2022-04-28 07:33:18, 胤煌科技 上海胤煌科技有限公司
中国药典2020
0902 澄清度检查法
澄清度检查法系将药品溶液与规定的浊度标准液相比较,用以检查溶液的澄清度。除另有规定外,应采用第一法进行检测。
品种项下规定的“澄清”,系指供试品溶液的澄清度与所用溶剂相同,或不超过0.5号浊度标准。“几乎澄清”,系指供试品溶液的浊度介于0.5号至1号浊度标准液的浊度之间。
第一法(目视法)
除另有规定外,按各品种项下规定的浓度要求,在室温条件下将用水稀释至一定浓度的供试品溶液与等量的浊度标准液分别置于配对的比浊用玻璃管(内径15-16 mm,平底,具塞,以无色、透明、中性硬质玻璃制成)中,在浊度标准液制备5 分钟后,在暗室内垂直置于伞棚灯下,照度为1000 lx,从水平方向观察、比较。除另有规定外外,供试品溶解后应立即检视。
第一法无法准确判定两者的澄清度差异时,改用第二法进行测定,并以其测定结果进行判定。
浊度标准存贮液的制备 称取于105℃干燥至恒重的硫酸肼1.00 g,置于100 ml量瓶中,加水适量使溶解,必要时可在40℃的水浴中温热溶解,并用水稀释至刻度,摇匀,放置4-6小时;取此溶液于等容量的10%乌洛托品溶液混合,摇匀,于25℃避光静置24小时,即得。该溶液置冷处避光保存,可在2个月内使用,用前摇匀。
浊度标准原液的制备 取浊度标准贮备液15.0 ml,置1000 ml量瓶中,加水稀释至刻度,摇匀,取适量,置1 cm吸收池中,照紫外-可见分光光度法(通则0401),在550 nm的波长处测定,其吸光度在0.12-0.15范围内,该溶液应在48小时内使用,用前摇匀。
浊度标准液制备 取浊度标准原液与水,按照下表配置,即得。浊度标准液应临用时制备,使用前充分摇匀。
第二法(浊度仪法)
供试品的浊度可采用浊度仪测定。溶液中不同大小、不同特性的微粒物质包括有色物质均可使入射光产生散射,通过测定透射光或者散射光的强度,可以检查供试品的浊度。仪器测定模式通常有三种类型,透射光式、散射光式和透射光-散射光比较测量模式(比率浊度模式)。
1.仪器的一般要求
采用散射光式浊度仪时,光源峰值波长为860 nm;测量范围应包含0.01-100NTU。在0-10NTU范围内分辨率应为0.01NTU;在10-100NTU范围内分辨率应为0.1NTU.
2.适用范围及检测原理
本法采用散射光式浊度仪,适用于低、中浊度无色供试品溶液的浊度测定(浊度值为100NTU以下的供试品。)因为高浊度的供试品会造成多次散射现象,时散射光强度迅速下降,导致散射光强度不能正确反映供试品的浊度值。0.5-4号浊度标准液的浊度值范围约为0-40NTU。
采用散射光式浊度仪测定时,入射光和测定的散射光呈90℃夹角,入射光强度和散射光强度关系式如下。
I=K’T I0
式中 I为散射光强度,单位为cd;
I0 为入射光强度,单位为cd;
K’为散射系数;
T为供试品溶液的浊度值,单位为NTU(NTU是基于福尔马肼浊度标准液液测定的散射浊度单位,福尔马肼浊度标准液即为第一法中的浊度标准贮备液)。
在入射光I0不变的情况下,散射光强度I与浊度值成正比。因此,可以将浊度测量转化为散射光强度的测量。
3.系统的适用性试验
仪器应定期(一般每月一次)对浊度标准液的线性和重复性进行考察,采用0.5号至4号浊度标准液进行浊度值测定,浊度标准液的测定解果(单位NTU)与浓度间应呈线性关系,线性方程的相关系数应不低于0.999;取0.5号至4号浊度标准液,重复测定5次,0.5号和1号浊度标准液测量浊度值的相对标准偏差应不大于5%,2-4号浊度标准液测量浊度值的相对标准偏差不大于2%。
4.测定法
按照仪器说明书要求并采用规定的浊度液进行仪器校正。溶液剂直接取样测定;原料药或者其它剂型按照个论项下的标准规定制备供试品溶液,临用时制备。分别取供试品溶液和相应浊度标准液进行测定,测定前应摇匀,并避免产生气泡,读取浊度值。供试品溶液浊度值不得大于相应浊度标准液的浊度值。
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美国药典USP44
<630> VISUAL COMPARISON 视觉比较
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.
