng)，透射入射光束強(qiáng)度降低的一種度量。懸浮物的量可以通過(guò)觀察透射光（比濁法）或散射光（濁度法）來(lái)測(cè)量。

log I₀/I_t = kbc = T

I₀=入射光強(qiáng)度

I_t=透射光強(qiáng)度

k=摩爾濁度系數(shù)

b=樣品池路徑長(zhǎng)度

c=濃度

T=濁度

•濁度（符號(hào)，τ）：在透射光濁度測(cè)量中，濁度是給定懸浮液的入射光束強(qiáng)度/單位長(zhǎng)度減少的量度。國(guó)際標(biāo)準(zhǔn)化組織將濁度定義為“由于存在未溶解物質(zhì)而導(dǎo)致液體透明度降低”。

•濁度測(cè)量單位：渾濁度單位用一個(gè)描述符表示，該描述符指示測(cè)量方法。

•散射光濁度計(jì)濁度單位（NTU）：當(dāng)使用散射光濁度法測(cè)量濁度時(shí)，濁度計(jì)以與入射光傳播方向成90°角的角度測(cè)量散射光，濁度單位稱(chēng)為散射光濁度法濁度單位（NTU）。NTU的大小是根據(jù)初級(jí)福爾馬肼標(biāo)準(zhǔn)品（一種將硫酸肼和六亞甲基四胺溶液混合在水中制成的懸浮液）產(chǎn)生的濁度定義的。更安全的聚合物微珠懸浮液現(xiàn)已上市，并被*為可接受的替代品。然而，所有這些標(biāo)準(zhǔn)都可以追溯到福爾馬肼。初級(jí)福爾馬肼標(biāo)準(zhǔn)溶液的濁度為4000 NTU。

其他*的濁度單位包括福爾馬肼比濁法單位（FTU）和福爾馬肼濁度法單位（FNU）。這些單位相當(dāng)于0-40 NTU范圍內(nèi)的NTU。

3. APPLICATIONS 應(yīng)用

Turbidimetric and nephelometric techniques have applications that include 1) concentration determination of solutions and/or suspensions (determination of several cations and anions by precipitating and suspending the resulting precipitate at well-controlled reaction parameters); 2) measurement of the degree of turbidity of turbid solutions or suspensions; 3) determination of weight-average molecular weights and dimensions of polydisperse systems in the molecular weight range from 1000 to several hundred million; 4) measurement of immunoassays’ reaction kinetics or kinetics of immunoprecipitations (rate nephelometry); 5) monitoring of cell and bacteria growth; and 6) particle size distribution determination of suspended material, particle counting, etc.

透射光比濁法和散射光濁度法技術(shù)的應(yīng)用包括1）溶液和/或懸浮液的濃度測(cè)定（通過(guò)在控制良好的反應(yīng)參數(shù)下沉淀和懸浮產(chǎn)生的沉淀物，來(lái)測(cè)定幾種陽(yáng)離子和陰離子）；2）測(cè)量混濁溶液或懸浮液的濁度；3）測(cè)定分子量在1000到數(shù)億之間的多分散體系的重均分子量和尺寸；4）測(cè)量免疫分析的反應(yīng)動(dòng)力學(xué)或免疫沉淀動(dòng)力學(xué)（比率散射濁度法）；5）監(jiān)測(cè)細(xì)胞和細(xì)菌的生長(zhǎng)；6）懸浮物粒度分布測(cè)定、顆粒計(jì)數(shù)等。

Rate nephelometry is widely used for vaccine components assays and/or quantitation of components in blood serum. It is also used for host cell protein qualification in recombinant biopharmaceuticals. When using the technique, the measurement of the change in the light-scattering response by antigen–antiserum or antigen-purified antibody complexes is used to calculate the amount of antigen (Ag) or antibody (Ab) responsible for the immunological Ab-Ag precipitation reaction or agglutination reaction. Often the antigens under consideration are linked covalently or adsorbed to polymeric microspheres to increase the scattering efficiency; the resulting technique is known as "particle-enhanced immunoassay". Although the technique is described as nephelometry, usually both scattered and transmitted light are measured using the ratio instruments.

比率散射濁度法廣泛用于疫苗成分分析和/或血清成分的定量。它還用于重組生物制藥中的宿主細(xì)胞蛋白質(zhì)鑒定。當(dāng)使用該技術(shù)時(shí)，通過(guò)測(cè)量抗原-抗血清或抗原純化抗體復(fù)合物的光散射反應(yīng)的變化，來(lái)計(jì)算導(dǎo)致免疫抗體-抗原沉淀反應(yīng)或凝集反應(yīng)的抗原（Ag）或抗體（Ab）的量。通?？紤]抗原共價(jià)連接或吸附在聚合物微球上，以提高散射效率；由此產(chǎn)生的技術(shù)被稱(chēng)為“顆粒增強(qiáng)免疫分析”。雖然這項(xiàng)技術(shù)被稱(chēng)為散射光濁度法，但通常散射光和透射光都是用比率儀器測(cè)量的。

Nephelometric measurements are more reliable in low turbidity ranges (relatively low concentration of the scattering medium). In this range, a linear relationship is observed between the sample concentration and the detector’s signal intensity expressed as NTU. As the concentration increases, so does the incidence of multiple scattering that deviates the response from the linearity. The maximum NTU value, which supports a reliable linearity relationship, is in the range of 1750–2000 NTUs. Turbidimetry is preferred for higher turbidity ranges (concentrations of the scattering media). To achieve consistent results, all measurement variables must be carefully controlled. Where such control is possible, extremely dilute suspensions may be measured.

散射光法濁度測(cè)量在低濁度范圍（散射介質(zhì)濃度相對(duì)較低）更可靠。在該范圍內(nèi)，觀察到樣品濃度與檢測(cè)器信號(hào)強(qiáng)度（以NTU表示）之間存在線(xiàn)性關(guān)系。隨著濃度的增加，多次散射的入射角也會(huì)增加，從而偏離線(xiàn)性響應(yīng)。支持可靠線(xiàn)性關(guān)系的最大NTU值在1750–2000 NTU范圍內(nèi)。透射光比濁法適用于更高的濁度范圍（散射介質(zhì)的濃度）。為了獲得一致的結(jié)果，必須仔細(xì)控制所有測(cè)量變量。在可能的情況下，可以測(cè)量極稀的懸浮液。

4. INSTRUMENTATION 儀器儀表

Instruments used for turbidimetric and nephelometric measurements are called turbidimeters and nephelometers, respectively. Generally, these instruments consist of a mercury lamp with filters for the strong green or blue lines, a shutter, a set of neutral filters with known transmittance, and a sensitive photomultiplier, which can be mounted fixed at 0° or at a 90° angle from the incident light propagation direction, or on an arm that can be rotated around the solution cell and set at any angle from −135° to 0° to +135° by a dial outside of the light-tight housing. Solution cells are of various shapes, such as square for measuring 90° scattering; semioctagonal for 45°, 90°, and 135° scattering; and cylindrical for scattering at all angles (see Figure 1).

用于透射光比濁法和散射光濁度法測(cè)量的儀器分別稱(chēng)為透射光濁度計(jì)和散射光濁度計(jì)。通常，這些儀器包括一個(gè)帶有濾光器的汞燈（用于強(qiáng)綠線(xiàn)或藍(lán)線(xiàn)）、一個(gè)快門(mén)、一組具有已知透射率的中性濾光器和一個(gè)靈敏的光電倍增管，該光電倍增管可安裝在與入射光傳播方向成0°或90°角的位置，或者在一個(gè)臂上，它可以圍繞溶液?jiǎn)卧D(zhuǎn)，并通過(guò)不透光外殼外的表盤(pán)設(shè)置為−135°到0°到+135°的任何角度。溶液池的形狀多種多樣，例如用于測(cè)量90°散射的正方形；45°、90°和135°散射為半八角形；圓柱形可適用于所有角度的散射（見(jiàn)圖1）。

Figure 1. Representative nephelometric (turbidimetric) instrument. Note that Detector 2 may be mounted on a movable arm.

圖1。代表性濁度儀。注意，探測(cè)器2可安裝在可移動(dòng)臂上。

Turbidity also can be measured with a standard photoelectric filter photometer or spectrophotometer, preferably with illumination in the blue portion of the spectrum. Nephelometric measurements require an instrument with a photocell placed so as to receive scattered, rather than transmitted, light. Because this is the same geometry used in fluorometers, they can be used as nephelometers by proper selection of filters. A ratio turbidimeter combines the technology of 90° nephelometry and turbidimetry. It contains photocells that receive and measure scattered light at a 90° angle from the sample as well as receiving and measuring the forward scatter in front of the sample. It also measures light transmitted directly through the sample. Linearity is attained by calculating the ratio of the 90° angle scattered light measurement to the sum of the forward scattered light measurement and the transmitted light measurement. The benefit of using a ratio turbidimetric system is that the measurement of stray light becomes negligible. In addition, the determination of turbidity of colored suspensions is done exclusively using turbidimetric or nephelometric instruments with ratio mode because this procedure compensates for the attenuation of light as the result of the suspension color. Typically, the light source in these instruments is a tungsten lamp with most of the light intensity at about 550 nm operating at the filament temperature of 2700 K. Other suitable light sources are also available. Typically, the detectors are ▲silicon diodes▲ _{(ERR 1-May-2019)} and photomultipliers. An alternative for eliminating the color effect involves using an infrared light-emitting diode as a light source, which yields an emission maximum centered at about 860 nm and a spectral bandwidth of 60 nm. When laser light sources are also used, especially in nephelometric instruments, the technique is commonly known as "laser nephelometry". The advantage of using laser nephelometers is the significant improvement in signal-to-noise ratio at very low detection levels. Usually the light source is a laser diode with a working wavelength at 660 nm. The high-power density of the laser beam gives rise to higher scattered intensity from smaller particles. Combined with a light trap, which absorbs the unscattered light, the system lowers the stray light significantly. When the use of a nephelometer or turbidimeter is indicated for a procedure in a monograph, instruments working in ratio mode may be used instead.