本试验的目的是提供特定浓度的样品溶液与已知浓度的标准溶液或一系列标准溶液的颜色和/或浊度的视觉比较细节。如果需要进行颜色或浊度比较,请遵循以下程序和条件进行这些测试。
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.
散射光浊度法和透射光比浊法是基于光散射现象原理的分析技术。光散射是一种物理现象,其中光束由于与足够小的物质粒子相互作用而改变其传播方向(称为偏转)。根据麦克斯韦电磁理论,散射发生的先决条件是悬浮颗粒的折射率必须不同于悬浮液体的折射率。差异越大,散射越强烈。光散射有两种类型:1)弹性散射,其中散射光和入射光的波长相同;2)非弹性光散射,其中散射光和入射光的波长不同。只有第一种光散射(弹性)与散射光浊度法和透射光比浊法有关。
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.
在透射光比浊法中,测量透射光的强度,并在入射光方向(即0°)测量散射导致的入射光强度的衰减,并与入射光强度进行比较(空白测量)。被测特性是悬浮颗粒散射效应的间接测量,称为浊度。悬浮样品对光的任何吸收都会导致光强度的额外衰减(参见<857> Ultraviolet-Visible Spectroscopy和<1857> Ultraviolet-Visible Spectroscopy—Theory and Practice)。因此,确保被测材料不会吸收测量波长处的光非常重要。实际上,控制吸收和浊度测定的方程式是相同的(尽管衰减常数的值不同)。在散射光浊度法中,测量与入射光传播方向成90°角的散射光强度。因此,散射光浊度法浊度测量是对悬浮物散射效应的直接测量。
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.
透射光比浊法和散射光浊度法技术的应用包括1)溶液和/或悬浮液的浓度测定(通过在控制良好的反应参数下沉淀和悬浮产生的沉淀物,来测定几种阳离子和阴离子);2)测量混浊溶液或悬浮液的浊度;3)测定分子量在1000到数亿之间的多分散体系的重均分子量和尺寸;4)测量免疫分析的反应动力学或免疫沉淀动力学(比率散射浊度法);5)监测细胞和细菌的生长;6)悬浮物粒度分布测定、颗粒计数等。
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.
比率散射浊度法广泛用于疫苗成分分析和/或血清成分的定量。它还用于重组生物制药中的宿主细胞蛋白质鉴定。当使用该技术时,通过测量抗原-抗血清或抗原纯化抗体复合物的光散射反应的变化,来计算导致免疫抗体-抗原沉淀反应或凝集反应的抗原(Ag)或抗体(Ab)的量。通常考虑抗原共价连接或吸附在聚合物微球上,以提高散射效率;由此产生的技术被称为“颗粒增强免疫分析”。虽然这项技术被称为散射光浊度法,但通常散射光和透射光都是用比率仪器测量的。
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.
散射光法浊度测量在低浊度范围(散射介质浓度相对较低)更可靠。在该范围内,观察到样品浓度与检测器信号强度(以NTU表示)之间存在线性关系。随着浓度的增加,多次散射的入射角也会增加,从而偏离线性响应。支持可靠线性关系的最大NTU值在1750–2000 NTU范围内。透射光比浊法适用于更高的浊度范围(散射介质的浓度)。为了获得一致的结果,必须仔细控制所有测量变量。在可能的情况下,可以测量极稀的悬浮液。
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).
用于透射光比浊法和散射光浊度法测量的仪器分别称为透射光浊度计和散射光浊度计。通常,这些仪器包括一个带有滤光器的汞灯(用于强绿线或蓝线)、一个快门、一组具有已知透射率的中性滤光器和一个灵敏的光电倍增管,该光电倍增管可安装在与入射光传播方向成0°或90°角的位置,或者在一个臂上,它可以围绕溶液单元旋转,并通过不透光外壳外的表盘设置为−135°到0°到+135°的任何角度。溶液池的形状多种多样,例如用于测量90°散射的正方形;45°、90°和135°散射为半八角形;圆柱形可适用于所有角度的散射(见图1)。
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.