濁度也可以用標(biāo)準(zhǔn)光電濾光光度計(jì)或分光光度計(jì)測(cè)量，最好是在光譜的藍(lán)色部分進(jìn)行照明。散射光濁度法測(cè)量需要一個(gè)裝有光電管的儀器，以便接收散射光，而不是透射光。由于這與熒光計(jì)中使用的幾何結(jié)構(gòu)相同，因此可通過(guò)適當(dāng)選擇濾光片將其用作濁度計(jì)。比率濁度計(jì)結(jié)合了90°散射光濁度法和透射光比濁法。它包含光電管，接收和測(cè)量與樣品成90°角的散射光，以及接收和測(cè)量樣品前面的前向散射光。它還測(cè)量直接穿過(guò)樣品的光。通過(guò)90°角散射光測(cè)量值，前向散射光測(cè)量值和透射光測(cè)量值之和，計(jì)算兩者的比值，可獲得線(xiàn)性度。使用比率濁度測(cè)量系統(tǒng)的好處是雜散光的測(cè)量變得可以忽略不計(jì)。此外，彩色懸浮液的濁度測(cè)定僅使用透射光比濁法濁度儀或濁度儀（帶比率模式）進(jìn)行，因?yàn)樵摮绦蜓a(bǔ)償了懸浮液顏色導(dǎo)致的光衰減。通常，這些儀器中的光源是鎢燈，在2700 K的燈絲溫度下工作，大部分光強(qiáng)約為550 nm。也可使用其他合適的光源。通常，探測(cè)器是▲硅二極管▲和光電倍增管。另一種消除顏色效應(yīng)的方法是使用紅外發(fā)光二極管作為光源，其最大發(fā)射中心約為860 nm，光譜帶寬為60 nm。當(dāng)激光光源也被使用時(shí)，尤其是在濁度測(cè)量?jī)x器中，這種技術(shù)通常被稱(chēng)為“激光濁度測(cè)量”。使用激光散射光濁度計(jì)的優(yōu)點(diǎn)是，在非常低的檢測(cè)水平下，信噪比顯著提高。通常光源是工作波長(zhǎng)為660 nm的激光二極管。激光束的高功率密度使較小粒子產(chǎn)生更高的散射強(qiáng)度。與吸收未散射光的光阱相結(jié)合，該系統(tǒng)可顯著降低雜散光。當(dāng)專(zhuān)著中的某個(gè)程序指示使用散射光濁度計(jì)或透射光濁度計(jì)時(shí)，可以使用在比率模式下工作的儀器。

5. FORMAZIN TURBIDITY STANDARDS福爾馬肼濁度標(biāo)準(zhǔn)

Formazin is the only known primary turbidity standard. All other standards are secondary and must be traced to formazin. The primary standard is defined in the ▲IUPAC Compendium of Chemical Terminology,▲ (ERR 1-May-2019) 2nd ed. (the Gold Book) as one that is prepared by the user from traceable materials using well-defined methodologies and conditions.

福爾馬肼是唯yi已知的主要濁度標(biāo)準(zhǔn)。 所有其他標(biāo)準(zhǔn)都是次要的，必須追溯到福爾馬肼。 主要標(biāo)準(zhǔn)在▲IUPAC Compendium of Chemical Terminology▲（ERR 1-May-2019）第 2 版（金書(shū)）中被定義為由用戶(hù)使用明確定義的方法和條件從可追溯的材料準(zhǔn)備的標(biāo)準(zhǔn)。

Formazin suspension has many features that ensure its suitability as a primary standard. It can be consistently and accurately prepared from reagent-grade starting materials. The suspension consists of random polymers with different lengths and of random configurations, which result in moieties of varying shapes and sizes ranging from less than 0.1 μm to more than 10 μm. Although the polymer chain length distribution has been shown to vary from preparation to preparation, the overall resulting turbidity has been very reproducible.

福爾馬肼懸浮液有許多特點(diǎn)，以確保其適合作為主要標(biāo)準(zhǔn)。它可以從試劑級(jí)的起始材料中始終如一、準(zhǔn)確地制備。該懸浮液由不同長(zhǎng)度和隨機(jī)構(gòu)型的聚合物組成，其組成的聚合物的形狀和尺寸從小于0.1 μm到大于10 μm不等。盡管聚合物鏈長(zhǎng)分布已被證明因制備而異，但總的濁度結(jié)果是可以很好地重現(xiàn)的。

5.1 Preparation of the Formazin Standards 福爾馬肼標(biāo)準(zhǔn)液的制備

[NOTE—All procedures described below must be performed at 20 ± 2° (see Volumetric Apparatus <31>.]

• Hydrazine sulfate solution: Dissolve 1.000 g of ACS grade hydrazine sulfate (N₂H₄·H₂SO₄) in particle-free water in a 100-mL Class A volumetric flask and dilute with particle-free water to volume. Allow this solution to stand for 4–6 h.

•Primary formazin standard: Dissolve 2.50 g of analytical grade hexamethylenetetramine [(CH₂)₆N₄] in 25.0 mL of particle-free water in a 100-mL flask. Add 25.0 mL of hydrazine sulfate solution using a Class A 25-mL “to deliver” pipette and mix thoroughly. Allow the preparation to stand for 48 h at 25 ± 1° before using. The resulting suspension is stable for 2 months.

•Formazin stock standard suspension 1: Using a 15-mL Class A “to deliver” pipette, transfer 15 mL of the Primary formazin standard to a 1-L volumetric flask, and dilute with particle-free water to volume and mix. The resulting suspension has a turbidity of 60 NTU.

• Formazin stock standard suspension 2: Using a 50-mL Class A “to deliver” pipette, transfer 50 mL of Primary formazin standard to a 200-mL volumetric flask, and dilute with particle-free water to volume and mix. The resulting suspension has a turbidity of 1000 NTUs.

• Formazin reference suspensions: Prepare by mixing in a 100-mL volumetric flask, portions of the respective Formazin stock standard suspension and particle-free water according to Table 1.

[注：以下所有的程序必須在20±2°的條件下進(jìn)行（參見(jiàn)<31 Volumetric Apparatus >）]

•硫酸肼溶液：將1.000 g ACS級(jí)硫酸肼（N₂H₄·H₂SO₄）溶解在100 mL 的A類(lèi)容量瓶中中，并用無(wú)顆粒水稀釋至刻度。讓該溶液靜置4-6小時(shí)。

•初級(jí)福爾馬肼標(biāo)準(zhǔn)液：將2.50 g分析級(jí)六亞甲基四胺[(CH₂)₆N₄]溶于25.0 mL無(wú)顆粒水中，裝入100 mL燒瓶。使用A類(lèi)25ml移液管加入25.0 mL硫酸肼溶液，并充分混合。使用前，讓制劑在25±1°的溫度下靜置48小時(shí)。由此產(chǎn)生的懸浮液可穩(wěn)定運(yùn)行2個(gè)月。

•福爾馬肼儲(chǔ)備標(biāo)準(zhǔn)懸浮液1：使用15 mL A類(lèi)移液管，將15 mL福爾馬肼初級(jí)標(biāo)準(zhǔn)液轉(zhuǎn)移至1 L容量瓶中，并用無(wú)顆粒水稀釋至刻度并混合。所得懸浮液的濁度為60 NTU。

•福爾馬肼儲(chǔ)備標(biāo)準(zhǔn)懸浮液2：使用50 mL A類(lèi)移液管，將50 mL福爾馬肼初級(jí)標(biāo)準(zhǔn)液轉(zhuǎn)移至200 mL容量瓶中，并用無(wú)顆粒水稀釋至刻度并混合。所得懸浮液的濁度為1000 NTU。

•福爾馬肼參考懸浮液：根據(jù)表1，在100 mL容量瓶中混合各份福爾馬肼儲(chǔ)備標(biāo)準(zhǔn)懸浮液和無(wú)顆粒水，制備福爾馬肼參考懸浮液。

6. QUALIFICATION OF TURBIDIMETERS AND NEPHELOMETERS 透射光式濁度儀與散射光式濁度儀的鑒定

The suitability of a specific instrument for a given procedure is ensured by a stepwise life cycle evaluation for the desired application from selection to instrument retirement. The qualification comprises three components: 1) installation qualification (IQ), 2) operational qualification (OQ), and 3) performance qualification (PQ) (see Analytical Instrument Qualification <1058>).

特定儀器對(duì)給定程序的適用性由從選擇到儀器報(bào)廢的預(yù)期應(yīng)用的逐步生命周期評(píng)估來(lái)確保。鑒定包括三個(gè)部分：1）安裝鑒定（IQ）、2）操作鑒定（OQ）和3）性能鑒定（PQ）（參見(jiàn)<1058>Analytical Instrument Qualification章節(jié)）。

The purpose of this section is to provide test methods and acceptance criteria to ensure that the instrument is suitable for its intended use (OQ), and that it will continue to function properly over extended time periods (PQ). As with any spectrometric device, a turbidimetric and nephelometric spectrometer must be qualified for both wavelength (x-axis, if not fixed) and photometric (y-axis, or signal axis) accuracy and precision, and meet the requirements for the stray light. OQ is carried out across the operational ranges required within the laboratory for both the absorbance and wavelength scales.

本節(jié)的目的是提供測(cè)試方法和驗(yàn)收標(biāo)準(zhǔn)，以確保儀器適合其預(yù)期用途（OQ），并在延長(zhǎng)的時(shí)間段（PQ）內(nèi)繼續(xù)正常工作。與任何光譜儀一樣，透射光式和散射光式濁度光譜儀必須具備波長(zhǎng)（x軸，如果不固定）和光度（y軸或信號(hào)軸）的準(zhǔn)確度及精度，并滿(mǎn)足雜散光的要求。OQ是在實(shí)驗(yàn)室內(nèi)吸光度和波長(zhǎng)標(biāo)度所需的操作范圍內(nèi)進(jìn)行的。

Acceptance criteria for critical instrument parameters that establish “fitness for purpose” are verified during IQ and OQ. Specifications for particular instruments and applications can vary depending on the analytical procedure used and the desired accuracy of the final result. Instrument vendors often have samples and test parameters available as part of the IQ/OQ package.

在IQ和OQ期間，驗(yàn)證確定“用途適用性”的關(guān)鍵儀器參數(shù)的驗(yàn)收標(biāo)準(zhǔn)。特定儀器和應(yīng)用的規(guī)格可能因使用的分析程序和最終結(jié)果的預(yù)期準(zhǔn)確度而異。儀器供應(yīng)商通常將樣品和測(cè)試參數(shù)作為IQ/OQ包的一部分提供。

Wherever possible in the procedures detailed as follows, primary reference standards or certified reference materials (CRMs) are to be used. Formazin is the only primary reference standard used in turbidimetry and nephelometry. All the other standards, including the CRMs, must be correlated to formazin. The CRMs should be obtained from a recognized accredited source and include independently verified traceable value assignments with associated calculated uncertainty. CRMs must be kept clean and free from dust. Recertification should be performed periodically to maintain the validity of the certification.

在以下詳述的程序中，應(yīng)盡可能使用主要參考標(biāo)準(zhǔn)或認(rèn)證參考材料（CRM）。福爾馬肼是比濁法法和濁度法中唯yi使用的主要參考標(biāo)準(zhǔn)。所有其他標(biāo)準(zhǔn)，包括CRM，必須與福爾馬肼相關(guān)。CRM應(yīng)從認(rèn)可的認(rèn)證來(lái)源獲得，并包括獨(dú)立驗(yàn)證的可追溯值分配及相關(guān)的計(jì)算不確定性。CRM必須保持清潔，無(wú)灰塵。應(yīng)定期進(jìn)行重新認(rèn)證，以保持認(rèn)證的有效性。

6.1 Calibration校準(zhǔn)

All of the turbidimetric and nephelometric instruments are calibrated against standards of known turbidity. The instrument must be calibrated using formazin turbidity standards prior to its first time use and at least every 3 months or as specified by the vendor. Calibration is performed using at least four formazin turbidity standards whose turbidity proportionally covers the range of interest. Many instrument manufactures provide calibration verification standards. They usually consist of sealed sample cells filled with a latex suspension or with metal oxide particles in polymer gel. These standards must be used only for checking the calibration in the time intervals between the instrument recommended calibrations.

所有透射光式濁度儀和散射光式濁度儀均根據(jù)已知濁度的標(biāo)準(zhǔn)進(jìn)行校準(zhǔn)。在首ci使用之前，必須使用福爾馬肼濁度標(biāo)準(zhǔn)液對(duì)儀器進(jìn)行校準(zhǔn)，至少每3個(gè)月或按照供應(yīng)商的規(guī)定進(jìn)行一次校準(zhǔn)。使用至少四種福爾馬肼濁度標(biāo)準(zhǔn)液進(jìn)行校準(zhǔn)，其濁度按比例覆蓋感興趣的范圍。許多儀器制造商提供校準(zhǔn)驗(yàn)證標(biāo)準(zhǔn)。它們通常由其中充滿(mǎn)聚合物凝膠中的金屬氧化物顆粒的密封樣品池或乳膠懸浮液組成。這些標(biāo)準(zhǔn)只能用于檢查儀器推薦校準(zhǔn)的時(shí)間間隔內(nèi)的校準(zhǔn)。

6.2 Stray Light雜散光

Stray light (stray radiant energy) is a very significant error source, especially for measurements in the range of the lower turbidity readings. It is defined as external light that reaches the detector without being scattered from the sample. There are several sources of stray light including the inherent cell surface imperfections, reflections from within the cell that are unaccounted for, optical system parts, light sources, and, to a smaller degree, the electronics fluctuations. Although there are many design features that instrument vendors use to minimize the stray light, a complete mitigation of the stray light cannot be achieved. Unlike spectrophotometric measurements, the stray light cannot be compensated for in turbidimetry. The stray light must be measured and the values should be within the specification range set by the vendor of the particular instrument or <0.15 NTUs for the measurement in the range of 0–10 NTUs and 0.5 NTUs for the measurements in the range of 10– 1100 NTUs, whichever is smaller.

雜散光(雜散光輻射能)是一個(gè)非常重要的誤差源，特別是在較低的濁度讀數(shù)范圍內(nèi)的測(cè)量。它被定義為到達(dá)探測(cè)器而不被樣品散射的外部光線(xiàn)。雜散光有幾種來(lái)源，包括電池表面固有缺陷、電池內(nèi)部未被解釋的反射、光學(xué)系統(tǒng)部件、光源，以及在較小程度上的電子波動(dòng)。盡管儀器供應(yīng)商使用了許多設(shè)計(jì)功能來(lái)最小化雜散光，但無(wú)法*緩解雜散光。與分光光度測(cè)量不同，濁度法無(wú)法補(bǔ)償雜散光。必須測(cè)量雜散光，其值應(yīng)在特定儀器供應(yīng)商設(shè)定的規(guī)格范圍內(nèi)，或在0-10 NTU范圍內(nèi)測(cè)量時(shí)小于0.15 NTU，在10-1100 NTU范圍內(nèi)測(cè)量時(shí)小于0.5 NTU，以較小者為準(zhǔn)。

6.3 Range of Measuring Capability測(cè)量能力的范圍

The instrument must be able to measure the turbidity in the range of 0.01–1100 NTUs or from 50%–200% of the target turbidity. To demonstrate the linearity for the intended measurements range, choose at least four appropriate reference suspensions from Table 1.

儀器必須能夠測(cè)量0.01–1100 NTU范圍內(nèi)或目標(biāo)濁度50%-200%范圍內(nèi)的濁度。為了證明預(yù)期測(cè)量范圍的線(xiàn)性，從表1中選擇至少四種合適的參考懸浮液。

6.4 Resolution 解決方案

Instrument resolution must be 0.01 NTU or less for the measurements range of 0–9.99 NTUs; 0.1 NTU or less for the measurements range of 10–99.9 NTUs; and 1 NTU for the measurements above 100 NTUs.

對(duì)于0-9.99 NTU的測(cè)量范圍，儀器分辨率必須小于等于0.01 NTU；測(cè)量范圍為10-99.9 NTU時(shí)，小于等于0.1 NTU；100 NTU以上的測(cè)量分辨率值為1 NTU。

6.5 Accuracy準(zhǔn)確度

The instrument reading accuracy must be ±10% of the reading + 0.01 NTU for the measurement range from 0–19.9 NTUs, and ±7.5% of the reading for the measurement range from 20–1100 NTUs.

對(duì)于0-19.9 NTU的測(cè)量范圍，儀器讀數(shù)準(zhǔn)確度必須為讀數(shù)+0.01 NTU的±10%，對(duì)于20-1100 NTU的測(cè)量范圍，儀器讀數(shù)準(zhǔn)確度必須為讀數(shù)的±7.5%。

6.6 Performance Qualification性能鑒定

The instrument PQ is accomplished periodically or as needed between the calibrations. Primary turbidity standards (formazin) or secondary calibration verification standards (latex suspensions or metal oxide particles in polymer gels contained in sealed sample cells) supplied by instrument manufacturers may be used.

定期或根據(jù)需要在校準(zhǔn)期之間完成儀器PQ?？墒褂脙x器制造商提供的一級(jí)濁度標(biāo)準(zhǔn)（福爾馬肼）或二級(jí)校準(zhǔn)驗(yàn)證標(biāo)準(zhǔn)（乳膠懸浮液或密封樣品池中聚合物凝膠中的金屬氧化物顆粒）。

7. PROCEDURE步驟

7.1 Turbidimetric Procedures 透射光比濁法測(cè)試步驟

SAMPLE CELL PREPARATION 樣品池準(zhǔn)備

The sample cells for sample measurements must be clean. Follow the sample cell or instrument manufacturer recommendations for cleaning the sample cells appropriately. For low turbidity measurements it is a good practice to use a single-indexed sample cell or a flow cell, which help ensure adequate precision and repeatability of the measurements. Using particle-free water, find the sample cell orientation in the sample cell holder that gives the lowest reading. For higher values of turbidity, different sample cells may be used. However, the sample cells must be matched (the difference in readings for a standard prepared at nominal sample concentration from two different sample cells must be within ±0.005 NTU or below the measurement precision requirement, whichever is lower).

用于樣品測(cè)量的樣品室必須清潔。按照樣品池或儀器制造商的建議適當(dāng)清潔樣品池。對(duì)于低濁度測(cè)量，最好使用一個(gè)單指數(shù)樣品池或流動(dòng)池，這有助于確保測(cè)量的足夠精度和可重復(fù)性。使用無(wú)顆粒水，在樣品池支架中找到讀數(shù)最di的樣品池方向。對(duì)于較高的濁度值，可使用不同的樣品池。然而，樣品池必須匹配（兩個(gè)不同樣品池在標(biāo)稱(chēng)樣品濃度下制備的標(biāo)準(zhǔn)品讀數(shù)差異必須在±0.005 NTU范圍內(nèi)或低于測(cè)量精度要求，以較低者為準(zhǔn)）。

SAMPLE PREPARATION樣品準(zhǔn)備

Prepare the samples as prescribed in the individual monograph. Carefully mix the samples thoroughly by swirling or inverting the volumetric flask slowly several times. Avoid shaking or stirring since it may introduce bubbles. Degassing the samples helps to improve the measurements. For degassing, the samples could stand for several minutes or a vacuum could be applied, or they could be gently sonicated using an ultrasonic bath. After degassing, let the samples stand for several minutes and mix again by carefully inverting two to three times. Transfer the sample to the sample cell and take the readings.

按照各專(zhuān)題中的規(guī)定制備樣品。通過(guò)緩慢旋轉(zhuǎn)或倒置容量瓶數(shù)次，仔細(xì)混合樣品。避免搖晃或攪拌，因?yàn)檫@可能會(huì)產(chǎn)生氣泡。對(duì)樣品進(jìn)行脫氣有助于改進(jìn)測(cè)量。對(duì)于脫氣，樣品可以靜置幾分鐘，或者可以施加真空，或者可以使用超聲波浴對(duì)其進(jìn)行輕輕的超聲波處理。脫氣后，讓樣品靜置幾分鐘，然后小心地反轉(zhuǎn)兩到三次，再次混合。將樣品轉(zhuǎn)移至樣品池并讀取讀數(shù)。

USE OF FLOW CELLS流動(dòng)池的使用

Flow cells are mainly used for low turbidity measurements for samples with small particles. When such cells are used, the sample is introduced by carefully pouring it down the interior edge of the inlet reservoir.

In practice, it is advisable to ensure that settling of the particles being measured is negligible. This is usually accomplished by including a protective colloid in the liquid-suspending medium. It is important that results be interpreted by a comparison of readings with those representing known concentrations of suspended matter, produced under precisely the same conditions.

流動(dòng)池主要用于小顆粒樣品的低濁度測(cè)量。當(dāng)使用這種樣品池時(shí)，通過(guò)小心地將樣品倒入進(jìn)水倉(cāng)的內(nèi)邊緣來(lái)引入樣品。

在實(shí)際過(guò)程中，建議確保被測(cè)顆粒的沉降可以忽略不計(jì)。這通常通過(guò)在液體懸浮介質(zhì)中加入保護(hù)膠體來(lái)實(shí)現(xiàn)。重要的是，通過(guò)將讀數(shù)與在*相同的條件下產(chǎn)生的已知懸浮物濃度的讀數(shù)進(jìn)行比較來(lái)解釋結(jié)果。

7.2 Nephelometric Procedures散射光濁度法步驟

Nephelometric procedures are performed similarly to turbidimetric procedures for both direct measurements and measurements in the ratio mode as described above.