浊度也可以用标准光电滤光光度计或分光光度计测量,最好是在光谱的蓝色部分进行照明。散射光浊度法测量需要一个装有光电管的仪器,以便接收散射光,而不是透射光。由于这与荧光计中使用的几何结构相同,因此可通过适当选择滤光片将其用作浊度计。比率浊度计结合了90°散射光浊度法和透射光比浊法。它包含光电管,接收和测量与样品成90°角的散射光,以及接收和测量样品前面的前向散射光。它还测量直接穿过样品的光。通过90°角散射光测量值,前向散射光测量值和透射光测量值之和,计算两者的比值,可获得线性度。使用比率浊度测量系统的好处是杂散光的测量变得可以忽略不计。此外,彩色悬浮液的浊度测定仅使用透射光比浊法浊度仪或浊度仪(带比率模式)进行,因为该程序补偿了悬浮液颜色导致的光衰减。通常,这些仪器中的光源是钨灯,在2700 K的灯丝温度下工作,大部分光强约为550 nm。也可使用其他合适的光源。通常,探测器是▲硅二极管▲和光电倍增管。另一种消除颜色效应的方法是使用红外发光二极管作为光源,其最大发射中心约为860 nm,光谱带宽为60 nm。当激光光源也被使用时,尤其是在浊度测量仪器中,这种技术通常被称为“激光浊度测量”。使用激光散射光浊度计的优点是,在非常低的检测水平下,信噪比显著提高。通常光源是工作波长为660 nm的激光二极管。激光束的高功率密度使较小粒子产生更高的散射强度。与吸收未散射光的光阱相结合,该系统可显著降低杂散光。当专著中的某个程序指示使用散射光浊度计或透射光浊度计时,可以使用在比率模式下工作的仪器。
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.
福尔马肼是唯一已知的主要浊度标准。所有其他标准都是次要的,必须追溯到福尔马肼。主要标准在▲IUPAC Compendium of Chemical Terminology▲(ERR 1-May-2019)第 2 版(金书)中被定义为由用户使用明确定义的方法和条件从可追溯的材料准备的标准。
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.
福尔马肼悬浮液有许多特点,以确保其适合作为主要标准。它可以从试剂级的起始材料中始终如一、准确地制备。该悬浮液由不同长度和随机构型的聚合物组成,其组成的聚合物的形状和尺寸从小于0.1 μm到大于10 μm不等。尽管聚合物链长分布已被证明因制备而异,但总的浊度结果是可以很好地重现的。
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>).
特定仪器对给定程序的适用性由从选择到仪器报废的预期应用的逐步生命周期评估来确保。鉴定包括三个部分:1)安装鉴定(IQ)、2)操作鉴定(OQ)和3)性能鉴定(PQ)(参见<1058>Analytical Instrument Qualification章节)。
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.
本节的目的是提供测试方法和验收标准,以确保仪器适合其预期用途(OQ),并在延长的时间段(PQ)内继续正常工作。与任何光谱仪一样,透射光式和散射光式浊度光谱仪必须具备波长(x轴,如果不固定)和光度(y轴或信号轴)的准确度及精度,并满足杂散光的要求。OQ是在实验室内吸光度和波长标度所需的操作范围内进行的。
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期间,验证确定“用途适用性”的关键仪器参数的验收标准。特定仪器和应用的规格可能因使用的分析程序和最终结果的预期准确度而异。仪器供应商通常将样品和测试参数作为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.
在以下详述的程序中,应尽可能使用主要参考标准或认证参考材料(CRM)。福尔马肼是比浊法法和浊度法中唯一使用的主要参考标准。所有其他标准,包括CRM,必须与福尔马肼相关。CRM应从认可的认证来源获得,并包括独立验证的可追溯值分配及相关的计算不确定性。CRM必须保持清洁,无灰尘。应定期进行重新认证,以保持认证的有效性。
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.