散射光濁度法步驟的執(zhí)行方式與透射光比濁法程序類(lèi)似，適用于直接測(cè)量和上述比率模式下的測(cè)量。

RATE NEPHELOMETRIC PROCEDURES比率模式散射光濁度法步驟

The overall procedure for monitoring the progress of the reaction consists of three well-defined steps: 1) record a baseline reading of the turbidity of the medium (blank); 2) record the turbidity after the first reagent (antigen) is added, which results in an increase of the turbidity until a plateau is reached; and 3) add the second reagent (antibody), which results in another turbidity increase and a second plateau followed by a final turbidity increase that continues until a third plateau is reached. The measurement zone is selected from the addition of the antibody until the third plateau, depending on the purpose of the assay and the respective component concentrations. Kinetic nephelometry and Endpoint nephelometry are two general procedures that are used for quantifying the immune complexes formed in the immunoassay methods (also known as immunonephelometry because the measured turbidity is due to immunocomplexes that are formed). For each procedure, there are several parameters that need to be optimized in each individual application. The main parameters are 1) with or without particle enhancement; 2) particle types, sizes, and respective optimum wavelength, if applicable; 3) monitoring reaction kinetic or endpoint; 4) antibody/antigen under consideration and, related to that, the optimum level of antigen loading; 5) buffers and other ionic species and respective optimal pH; 6) type and concentration of polymers used to modify the solubility of proteins; and 7) temperature and other environmental factors. Generally these parameters are optimized during the method development and the values are given in specific monograph(s) and/or chapter(s) as applicable.

監(jiān)測(cè)反應(yīng)進(jìn)程的總體程序包括三個(gè)明確定義的步驟：1）記錄介質(zhì)濁度的基線(xiàn)讀數(shù)（空白）；2） 在添加一種試劑（抗原）后，記錄濁度，這會(huì)導(dǎo)致濁度增加，直到達(dá)到一個(gè)穩(wěn)定期；3）添加第二種試劑（抗體），這會(huì)導(dǎo)致另一個(gè)濁度增加和第二個(gè)穩(wěn)定期，然后是最終濁度增加，直到達(dá)到第三個(gè)穩(wěn)定器。根據(jù)分析目的和各自的組分濃度，從添加抗體到第三個(gè)穩(wěn)定期中間選擇測(cè)量區(qū)。動(dòng)力學(xué)散射比濁法和終點(diǎn)散射比濁法是兩種通用程序，用于量化免疫分析方法中形成的免疫復(fù)合物（也稱(chēng)為免疫散射比濁法，因?yàn)闇y(cè)得的濁度是由形成的免疫復(fù)合物引起的）。對(duì)于每一個(gè)步驟，都有幾個(gè)參數(shù)需要在每個(gè)單獨(dú)的應(yīng)用中進(jìn)行優(yōu)化。主要參數(shù)為1）有無(wú)粒子增強(qiáng)；2） 顆粒類(lèi)型、尺寸和各自的最佳波長(zhǎng)（如適用）；3） 監(jiān)測(cè)反應(yīng)動(dòng)力學(xué)或終點(diǎn)；4） 考慮中的抗體/抗原，以及與之相關(guān)的抗原負(fù)載的最佳水平；5） 緩沖液和其他離子種類(lèi)以及各自的最佳pH值；6） 用于改變蛋白質(zhì)溶解度的聚合物的類(lèi)型和濃度；7）溫度和其他環(huán)境因素。通常，這些參數(shù)在方法開(kāi)發(fā)過(guò)程中進(jìn)行了優(yōu)化，具體的專(zhuān)著和/或章節(jié)（如適用）中給出了這些值。

Kinetic nephelometry: The kinetic nephelometry is advantageous compared to the endpoint nephelometry mainly because of the capability to take a sample blank reading in addition to a reagent blank reading. This procedure assesses the rate of the immunocomplex formation based on the increased intensity response of the scattered light of the chosen wavelength. The reaction kinetic may be monitored continuously or a certain number of data points may be taken, depending on the time response of the instrument used and the type of application. At times it may involve only two data points; however, proper care must be exercised because the choice of point selection can influence the overall accuracy in cases where differences in reaction kinetics exist between samples and calibrating standards. Careful consideration should be given to the appropriate choice of specificity control strategy.

動(dòng)力學(xué)散射比濁法：與終點(diǎn)散射比濁法相比，動(dòng)力學(xué)散射比濁法具有優(yōu)勢(shì)，主要是因?yàn)槌嗽噭┛瞻鬃x數(shù)外，還能夠讀取樣品空白讀數(shù)。該程序基于所選波長(zhǎng)的散射光的增強(qiáng)強(qiáng)度響應(yīng)來(lái)評(píng)估免疫復(fù)合物的形成速率。根據(jù)所用儀器的時(shí)間響應(yīng)和應(yīng)用類(lèi)型，可連續(xù)監(jiān)測(cè)反應(yīng)動(dòng)力學(xué)，或采集一定數(shù)量的數(shù)據(jù)點(diǎn)。有時(shí)它可能只涉及兩個(gè)數(shù)據(jù)點(diǎn)；但是在樣品和校準(zhǔn)標(biāo)準(zhǔn)之間存在反應(yīng)動(dòng)力學(xué)差異的情況下，選擇點(diǎn)可能會(huì)影響整體準(zhǔn)確度。應(yīng)仔細(xì)考慮特異性控制策略的適當(dāng)選擇。

Endpoint nephelometry: In this method, an initial measurement is performed before adding the reagent, which represents the blank reading. A second measurement is performed after the immune complex is formed after approximately 60 min. The difference between these two measurements is proportional to the content of the component being assayed.

終點(diǎn)散射比濁法：在該方法中，在添加試劑之前進(jìn)行初始測(cè)量，這代表空白讀數(shù)。大約60分鐘后，在免疫復(fù)合物形成后進(jìn)行第二次測(cè)量。這兩次測(cè)量之間的差異與所分析成分的含量成正比。

8. VALIDATION AND VERIFICATION 驗(yàn)證與核查

8.1 Validation 驗(yàn)證

Validation is required when a nephelometric/turbidimetric method is intended for use as an alternative to the official procedure for testing an official article. The objective of nephelometric/turbidimetric method validation is to demonstrate that the measurement is suitable for its intended purpose, including quantitative determination of the main component in a drug substance or a drug product (Category I assays), quantitative determination of impurities or limit tests (Category II), and identification tests (Category IV). Depending on the category of the test (see Validation of Compendial Procedures <1225>, Table 2), the analytical method validation process for nephelometry/turbidimetry requires testing for accuracy, precision, specificity, detection limit (DL), quantitation limit (QL), linearity, range, and robustness. These analytical performance characteristics apply to externally standardized procedures and those that use standard additions.

當(dāng)散射比濁法/透射濁度法擬用作官fang物品測(cè)試程序的替代方法時(shí)，需要進(jìn)行驗(yàn)證。當(dāng)散射比濁法/透射濁度法驗(yàn)證的目的是證明測(cè)量適用于其預(yù)期目的，包括原料藥或藥品中主要成分的定量測(cè)定（I類(lèi)分析）、雜質(zhì)的定量測(cè)定或限度試驗(yàn)（II類(lèi)）以及鑒定試驗(yàn)（IV類(lèi)）。根據(jù)試驗(yàn)的類(lèi)別（參見(jiàn)<1225>Validation of Compendial Procedures，表2），透射濁度法/散射比濁法的分析方法驗(yàn)證過(guò)程需要對(duì)準(zhǔn)確度、精密度、特異性、檢測(cè)限（DL）、定量限（QL）、線(xiàn)性、范圍和穩(wěn)健性進(jìn)行試驗(yàn)。這些分析性能特征適用于外部標(biāo)準(zhǔn)化程序和那些使用標(biāo)準(zhǔn)添加的程序。

Validation of Compendial Procedures <1225> provides definitions and general guidance on analytical procedures validation without indicating specific validation criteria for each characteristic. The intention of the following sections is to provide the user with specific validation criteria that represent the minimum expectations for this technology. For each particular application, tighter criteria may be needed in order to demonstrate suitability for the intended use.

<1225>Validation of Compendial Procedures章節(jié)提供了分析程序驗(yàn)證的定義和一般指南，但沒(méi)有說(shuō)明每個(gè)特征的具體驗(yàn)證標(biāo)準(zhǔn)。以下各節(jié)的目的是向用戶(hù)提供具體的驗(yàn)證標(biāo)準(zhǔn)，這些標(biāo)準(zhǔn)代表了對(duì)該技術(shù)的最di期望。對(duì)于每個(gè)特定應(yīng)用，可能需要更嚴(yán)格的標(biāo)準(zhǔn)，以證明其適用于預(yù)期用途。

ACCURACY 準(zhǔn)確度

For Category I, II, and III procedures, accuracy can be determined by conducting recovery studies with the appropriate matrix spiked with known concentrations of the analyte. Analysts can also compare the assay results obtained using the nephelometric/ turbidimetric procedure under validation to those from an established analytical procedure.

Validation criteria: 98.0%–102.0% mean recovery for the drug substances, 95.0%–105.0% mean recovery for the drug product assay, and 80.0%–120.0% mean recovery for the impurity analysis. These criteria are met throughout the specified range.

對(duì)于I類(lèi)、II類(lèi)和III類(lèi)程序，可通過(guò)使用加入已知分析物濃度的適當(dāng)基質(zhì)進(jìn)行回收研究來(lái)確定準(zhǔn)確度。分析員還可以將使用驗(yàn)證中的散射光濁度法/透射光比濁法程序獲得的分析結(jié)果與已建立的分析程序獲得的結(jié)果進(jìn)行比較。

驗(yàn)證標(biāo)準(zhǔn)：原料藥的平均回收率為98.0%–102.0%，藥品分析的平均回收率為95.0%–105.0%，雜質(zhì)分析的平均回收率為80.0%–120.0%。這些標(biāo)準(zhǔn)在整個(gè)規(guī)定范圍內(nèi)都得到滿(mǎn)足。

PRECISION精度

Repeatability: The repeatability of the analytical procedure is assessed by measuring the concentrations of six independently prepared sample solutions at of the assay test concentration. Alternatively, it can be assessed by measuring the concentrations of three replicates of three separate sample solutions at different concentrations. The three concentrations should be close enough so that the repeatability is constant across the concentration range. If this is done, the repeatability at the three concentrations is pooled for comparison to the acceptance criteria.

Validation criteria: The relative standard deviation is NMT 1.0% for the drug substance, NMT 2.0% for the drug product assay, and NMT 20.0% for the impurity analysis.

重復(fù)性：通過(guò)測(cè)量六種獨(dú)立制備的樣品溶液在分析試驗(yàn)濃度下的濃度來(lái)評(píng)估分析程序的重復(fù)性?；蛘?，可以通過(guò)測(cè)量三種不同濃度的單獨(dú)樣品溶液的三個(gè)重復(fù)的濃度來(lái)評(píng)估。三種濃度應(yīng)足夠接近，以便在整個(gè)濃度范圍內(nèi)重復(fù)性保持恒定。如果這樣做，將三種濃度下的重復(fù)性匯總，以與驗(yàn)收標(biāo)準(zhǔn)進(jìn)行比較。

驗(yàn)證標(biāo)準(zhǔn)：原料藥的相對(duì)標(biāo)準(zhǔn)偏差為NMT 1.0%，藥品分析的相對(duì)標(biāo)準(zhǔn)偏差為NMT 2.0%，雜質(zhì)分析的相對(duì)標(biāo)準(zhǔn)偏差為NMT 20.0%。

Intermediate precision: The effect of random events on the analytical precision of the method must be established. Typical variables include performing the analysis on different days, using different instrumentation, and/or having the method performed by two or more analysts. At a minimum, any combination of at least two of these factors totaling six experiments will provide an estimation of intermediate precision.

Validation criteria: The relative standard deviation is NMT 1.5% for the drug substance, NMT 3.0% for the drug product assay, and NMT 25.0% for the impurity analysis.

中間精度：必須確定隨機(jī)事件對(duì)方法分析精度的影響。典型的變量包括在不同的日期使用不同的儀器進(jìn)行分析，和/或由兩名或兩名以上的分析員進(jìn)行分析。至少，這些因素中的至少兩個(gè)的組合，總共6個(gè)實(shí)驗(yàn)，將提供中等精度的評(píng)估。

驗(yàn)證標(biāo)準(zhǔn)：原料藥的相對(duì)標(biāo)準(zhǔn)偏差為NMT 1.5%，藥品分析的相對(duì)標(biāo)準(zhǔn)偏差為NMT 3.0%，雜質(zhì)分析的相對(duì)標(biāo)準(zhǔn)偏差為NMT 25.0%。

SPECIFICITY 特異性

In nephelometric/turbidimetric measurements, specificity is demonstrated by the lack of interference from other components present in the matrix (other components of the matrix produce a true solution).

在散射光濁度法/透射光比濁法的濁度測(cè)量中，特異性通過(guò)基質(zhì)中其他成分的干擾（基質(zhì)的其他成分產(chǎn)生真實(shí)溶液）的缺乏來(lái)證明。

DETECTION LIMIT 檢測(cè)限

The DL can be estimated by calculating the concentration of a solution that would give the signal-to-noise ratio of ≥3.3. The estimated DL must be confirmed by analyzing samples at the calculated concentration.

可以通過(guò)計(jì)算溶液的濃度來(lái)估計(jì)檢測(cè)限DL，該濃度將給出信號(hào)的信噪比≥3.3. 必須通過(guò)分析計(jì)算濃度下的樣品來(lái)確認(rèn)估計(jì)的DL。

QUANTITATION LIMIT 定量限

The QL can be estimated by calculating the concentration of a solution that would give the signal-to-noise ratio of ≥10.0. The estimated QL must be confirmed by analyzing samples at the calculated concentration. Measurement of a test solution prepared from a representative sample matrix spiked at the required QL concentration must be performed to confirm sufficient sensitivity and adequate precision. The observed signal-to-noise ratio at the required QL should be >10.

Validation criteria: For the estimated limit of quantitation to be considered valid, the measured concentration must be accurate and precise at a level ≤50% of the specification.

定量限QL可以通過(guò)計(jì)算溶液的濃度來(lái)估算，該濃度將給出信號(hào)的信噪比≥10.0. 必須通過(guò)分析計(jì)算濃度下的樣品來(lái)確認(rèn)估算的QL。必須對(duì)以所需QL濃度添加的代表性樣品基質(zhì)制備的試液進(jìn)行測(cè)量，以確認(rèn)其具有足夠的靈敏度和精度。在所需QL下觀察到的信噪比應(yīng)大于10。

驗(yàn)證標(biāo)準(zhǔn):估計(jì)的定量限被認(rèn)為是有效的，測(cè)量的濃度必須是準(zhǔn)確的，并且在≤50%的規(guī)格水平上是精確的。

LINEARITY線(xiàn)性

A linear relationship between the analyte concentration and measured turbidity response must be demonstrated by preparation of at least four standard solutions at concentrations encompassing the anticipated concentration of the test solution. The standard curve is then evaluated using appropriate statistical methods such as a least-squares regression. Deviation from linearity results from instrumental or sample factors, or both, can be reduced to acceptable levels by reducing or increasing the analyte concentration, thereby respectively decreasing or increasing the turbidity readings to within the nephelometer/turbidimeter instrument linearity range.

Validation criteria: The correlation coefficient (R) must be NLT 0.995 for Category I assays and NLT 0.99 for Category II quantitative tests.

分析物濃度和測(cè)得的濁度響應(yīng)之間的線(xiàn)性關(guān)系必須通過(guò)制備至少四種標(biāo)準(zhǔn)溶液來(lái)證明，其濃度包括試驗(yàn)溶液的預(yù)期濃度。然后使用適當(dāng)?shù)慕y(tǒng)計(jì)方法（如最小二乘回歸）評(píng)估標(biāo)準(zhǔn)曲線(xiàn)。通過(guò)降低或增加分析物濃度，可將儀器或樣品因素或兩者的線(xiàn)性偏差降低至可接受水平，從而分別將濁度讀數(shù)降低或增加至透射光法濁度計(jì)/散射光濁度計(jì)儀器線(xiàn)性范圍內(nèi)。

驗(yàn)證標(biāo)準(zhǔn)：對(duì)于I類(lèi)分析，相關(guān)系數(shù)（R）必須為NLT 0.995，對(duì)于II類(lèi)定量測(cè)試，相關(guān)系數(shù)（R）必須為NLT 0.99。

RANGE 范圍

The operational range of an analytical instrument (and the analytical procedure as a whole) is the interval between the upper and lower concentrations (amounts) of analyte in the sample (including these concentrations) for which it has been demonstrated that the instrumental response function has a suitable level of precision, accuracy, and linearity.

Validation criteria: For Category I tests, the validation range for 100.0% centered acceptance criteria is 80.0%–120.0%. For non-centered acceptance criteria, the validation range is 10.0% below the lower limit to 10.0% above the upper limit. For Category II tests, the validation range covers 50.0%–120.0% of the acceptance criteria.

分析儀器（以及整個(gè)分析程序）的操作范圍是樣品中分析物的上下濃度（數(shù)量）（包括這些濃度）之間的間隔，已證明儀器響應(yīng)函數(shù)具有適當(dāng)?shù)木取?zhǔn)確度和線(xiàn)性水平。

驗(yàn)證標(biāo)準(zhǔn)：對(duì)于I類(lèi)試驗(yàn)，100.0%中心驗(yàn)收標(biāo)準(zhǔn)的驗(yàn)證范圍為80.0%–120.0%。對(duì)于非中心驗(yàn)收標(biāo)準(zhǔn)，驗(yàn)證范圍為下限以下10.0%到上限以上10.0%。對(duì)于II類(lèi)試驗(yàn)，驗(yàn)證范圍涵蓋驗(yàn)收標(biāo)準(zhǔn)的50.0%–120.0%。

ROBUSTNESS穩(wěn)健性

The reliability of an analytical measurement is demonstrated by deliberate changes to experimental parameters. For nephelometry/turbidimetry this can include, for example, measuring the stability of the analyte under specified storage conditions, varying pH, and adding possible interfering species. Robustness is determined concurrently using a suitable design for the experimental procedure.

分析測(cè)量的可靠性通過(guò)有意改變實(shí)驗(yàn)參數(shù)來(lái)證明。 對(duì)于散射光濁度法/透射光比濁法，這可以包括：測(cè)量分析物在特定儲(chǔ)存條件、變化的 pH 值和添加可能的干擾物質(zhì)下的穩(wěn)定性。使用適合實(shí)驗(yàn)程序的設(shè)計(jì)，同時(shí)確保穩(wěn)健性。

8.2 Verification核查

Current U.S. Good Manufacturing Practices regulations [21 CFR 211.194(a)(2)] indicate that users of analytical procedures described in the U.S. Pharmacopeia and National Formulary are not required to validate these procedures if provided in a monograph. Instead, they simply must verify their suitability under actual conditions of use.

現(xiàn)行的《美國(guó)生產(chǎn)規(guī)范條例》[21 CFR 211.194（a）（2）]表明，如果專(zhuān)論中提供了這些程序，則美國(guó)藥典和國(guó)家處方集中描述的分析程序的用戶(hù)無(wú)需驗(yàn)證這些程序。相反，他們只需驗(yàn)證其在實(shí)際使用條件下的適用性。

The objective of nephelometric/turbidimetric procedure verification is to demonstrate the suitability of a test procedure under actual conditions of use. Performance characteristics that verify the suitability of a nephelometric/turbidimetric procedure are similar to those required for any analytical procedure. A discussion of the applicable general principles is found in Verification of Compendial Procedures <1226>. Verification is usually performed using a reference material and a well-defined matrix. Verification of compendial nephelometric/turbidimetric procedures includes, at minimum, the execution of the validation parameters for specificity, accuracy, precision, and QL, when appropriate, as indicated in 8.1 Validation.

散射光濁度法/透射光比濁法程序驗(yàn)證的目的是證明測(cè)試程序在實(shí)際使用條件下的適用性。驗(yàn)證散射光濁度法/透射光比濁法程序適用性的性能特征與任何分析程序所需的性能特征相似。適用的一般原則的討論見(jiàn)<1226>Verification of Compendial Procedure 章節(jié)。通常使用參考材料和明確定義的基質(zhì)進(jìn)行驗(yàn)證。 藥典散射光濁度法/透射光比濁法程序的驗(yàn)證至少包括對(duì)特異性、準(zhǔn)確度、精密度和 QL 的驗(yàn)證參數(shù)的執(zhí)行（如 8.1 驗(yàn)證中所述）。

歐洲藥典EP10.0

2.2.1. CLARITY AND DEGREE OF OPALESCENCE OF LIQUIDS 液體的澄清度和乳光度

Opalescence is the effect of light being absorbed or scattered by submicroscopic particles or optical density inhomogeneities. The absence of any particles or inhomogeneities in a solution results in a clear solution.

光被亞微觀粒子吸收或散射、或光密度不均勻的產(chǎn)生的效果即為乳光。溶液中不存在任何粒子或不均勻性，就會(huì)得到清澈的溶液。

A liquid is considered clear if its clarity is the same as that of water R or of the solvent used, or if its opalescence is not more pronounced than that of reference suspension I (see Table 2.2.1.-1), when examined under the conditions described below.