所有透射光式浊度仪和散射光式浊度仪均根据已知浊度的标准进行校准。在首次使用之前,必须使用福尔马肼浊度标准液对仪器进行校准,至少每3个月或按照供应商的规定进行一次校准。使用至少四种福尔马肼浊度标准液进行校准,其浊度按比例覆盖感兴趣的范围。许多仪器制造商提供校准验证标准。它们通常由其中充满聚合物凝胶中的金属氧化物颗粒的密封样品池或乳胶悬浮液组成。这些标准只能用于检查仪器推荐校准的时间间隔内的校准。
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.
杂散光(杂散光辐射能)是一个非常重要的误差源,特别是在较低的浊度读数范围内的测量。它被定义为到达探测器而不被样品散射的外部光线。杂散光有几种来源,包括电池表面固有缺陷、电池内部未被解释的反射、光学系统部件、光源,以及在较小程度上的电子波动。尽管仪器供应商使用了许多设计功能来最小化杂散光,但无法完全缓解杂散光。与分光光度测量不同,浊度法无法补偿杂散光。必须测量杂散光,其值应在特定仪器供应商设定的规格范围内,或在0-10 NTU范围内测量时小于0.15 NTU,在10-1100 NTU范围内测量时小于0.5 NTU,以较小者为准。
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.
仪器必须能够测量0.01–1100 NTU范围内或目标浊度50%-200%范围内的浊度。为了证明预期测量范围的线性,从表1中选择至少四种合适的参考悬浮液。
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.
对于0-9.99 NTU的测量范围,仪器分辨率必须小于等于0.01 NTU;测量范围为10-99.9 NTU时,小于等于0.1 NTU;100 NTU以上的测量分辨率值为1 NTU。
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.
对于0-19.9 NTU的测量范围,仪器读数准确度必须为读数+0.01 NTU的±10%,对于20-1100 NTU的测量范围,仪器读数准确度必须为读数的±7.5%。
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.
定期或根据需要在校准期之间完成仪器PQ。可使用仪器制造商提供的一级浊度标准(福尔马肼)或二级校准验证标准(乳胶悬浮液或密封样品池中聚合物凝胶中的金属氧化物颗粒)。
SAMPLE CELL PREPARATION 样品池准备
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).
用于样品测量的样品室必须清洁。按照样品池或仪器制造商的建议适当清洁样品池。对于低浊度测量,最好使用一个单指数样品池或流动池,这有助于确保测量的足够精度和可重复性。使用无颗粒水,在样品池支架中找到读数最低的样品池方向。对于较高的浊度值,可使用不同的样品池。然而,样品池必须匹配(两个不同样品池在标称样品浓度下制备的标准品读数差异必须在±0.005 NTU范围内或低于测量精度要求,以较低者为准)。
SAMPLE PREPARATION样品准备
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.
按照各专题中的规定制备样品。通过缓慢旋转或倒置容量瓶数次,仔细混合样品。避免摇晃或搅拌,因为这可能会产生气泡。对样品进行脱气有助于改进测量。对于脱气,样品可以静置几分钟,或者可以施加真空,或者可以使用超声波浴对其进行轻轻的超声波处理。脱气后,让样品静置几分钟,然后小心地反转两到三次,再次混合。将样品转移至样品池并读取读数。
USE OF FLOW CELLS流动池的使用
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.
流动池主要用于小颗粒样品的低浊度测量。当使用这种样品池时,通过小心地将样品倒入进水仓的内边缘来引入样品。
在实际过程中,建议确保被测颗粒的沉降可以忽略不计。这通常通过在液体悬浮介质中加入保护胶体来实现。重要的是,通过将读数与在完全相同的条件下产生的已知悬浮物浓度的读数进行比较来解释结果。
Nephelometric procedures are performed similarly to turbidimetric procedures for both direct measurements and measurements in the ratio mode as described above.
散射光浊度法步骤的执行方式与透射光比浊法程序类似,适用于直接测量和上述比率模式下的测量。
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.