在下述條件下檢查時(shí)，如果液體的透明度與水或所用溶劑的透明度相同，或者其乳光不比參考懸浮液I（見(jiàn)表2.2.1.-1）的乳光更明顯，則認(rèn)為液體是透明的。

Requirements in monographs are expressed in terms of the visual method by comparing with the defined reference suspensions (see Table 2.2.1.-1). However, instrumental methods may also be used for determining compliance with monograph requirements once the suitability of the instrument has been established as described below and calibration with reference suspensions I-IV and with water R or the solvent used has been performed.

通過(guò)與規(guī)定的參考懸浮液進(jìn)行比較（見(jiàn)表2.2.1.-1），以目視法表達(dá)專(zhuān)著中的要求。然而，一旦儀器的適用性如下所述建立，儀器方法也可用于確定是否符合專(zhuān)論要求，并使用參考懸浮液I-IV和水或所用溶劑進(jìn)行校準(zhǔn)。

VISUAL METHOD目視法

Using identical test-tubes of colourless, transparent, neutral glass with a flat base and an internal diameter of 15-25 mm, compare the liquid to be examined with a reference suspension freshly prepared as described below. Ensure that the depths of the layers in the 2 test-tubes are the same (about 40 mm).

使用相同的無(wú)色透明中性玻璃試管，底座平坦，內(nèi)徑為15-25 mm，將待檢液體與下述新制備的參考懸浮液進(jìn)行比較。確保兩個(gè)試管中各層的深度相同（約40 mm）。

Compare the liquids in diffused daylight 5 min after preparation of the reference suspension, viewing vertically against a black background.

制備參考懸浮液5分鐘后，在漫射日光下比較液體，在黑色背景下垂直觀察。

System suitability. The diffusion of light must be such that reference suspension I can readily be distinguished from water R, and that reference suspension II can readily be distinguished from reference suspension I (see Table 2.2.1.-1).

系統(tǒng)適用性。光的擴(kuò)散必須確保參考懸浮液I可以很容易地與水區(qū)分開(kāi)，并且參考懸浮液II可以很容易地與參考懸浮液I區(qū)分開(kāi)（見(jiàn)表2.2.1.-1）。

INSTRUMENTAL METHOD 儀器法

The instrumental assessment of clarity and opalescence provides a more discriminatory test that does not depend on the visual acuity of the analyst. Numerical results are more useful for process control and quality monitoring, especially in stability studies. For example, previous numerical data on stability can be extrapolated to determine whether a given batch of a preparation will exceed shelf-life limits prior to the expiry date.

儀器法評(píng)估給透明度和乳光度的提供了一種更具辨別力的測(cè)試，它不依賴(lài)于分析人員的視力。 數(shù)值結(jié)果對(duì)于過(guò)程控制和質(zhì)量監(jiān)控更有用，尤其是在穩(wěn)定性研究中。 例如，可以從以前關(guān)于穩(wěn)定性的數(shù)字?jǐn)?shù)據(jù)外推，來(lái)確定給定批次的制劑是否會(huì)在有效期之前超過(guò)保質(zhì)期限制。

TURBIDIMETRY AND NEPHELOMETRY比濁法和濁度法

When a suspension is viewed at right angles to the direction of the incident light, the system appears opalescent due to the scattering of light by the particles of the suspension (Tyndall effect). A certain portion of the light beam entering a turbid liquid is transmitted, another portion is absorbed and the remaining portion is scattered by the suspended particles. The light-scattering effect of suspended particles can be measured either indirectly by observation of the transmitted light (turbidimetry) or directly by measuring the scattered light (nephelometry). Turbidimetry and nephelometry are more reliable in low turbidity ranges, where there is a linear relationship between turbidity values and detector signals. As the degree of turbidity increases, not all the particles are exposed to the incident light and the scattered or the transmitted radiation of other particles is hindered on its way to the detector.

當(dāng)以與入射光方向成直角的角度觀察懸浮液時(shí)，由于懸浮液顆粒對(duì)光的散射（丁達(dá)爾效應(yīng)），系統(tǒng)呈現(xiàn)乳白色。進(jìn)入混濁液體的光束的一部分被透射，另一部分被吸收，其余部分被懸浮顆粒散射。懸浮顆粒的光散射效應(yīng)可以通過(guò)觀察透射光（比濁法）間接測(cè)量，也可以通過(guò)測(cè)量散射光（濁度法）直接測(cè)量。比濁法和濁度法在低濁度范圍內(nèi)更可靠，濁度值和檢測(cè)器信號(hào)之間存在線(xiàn)性關(guān)系。隨著濁度的增加，并非所有粒子都暴露在入射光下，其他粒子的散射或透射輻射在到達(dá)探測(cè)器的過(guò)程中會(huì)受到阻礙。

For quantitative measurements, the construction of calibration curves is essential. Linearity must be based on at least 4 levels of concentrations. Reference suspensions must show a sufficiently stable degree of turbidity and must be produced under well-defined conditions.

對(duì)于定量測(cè)量，校準(zhǔn)曲線(xiàn)的構(gòu)建至關(guān)重要。線(xiàn)性必須基于至少4個(gè)濃度水平。參考懸浮液必須顯示足夠穩(wěn)定的濁度，并且必須在明確的條件下產(chǎn)生。

MEASUREMENTS IN RATIO MODE比率模式下的測(cè)量

The determination of opalescence of coloured liquids is done using instruments with ratio mode, since colour provides a negative interference, attenuating both incident and scattered light and lowering the turbidity value. The effect is so great, even for moderately coloured samples, that conventional nephelometers cannot be used.

由于顏色會(huì)產(chǎn)生負(fù)干擾，衰減入射光和散射光，降低濁度值，因此使用具有比率模式的儀器測(cè)定有色液體的乳光。 這種影響是如此之大，即使是中等顏色的樣品，以至于不能使用傳統(tǒng)的濁度計(jì)。

In turbidimetry or nephelometry with ratio mode, the ratio of the transmission measurement to the 90° scattered light measurement is determined. This procedure compensates for the light that is diminished by the colour of the sample. Instruments with ratio mode use as light source a tungsten lamp with spectral sensitivity at about 550 nm operating at a filament colour temperature of 2700 K. Other suitable light sources may also be used. Silicon photodiodes and photomultipliers are commonly used as detectors and record changes in light scattered or transmitted by the sample. The light scattered at 90 ± 2.5° is measured by the primary detector. Other detectors measure back and forward scatter (reflected light) as well as transmitted light. The results are obtained by calculating the ratio of the 90° scattered light measured to the sum of the components of forward scattered and transmitted light values.

在比濁法或濁度法中，通過(guò)比率模式，確定透射測(cè)量與90°散射光測(cè)量的比率。該程序補(bǔ)償因樣品顏色而減弱的光線(xiàn)。具有比率模式的儀器使用光譜靈敏度約為550 nm的鎢燈作為光源，在2700 K的燈絲色溫下工作。也可以使用其他合適的光源。硅光電二極管和光電倍增管常用作探測(cè)器，記錄樣品散射或透射光的變化。由主探測(cè)器測(cè)量90±2.5°處的散射光。其他探測(cè)器測(cè)量前后散射（反射光）以及透射光。通過(guò)計(jì)算測(cè)得的90°散射光與前向散射光和透射光值分量之和的比值，可以獲得結(jié)果。

The instruments used are calibrated against standards of known turbidity and are capable of automatic measurement of turbidity. The test results are obtained directly from the instrument and compared to the specifications in the individual monograph.

使用的儀器根據(jù)已知濁度標(biāo)準(zhǔn)進(jìn)行校準(zhǔn)，并能夠自動(dòng)測(cè)量濁度。測(cè)試結(jié)果直接從儀器中獲得，并與各專(zhuān)著中的規(guī)范進(jìn)行比較。

Alternatively, the influence of the colour of the sample may also be eliminated by using an infrared light-emitting diode (IR LED) having an emission maximum at 860 nm with a 60 nm spectral bandwidth as the light source of the instrument.

或者，也可以通過(guò)使用最大發(fā)射波長(zhǎng)為860nm、光譜帶寬為60nm的紅外發(fā)光二極管（IR LED）作為儀器光源來(lái)消除樣品顏色的影響。

INSTRUMENT REQUIREMENTS儀器要求

Instruments complying with the following characteristics and verified using reference suspensions as described below may be used instead of visual examination for determination of compliance with monograph requirements.

可使用符合以下特征并使用下述參考懸浮液驗(yàn)證的儀器代替目視檢查，以確定是否符合專(zhuān)論要求。

– Measuring unit: NTU (nephelometric turbidity units). NTU is based on the turbidity of a primary standard of formazin. FTU (formazin turbidity units) or FNU (formazin nephelometric units) are also used, and are equivalent to NTU in regions of low turbidity (up to 40 NTU). These units are used in all 3 instrumental methods (nephelometry, turbidimetry and in ratio mode).

– Measuring range: 0.01-1100 NTU.

– Resolution: 0.01 NTU within the range 0-9.99 NTU; 0.1 NTU within the range 10.0-99.9 NTU; and 1 NTU for the range > 100 NTU.

– Accuracy: ± (10 per cent of reading + 0.01 NTU) with in the range 0-20 NTU; ± 7.5 per cent within the range 20-1100 NTU.

– Repeatability: ± 0.05 NTU within the range0-20 NTU; ± 2 per cent of the reading within the range 20-1100 NTU.

測(cè)量單位：NTU（濁度測(cè)量單位）。NTU是基于福爾馬肼一級(jí)標(biāo)準(zhǔn)品的濁度。也可使用FTU（福爾馬肼濁度單位）或FNU（福爾馬肼濁度單位），相當(dāng)于NTU的在低濁度區(qū)域（最高40 NTU）。這些單位適用于所有3種儀器方法（比濁法、濁度法和比率模式）。

–測(cè)量范圍：0.01-1100 NTU

–分辨率：0-9.99 NTU范圍內(nèi)為0.01 NTU；10.0-99.9 NTU范圍內(nèi)為0.1 NTU；對(duì)于大于100 NTU的范圍，則為1 NTU

–準(zhǔn)確度：范圍在0-20 NTU之間，讀數(shù)準(zhǔn)確度偏差為±（讀數(shù)的10%+0.01 NTU）；范圍在20-1100 NTU時(shí)，讀數(shù)準(zhǔn)確偏差為±7.5%。

–重復(fù)性：在0-20 NTU范圍內(nèi)重復(fù)性為±0.05 NTU；在20-1100 NTU范圍內(nèi)讀數(shù)重復(fù)性為±2%。

Instruments with measuring range or resolution, accuracy and repeatability capabilities other than those mentioned above may be used provided they are sufficiently validated and are capable for the intended use.

測(cè)量范圍或分辨率、精度和重復(fù)性能力不同于上述測(cè)量范圍或分辨率、精度和重復(fù)性能力的儀器經(jīng)過(guò)有效驗(yàn)證，也能夠應(yīng)用于預(yù)期用途。

CONTROL OF INSTRUMENT PERFORMANCE儀器性能的控制

– Calibration: performed with at least 4 reference suspensions of formazin covering the measuring range of interest. Reference suspensions described in this chapter or suitable reference standards calibrated against the primary reference suspensions may be used.

–校準(zhǔn)：使用至少4種福爾馬肼參考懸浮液進(jìn)行校準(zhǔn)，覆蓋感興趣的測(cè)量范圍?？墒褂帽菊滤龅膮⒖紤腋∫夯蚋鶕?jù)主要參考懸浮液校準(zhǔn)的適當(dāng)參考標(biāo)準(zhǔn)。

– Stray light: < 0.15 NTU within the range 0-10 NTU; < 0.5 NTU within the range 10-1100 NTU. Stray light is defined as that light that reaches the nephelometric detector without being a result of scatter from the sample. Stray light is always a positive interference and is a significant source of error in low-range turbidity measurements. Sources of stray light include: imperfections in and scratches on sample cells, internal reflections of the optical system, contamination of the optics or sample cell chamber with dust, and electronic noise. Instrument design can also affect stray light. The influence of stray light becomes negligible in ratio mode measurements.

雜散光：在0-10NTU范圍內(nèi)<0.15 NTU；在10-1100 NTU范圍內(nèi)<0.5 NTU。雜散光是指到達(dá)濁度檢測(cè)器的光，而不是樣品散射的結(jié)果。雜散光總是一種正干擾，是低范圍濁度測(cè)量中的一個(gè)重要誤差源。雜散光的來(lái)源包括：樣品池中的缺陷和劃痕、光學(xué)系統(tǒng)的內(nèi)部反射、光學(xué)元件或樣品池被灰塵污染，以及電子噪聲。儀器設(shè)計(jì)也會(huì)影響雜散光。在比率模式測(cè)量中，雜散光的影響可以忽略不計(jì)。

The test methodology for the specific substance/product to be analysed must also be verified to demonstrate its analytical capability. The instrument and methodology shall be consistent with the attributes of the substance to be examined.

還必須驗(yàn)證待分析特定物質(zhì)/產(chǎn)品的試驗(yàn)方法，以證明其分析能力。儀器和方法應(yīng)與待檢物質(zhì)的屬性一致。

Measurements of standards and samples should be carried out under the same temperature conditions, preferably between 20 °C and 25 °C.

標(biāo)準(zhǔn)品和樣品的測(cè)量應(yīng)在相同的溫度條件下進(jìn)行，最好在20 °C和25 °C之間。

REFERENCE SUSPENSIONS 參考懸浮液

Formazin has several desirable characteristics that make it an excellent turbidity standard. It can be reproducibly prepared from assayed raw materials. The physical characteristics make it a desirable light-scatter calibration standard. The formazin polymer consists of chains of different lengths, which fold into random configurations. This results in a wide variety of particle shapes and sizes, which allows the analysis of different particle sizes and shapes that are found in real samples. Stabilised formazin suspensions that can be used to prepare stable, diluted turbidity standards are commercially available and may be used after comparison with the standards prepared as described.

福爾馬肼有幾個(gè)理想的特性，使其成為一個(gè)優(yōu)秀的濁度液標(biāo)準(zhǔn)。它可以從經(jīng)過(guò)分析的原材料中重復(fù)制備。其物理特性使其成為理想的光散射校準(zhǔn)標(biāo)準(zhǔn)。福爾馬肼聚合物由不同長(zhǎng)度的鏈組成，這些鏈折疊成隨機(jī)構(gòu)型。這會(huì)產(chǎn)生各種各樣形狀和尺寸的顆粒，從而可以分析真實(shí)樣品中發(fā)現(xiàn)的不同顆粒大小和形狀?？捎糜谥苽浞€(wěn)定稀釋濁度標(biāo)準(zhǔn)品的穩(wěn)定福爾馬肼懸浮液是可商購(gòu)的，并可在與所述制備的標(biāo)準(zhǔn)品進(jìn)行比較后使用。

All steps of the preparation of reference suspensions as described below are carried out at 25 ± 3 °C.

下述參考懸浮液制備的所有步驟均在25±3°C下進(jìn)行。

Hydrazine sulfate solution. Dissolve 1.0 g of hydrazine sulfate R in water R and dilute to 100.0 mL with the same solvent. Allow to stand for 4-6 h.

硫酸肼溶液。 將 1.0 g 硫酸肼溶解在水中，并用相同的溶劑稀釋至100.0 mL。 靜置 4-6 小時(shí)。

Primary opalescent suspension (formazin suspension). In a 100 mL ground-glass-stoppered flask, dissolve 2.5 g of hexamethylenetetramine R in 25.0 mL of water R. Add 25.0 mL of the hydrazine sulfate solution. Mix and allow to stand for 24 h. This suspension is stable for 2 months, provided it is stored in a glass container free from surface defects. The suspension must not adhere to the glass and must be mixed thoroughly before use.

初級(jí)乳白色懸浮液（福爾馬肼懸浮液）。在100 mL磨砂玻璃塞燒瓶中，將2.5g六亞甲基四胺溶解在25.0 mL水中。添加25.0 mL硫酸肼溶液。混合并靜置24小時(shí)。如果該懸浮液儲(chǔ)存在無(wú)表面缺陷的玻璃容器中，則可穩(wěn)定2個(gè)月。懸浮液不得粘附在玻璃上，使用前必須*混合。

Standard of opalescence. Dilute 15.0 mL of the primary opalescent suspension to 1000.0 mL with water R. This suspension is freshly prepared and may be stored for up to 24 h.

乳白色的標(biāo)準(zhǔn)濁度液。用水將15.0 mL初級(jí)乳白色懸浮液稀釋至1000.0 mL。該懸浮液是新制備的，可儲(chǔ)存24小時(shí)。

Reference suspensions. Prepare the reference suspensions according to Table 2.2.1.-1. Mix and shake before use.

參考懸浮液。根據(jù)表2.2.1-1制備參考懸浮液。使用前混合并搖勻。

Measurements of reference suspensions I-IV in ratio mode show a linear relationship between the concentrations and measured NTU values (see Table 2.2.1.-2).

在比率模式下，參考懸浮液I-IV的測(cè)量結(jié)果顯示，濃度與測(cè)量的NTU值之間存在線(xiàn)性關(guān)系（見(jiàn)表2.2.1.-2）。

日本藥典17版

2.61 Turbidity Measurement 濁度測(cè)量

Turbidity measurement is used to determine the turbidity (degree of opalescence) for the decision whether the article to be examined complies with the clarity requirement stated in the Purity.

As a rule, the visual method is specified for the requirement in individual monograph.

濁度測(cè)量用于確定濁度（乳光度），以決定待檢查的物品是否符合純度中規(guī)定的透明度要求。

作為一項(xiàng)規(guī)則，目視法是針對(duì)個(gè)別專(zhuān)論中的要求說(shuō)明的。

1. Visual method目視法

This is used to determine the degree of opalescence with white (or faintly-colored) fine particles. So the degree of opalescence of a colored sample is liable to be determined lower that it is difficult to compare the degree correctly without using similarly colored reference suspension.

這是用來(lái)確定乳白色(或淡色)細(xì)顆粒的乳光程度。因此，有色樣品的乳光度容易被測(cè)定得較低，因此，如果不使用類(lèi)似顏色的參考懸浮液，就很難正確地比較其乳光度。

1.1. Reference suspensions參考懸浮液

Pipet 5 mL, 10 mL, 30 mL and 50 mL of formazin opalescence standard solution, dilute them separately to exactly 100 mL with water, and use these solutions so obtained as Reference suspensions I, II, III and IV, respectively. Shake before use. Degrees of opalescence of Reference suspensions I, II, III and IV are equivalent to 3 NTU, 6 NTU, 18 NTU and 30 NTU, respectively.

用移液管分別吸取5 mL、10 mL、30 mL、50 mL福爾馬肼標(biāo)準(zhǔn)液，用水分別稀釋至100 mL，分別作為參比懸液I、II、III、IV。在使用前搖晃。參考懸浮液I、II、III和IV的乳光度分別相當(dāng)于3 NTU、6 NTU、18 NTU和30 NTU。

1.2. Procedure步驟

Place sufficient of the test solution, water or the solvent to prepare the test solution and, where necessary, newly prepared Reference suspensions in separate flat-bottomed test tubes, 15 – 25 mm in inside diameter and of colorless and transparent, to a depth of 40 mm, and compare the contents of the tubes against a black background by viewing in diffused light down the vertical axes of the tubes. The diffused light must be such that Reference suspension I can be readily distinguished from water, and that Reference suspension II can readily be distinguished from Reference suspension I.

取足夠的待測(cè)溶液、水或溶劑，以準(zhǔn)備測(cè)試溶液，必要時(shí)，將新制備的參考懸浮液置于獨(dú)立的平底試管中，試管內(nèi)徑15 - 25mm，無(wú)色透明，深度40 mm。然后在一個(gè)黑色的背景下通過(guò)漫射光下垂直于管軸進(jìn)行觀察，比較管內(nèi)的內(nèi)容。漫射光必須能使參考懸浮體I容易與水區(qū)分開(kāi)來(lái)，參考懸浮體II容易與參考懸浮體I區(qū)分開(kāi)來(lái)。

In this test Reference suspensions are used when the clarity of the test solution is obscurely and it is not easy to determine that its degree of opalescence is similar or not similar to water or to the solvent used to prepare the test solution.

在此測(cè)試中，當(dāng)測(cè)試溶液的透明度模糊不清，并且不容易確定其乳光度與水或與用于制備測(cè)試溶液的溶劑是否相似時(shí)，使用參考懸浮液。

1.3. Interpretation注釋

A liquid is considered “clear” when its clarity is the same as that of water or of the solvent used to prepare the liquid or its turbidity is not more pronounced than that of Reference suspension I. If the turbidity of the liquid is more than that of Reference suspension I, consider as follows: When the turbidity is more than that of Reference suspension I but not more than that of Reference suspension II, express “it is not more than Reference suspension II”. In the same way, when the turbidity is more than that of Reference suspension II but not more than that of Reference suspension III, express “it is not more than Reference suspension III”, and when the turbidity is more than that of Reference suspension III but not more than that of Reference suspension IV, express “it is not more than Reference suspension IV”. When the turbidity is more than that of Reference suspension IV, express “it is more than Reference suspension IV”.