监测反应进程的总体程序包括三个明确定义的步骤:1)记录介质浊度的基线读数(空白);2) 在添加第一种试剂(抗原)后,记录浊度,这会导致浊度增加,直到达到一个稳定期;3)添加第二种试剂(抗体),这会导致另一个浊度增加和第二个稳定期,然后是最终浊度增加,直到达到第三个稳定器。根据分析目的和各自的组分浓度,从添加抗体到第三个稳定期中间选择测量区。动力学散射比浊法和终点散射比浊法是两种通用程序,用于量化免疫分析方法中形成的免疫复合物(也称为免疫散射比浊法,因为测得的浊度是由形成的免疫复合物引起的)。对于每一个步骤,都有几个参数需要在每个单独的应用中进行优化。主要参数为1)有无粒子增强;2) 颗粒类型、尺寸和各自的最佳波长(如适用);3) 监测反应动力学或终点;4) 考虑中的抗体/抗原,以及与之相关的抗原负载的最佳水平;5) 缓冲液和其他离子种类以及各自的最佳pH值;6) 用于改变蛋白质溶解度的聚合物的类型和浓度;7)温度和其他环境因素。通常,这些参数在方法开发过程中进行了优化,具体的专著和/或章节(如适用)中给出了这些值。
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.
动力学散射比浊法:与终点散射比浊法相比,动力学散射比浊法具有优势,主要是因为除了试剂空白读数外,还能够读取样品空白读数。该程序基于所选波长的散射光的增强强度响应来评估免疫复合物的形成速率。根据所用仪器的时间响应和应用类型,可连续监测反应动力学,或采集一定数量的数据点。有时它可能只涉及两个数据点;但是在样品和校准标准之间存在反应动力学差异的情况下,选择点可能会影响整体准确度。应仔细考虑特异性控制策略的适当选择。
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.
终点散射比浊法:在该方法中,在添加试剂之前进行初始测量,这代表空白读数。大约60分钟后,在免疫复合物形成后进行第二次测量。这两次测量之间的差异与所分析成分的含量成正比。
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.
当散射比浊法/透射浊度法拟用作官方物品测试程序的替代方法时,需要进行验证。当散射比浊法/透射浊度法验证的目的是证明测量适用于其预期目的,包括原料药或药品中主要成分的定量测定(I类分析)、杂质的定量测定或限度试验(II类)以及鉴定试验(IV类)。根据试验的类别(参见<1225>Validation of Compendial Procedures,表2),透射浊度法/散射比浊法的分析方法验证过程需要对准确度、精密度、特异性、检测限(DL)、定量限(QL)、线性、范围和稳健性进行试验。这些分析性能特征适用于外部标准化程序和那些使用标准添加的程序。
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章节提供了分析程序验证的定义和一般指南,但没有说明每个特征的具体验证标准。以下各节的目的是向用户提供具体的验证标准,这些标准代表了对该技术的最低期望。对于每个特定应用,可能需要更严格的标准,以证明其适用于预期用途。
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.
定量限QL可以通过计算溶液的浓度来估算,该浓度将给出信号的信噪比≥10.0. 必须通过分析计算浓度下的样品来确认估算的QL。必须对以所需QL浓度添加的代表性样品基质制备的试液进行测量,以确认其具有足够的灵敏度和精度。在所需QL下观察到的信噪比应大于10。
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.
分析物浓度和测得的浊度响应之间的线性关系必须通过制备至少四种标准溶液来证明,其浓度包括试验溶液的预期浓度。然后使用适当的统计方法(如最小二乘回归)评估标准曲线。通过降低或增加分析物浓度,可将仪器或样品因素或两者的线性偏差降低至可接受水平,从而分别将浊度读数降低或增加至透射光法浊度计/散射光浊度计仪器线性范围内。
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.
分析仪器(以及整个分析程序)的操作范围是样品中分析物的上下浓度(数量)(包括这些浓度)之间的间隔,已证明仪器响应函数具有适当的精度、准确度和线性水平。
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.
分析测量的可靠性通过有意改变实验参数来证明。对于散射光浊度法/透射光比浊法,这可以包括:测量分析物在特定储存条件、变化的 pH 值和添加可能的干扰物质下的稳定性。使用适合实验程序的设计,同时确保稳健性。
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.