當(dāng)液體的澄清度與水或與用于制備液體的溶劑的澄清度相同或其濁度不比參比懸浮液 I 更明顯時(shí)，該液體被視為“澄清”。如果液體的濁度大于參考懸浮液I，考慮如下：當(dāng)濁度大于參考懸浮液I但不超過(guò)參考懸浮液II時(shí)，表示“不超過(guò)參考懸浮液II”。 同理，當(dāng)濁度大于參比懸濁液Ⅱ但不大于參比懸濁液Ⅲ時(shí)，表示“不大于參比懸濁液Ⅲ”，當(dāng)濁度大于參比懸濁液Ⅲ時(shí) 但不超過(guò)參考懸浮液IV，表示“不超過(guò)參考懸浮液IV”。 當(dāng)濁度大于參考懸浮液IV時(shí)，表示“大于參考懸浮液IV”。

1.4. Reagent solutions試劑溶液

Formazin opalescence standard solution: To exactly 3 mL of formazin stock suspension add water to make exactly 200 mL. Use within 24 hours after preparation. Shake thoroughly before use. Degrees of opalescence of this standard solution is equivalent to 60 NTU.

福爾馬津乳光標(biāo)準(zhǔn)溶液：準(zhǔn)確地取 3 mL福爾馬肼儲(chǔ)備懸浮液，加水至 200 mL。 配制后24小時(shí)內(nèi)使用。使用前*搖勻。此標(biāo)準(zhǔn)溶液的乳光度相當(dāng)于 60 NTU。

2. Photoelectric photometry光電光度法

The turbidity can also be estimated by instrumental measurement of the light absorbed or scattered on account of submicroscopic optical density inhomogeneities of opalescent solutions and suspensions. The photoelectric photometry is able to provide more objective determination than the visual method. Though they can determine the turbidity by measuring the scattered or transmitted light, the measuring system and light source must be specified in individual test method, and for the comparison of observed data, the same measuring system and light source should be used.

由于乳光溶液和懸浮液的亞顯微光密度不均勻性，還可以通過(guò)儀器測(cè)量吸收或散射的光來(lái)估計(jì)濁度。光電光度法比目測(cè)法能夠提供更客觀的測(cè)定。 雖然他們可以通過(guò)測(cè)量散射光或透射光來(lái)確定濁度，但必須在單獨(dú)的測(cè)試方法中標(biāo)明測(cè)量系統(tǒng)和光源，并且為了比較觀察數(shù)據(jù)，應(yīng)使用相同的測(cè)量系統(tǒng)和光源。

In each case, the linear relationship between turbidity and concentration must be demonstrated by constructing a calibration curve using at least 4 concentrations. For colored samples, the turbidity value is liable to be estimated lower because of attenuating both incident and scattered lights due to the absorption by the color, and the transmission-dispersion method is principally used.

在每種情況下，濁度和濃度之間的線(xiàn)性關(guān)系必須通過(guò)使用至少4種濃度構(gòu)建校準(zhǔn)曲線(xiàn)來(lái)證明。對(duì)于有顏色的樣品，由于顏色的吸收，入射光和散射光都被衰減，濁度值容易被估計(jì)得較低，主要采用透射-色散法。

2.1. Turbidimetry透射光比濁法

When a light passes through a turbid liquid the transmitted light is decreased by scattering with the particles dispersed in the liquid. A linear relationship is observed between turbidity and concentration when the particles with a constant size are uniformly dispersed, the size is small and the suspension is not higher concentration. The turbidity can be measured by Ultraviolet-visual Spectrophotometry <2.24> using spectrophotometer or photoelectric photometer. The turbidity of the sample in higher concentration can also be measured, however, it is susceptible to the color of the sample, and the measurement is usually performed at around 660 nm to avoid possible disturbance occurred from the absorption by the color.

當(dāng)光通過(guò)混濁液體時(shí)，透射光通過(guò)分散在液體中的顆粒散射而減少。當(dāng)粒徑恒定的顆粒分散均勻、粒徑較小且懸浮液濃度不高時(shí)，濁度與濃度呈線(xiàn)性關(guān)系。濁度可以通過(guò)紫外分光光度法<2.24>使用分光光度計(jì)或光電光度計(jì)進(jìn)行測(cè)量。較高濃度的樣品的濁度也可以測(cè)量，但它易受樣品顏色的影響，通常在660 nm左右進(jìn)行測(cè)量，以避免顏色吸收可能產(chǎn)生的干擾。

2.2. Nephelometry散射光濁度法

When a suspension is viewed at right angles to the direction of the incident light, it appears opalescent due to the refraction of light from the particles of the suspension (Tyndall effect). A certain portion of the light entering a turbid liquid is transmitted, another portion is absorbed and the remaining portion is scattered by the suspended particles. The scattered light measuring method shows the linear relationship between the nephelometric turbidity units (NTU) values and relative detector signals in a low turbidity range. As the degree of turbidity increases, not all the particles are exposed to the incident light and the scattered radiation of other particles is hindered on its way to the detector.

當(dāng)懸浮物與入射光方向成直角時(shí)，由于懸浮物粒子的光線(xiàn)折射(丁達(dá)爾效應(yīng))，懸浮物呈現(xiàn)乳白色。進(jìn)入混濁液體的光，一部分被透射，一部分被吸收，剩下的部分被懸浮的粒子散射。散射光測(cè)量方法顯示了低濁度范圍內(nèi)散射濁度單位(NTU)值與相對(duì)檢測(cè)器信號(hào)之間的線(xiàn)性關(guān)系。隨著濁度的增加，并不是所有的粒子都暴露在入射光下，其他粒子的散射輻射在到達(dá)探測(cè)器的過(guò)程中會(huì)受到阻礙。

2.3. Ratio Turbidimetry比率濁度法

This method measures both scattered and transmitted light values at the same time, and the turbidity is determined from the ratio of the scattered light value to the transmitted light value. This procedure compensates for the light that is diminished by the color of the sample and eliminates the influence of the color. When the measurement is performed by using an integrating sphere, it is particularly called the integrating sphere method, which measures the total transmitted light value as well as the scattered light value occurred with the suspended particles, and the turbidity can be determined from the ratio of them.

該方法同時(shí)測(cè)量散射光值和透射光值，濁度由散射光值與透射光值的比值確定。此程序可補(bǔ)償因樣品顏色而減弱的光線(xiàn)，并消除顏色的影響。當(dāng)用積分球進(jìn)行測(cè)量時(shí)，特別稱(chēng)為積分球法，它測(cè)量懸浮粒子的總透射光值和散射光值，由它們的比值可以確定濁度。

2.4. Application of photoelectric photometry for monograph requirements

光電光度法在專(zhuān)著要求中的應(yīng)用

The turbidity of the test solution, determined by the photoelectric photometry, can be used as an indicating standard for the conformity to the clarity requirements by converting into NTU by using turbidity known reference solutions such as Reference suspensions I – IV, if needed, and water or the solvent used. In an automatically compensable apparatus being calibrated with turbidity known reference solutions, the measuring result is given in NTU and it can be compared directly with required specified value.

由光電光度法測(cè)定的測(cè)試溶液的濁度，可以作為指示標(biāo)準(zhǔn)，通過(guò)使用濁度已知的參考溶液，如參考懸浮液 I-IV，如果需要，水和使用的溶劑液也可以，將其以NTU為單位的數(shù)據(jù)轉(zhuǎn)出。在使用濁度已知參考溶液校準(zhǔn)的自動(dòng)補(bǔ)償裝置中，測(cè)量結(jié)果以 NTU 為單位給出，并且可以直接與所需的規(guī)定值進(jìn)行比較。

NTU is often used as the unit in the turbidity determinations. It is the unit used in the case when the turbidity is estimated by the instrument which measures the 90 ± 30°scattered light against the incident light intensity, using tungsten lamp, and in the case the estimation is performed by the instrument which measures the 90 ± 2.5°scattered light against the incident light intensity using 860 nm infrared light, FNU is used as the unit. FNU is equivalent to NTU at a range of smaller measurements (less than 40 NTU). For the unit of formazin concentration, FTU is also used, which is defined as a suspension of 1 mg formazin in 1L of purified water is 1 FTU.

在濁度測(cè)定中經(jīng)常使用NTU作為單位。它是測(cè)量用90±30°散射光對(duì)入射光強(qiáng)度的得到濁度信息時(shí)使用的單位，使用鎢燈，采用860 nm紅外光測(cè)量90±2.5°的散射光對(duì)入射光強(qiáng)度，此時(shí)以FNU為單元。在較小的測(cè)量范圍內(nèi)(小于40 NTU)，FNU相當(dāng)于NTU。福爾馬肼的濃度單位也用FTU，即1L純凈水中1 mg 福爾馬肼的懸浮液為1 FTU。

Formazin stock suspension. To 25 mL of hexamethylenetetramine TS add 25 mL of hydrazinium sulfate TS, mix, and use after allowing to stand at room temperature for 24 hours. Store in a glass container free from surface defects. Use within 2 months. Shake thoroughly before use. The turbidity of this suspension is equivalent to 4000 NTU.

福爾馬肼貯備懸浮液：向 25 mL六亞甲基四胺中加入25 mL硫酸肼，混勻，室溫靜置 24 小時(shí)后使用。 儲(chǔ)存在沒(méi)有表面缺陷的玻璃容器中。2個(gè)月內(nèi)使用。使用前*搖勻。這種懸浮液的濁度相當(dāng)于4000 NTU。

Formazin opalescence standard solution. To 15 mL of formazin stock suspension add water to make 1000 mL. Use within 24 hours after preparation. Shake thoroughly before use.

福爾馬肼標(biāo)準(zhǔn)液：向 15 mL 的福爾馬肼貯備懸浮液中加水至 1000 mL。配制后24小時(shí)內(nèi)使用。使用前*搖勻。

解決方案：

上海胤煌科技針對(duì)藥劑的澄清度檢查推出了以下產(chǎn)品，符合各國(guó)藥典的溶液澄清度檢查規(guī)范。

1、澄清度檢查專(zhuān)用傘棚燈

HN-100A型和HN-200A型澄清度檢查專(zhuān)用傘棚燈符合各國(guó)藥典中目視法檢測(cè)溶液澄清度的儀器要求，具有光林帶型光源，能有效減少目視過(guò)程中光對(duì)眼睛的刺激，其照度可達(dá)5000 Lux。其中HN-200A型專(zhuān)用傘棚燈增加了RGB三色光源，可以對(duì)有色樣品進(jìn)行澄清度檢測(cè)。

2、YH-CLS-1201澄清度檢查分析儀

此儀器采用全彩液晶觸摸屏進(jìn)行操作控制，可以直接檢測(cè)注射用原料藥和注射劑的澄清度，并具備四級(jí)權(quán)限管理和審計(jì)追蹤功能，*GMP的數(shù)據(jù)完整性要求，是液體一致性評(píng)價(jià)的有效儀器。