现行的《美国生产规范条例》[21 CFR 211.194(a)(2)]表明,如果专论中提供了这些程序,则美国药典和国家处方集中描述的分析程序的用户无需验证这些程序。相反,他们只需验证其在实际使用条件下的适用性。
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.
散射光浊度法/透射光比浊法程序验证的目的是证明测试程序在实际使用条件下的适用性。验证散射光浊度法/透射光比浊法程序适用性的性能特征与任何分析程序所需的性能特征相似。适用的一般原则的讨论见<1226>Verification of Compendial Procedure 章节。通常使用参考材料和明确定义的基质进行验证。药典散射光浊度法/透射光比浊法程序的验证至少包括对特异性、准确度、精密度和 QL 的验证参数的执行(如 8.1 验证中所述)。
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欧洲药典EP10.0
2.2.1. CLARITY AND DEGREE OF OPALESCENCE OF LIQUIDS 液体的澄清度和乳光度
VISUAL METHOD目视法
INSTRUMENTAL METHOD 仪器法
TURBIDIMETRY AND NEPHELOMETRY透射光比浊法和散射光浊度法
MEASUREMENTS IN RATIO MODE比率模式下的测量
INSTRUMENT REQUIREMENTS仪器要求
– 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.
– 测量单位:NTU(浊度测量单位)。NTU是基于福尔马肼一级标准品的浊度。也可使用FTU(福尔马肼浊度单位)或FNU(福尔马肼浊度单位),相当于NTU的在低浊度区域(最高40 NTU)。这些单位适用于所有3种仪器方法(比浊法、浊度法和比率模式)。
–测量范围:0.01-1100 NTU
–分辨率:0-9.99 NTU范围内为0.01 NTU;10.0-99.9 NTU范围内为0.1 NTU;对于大于100 NTU的范围,则为1 NTU
–准确度:范围在0-20 NTU之间,读数准确度偏差为±(读数的10%+0.01 NTU);范围在20-1100 NTU时,读数准确偏差为±7.5%。
–重复性:在0-20 NTU范围内重复性为±0.05 NTU;在20-1100 NTU范围内读数重复性为±2%。
CONTROL OF INSTRUMENT PERFORMANCE仪器性能的控制
– 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.
–校准:使用至少4种福尔马肼参考悬浮液进行校准,覆盖感兴趣的测量范围。可使用本章所述的参考悬浮液或根据主要参考悬浮液校准的适当参考标准。
– 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.
杂散光:在0-10NTU范围内<0.15 NTU;在10-1100 NTU范围内<0.5 NTU。杂散光是指到达浊度检测器的光,而不是样品散射的结果。杂散光总是一种正干扰,是低范围浊度测量中的一个重要误差源。杂散光的来源包括:样品池中的缺陷和划痕、光学系统的内部反射、光学元件或样品池被灰尘污染,以及电子噪声。仪器设计也会影响杂散光。在比率模式测量中,杂散光的影响可以忽略不计。
REFERENCE SUSPENSIONS 参考悬浮液
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,参考悬浮液I-IV的测量结果显示,浓度与测量的NTU值之间存在线性关系(见表2.2.1.-2)。
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日本药典JP17
2.61 Turbidity Measurement 浊度测量
1. Visual method目视法
1.1. Reference suspensions参考悬浮液
1.2. Procedure步骤
1.3. Interpretation注释
1.4. Reagent solutions试剂溶液
2. Photoelectric photometry光电光度法
2.1. Turbidimetry透射光比浊法
2.2. Nephelometry散射光浊度法
2.3. Ratio Turbidimetry比率浊度法
2.4. Application of photoelectric photometry for monograph requirements
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.
福尔马肼贮备悬浮液:向 25 mL六亚甲基四胺中加入25 mL硫酸肼,混匀,室温静置 24 小时后使用。储存在没有表面缺陷的玻璃容器中。2个月内使用。使用前彻底摇匀。这种悬浮液的浊度相当于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.
福尔马肼标准液:向 15 mL 的福尔马肼贮备悬浮液中加水至 1000 mL。配制后24小时内使用。使用前彻底摇匀。
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解决方案:
1、澄清度检查专用伞棚灯
2、YH-CLS-1201澄清度检查分析仪
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