主题:【第三届原创参赛】已完结Guidance for Industry Bioanalytical Method Validation尝试翻译

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B.  Accuracy, Precision, and Recovery 准确度,精密度和回收率
The accuracy of an analytical method describes the closeness of mean test results obtained by the method to the true value (concentration) of the analyte.  Accuracy is determined by replicate analysis of samples containing known amounts of the analyte.  Accuracy should be measured using a minimum of five determinations per concentration.  A minimum of three concentrations in the range of expected concentrations is recommended.  The mean value should be within 15% of the actual value except at LLOQ, where it should not deviate by more than 20%.  The deviation of the mean from the true value serves as the measure of accuracy.
分析方法的准确度描述了由风发获得的测试结果平均值与被测物真值(浓度)的接近程度。准确度由重复分析含有已知量的被分析物的样品来确定。准确度应使至少用每个浓度测定5个数据的样品来确定,预期浓度范围内推荐至少3个浓度测定。平均值除LLOQ外,应是真值的±15%,LLOQ处应不得偏离20%。平均值与真值的偏离就是准确度的测定。
The precision of an analytical method describes the closeness of individual measures of an analyte when the procedure is applied repeatedly to multiple aliquots of a single homogeneous volume of biological matrix.  Precision should be measured using a minimum of five determinations per concentration.  A minimum of three concentrations in the range of expected concentrations is recommended.  The precision determined at each concentration level should not exceed 15% of the coefficient of variation (CV) except for the LLOQ, where it should not exceed 20% of the CV.  Precision is further subdivided into within-run, intra-batch precision or repeatability, which assesses precision during a single analytical run, and between-run, inter-batch precision or repeatability, which measures precision with time, and may involve different analysts, equipment, reagents, and laboratories.
分析方法的精密度描述了重复对一个生物基质的多个单一均匀等分体积进行重复分析时被分析物单次测定的接近程度。精密度应使至少用每个浓度测定5个数据的样品来确定。预期浓度范围内推荐至少3个浓度测定。在每个浓度测定得到的精密度除LLOQ外,其CV值不得超过±15%,LLOQ处CV值不得超过±20%。精密度进一步分成,同批样品同次处理,不同时间处理不同时间分析或重复性,评估精密度从单次分析,中间精密度,重现性,这可能包括时间,不同分析人,设备,试剂和实验室。这段文字的理解建议大家看看(臨床前藥物動力學試驗 林君榮 台大醫學院 臨床藥學研究所 助理教授的这篇文章)
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B.  Accuracy, Precision, and Recovery 准确度,精密度和回收率
The accuracy of an analytical method describes the closeness of mean test results obtained by the method to the true value (concentration) of the analyte.  Accuracy is determined by replicate analysis of samples containing known amounts of the analyte.  Accuracy should be measured using a minimum of five determinations per concentration.  A minimum of three concentrations in the range of expected concentrations is recommended.  The mean value should be within 15% of the actual value except at LLOQ, where it should not deviate by more than 20%.  The deviation of the mean from the true value serves as the measure of accuracy.

分析方法的准确度描述了由风发获得的测试结果平均值与被测物真值(浓度)的接近程度。准确度由重复分析含有已知量的被分析物的样品来确定。准确度应使至少用每个浓度测定5个数据的样品来确定,预期浓度范围内推荐至少3个浓度测定。平均值除LLOQ外,应是真值的±15%,LLOQ处应不得偏离20%。平均值与真值的偏离就是准确度的测定。

The precision of an analytical method describes the closeness of individual measures of an analyte when the procedure is applied repeatedly to multiple aliquots of a single homogeneous volume of biological matrix.  Precision should be measured using a minimum of five determinations per concentration.  A minimum of three concentrations in the range of expected concentrations is recommended.  The precision determined at each concentration level should not exceed 15% of the coefficient of variation (CV) except for the LLOQ, where it should not exceed 20% of the CV.  Precision is further subdivided into within-run, intra-batch precision or repeatability, which assesses precision during a single analytical run, and between-run, inter-batch precision or repeatability, which measures precision with time, and may involve different analysts, equipment, reagents, and laboratories.

分析方法的精密度描述了重复对一个生物基质的多个单一均匀等分体积进行重复分析时被分析物单次测定的接近程度。精密度应使至少用每个浓度测定5个数据的样品来确定。预期浓度范围内推荐至少3个浓度测定。在每个浓度测定得到的精密度除LLOQ外,其CV值不得超过±15%,LLOQ处CV值不得超过±20%。精密度进一步分成,同批样品同次处理,不同时间处理不同时间分析或重复性,评估精密度从单次分析,中间精密度,重现性,这可能包括时间,不同分析人,设备,试剂和实验室。这段文字的理解建议大家看看(臨床前藥物動力學試驗 林君榮 台大醫學院 臨床藥學研究所 助理教授的这篇文章)
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The recovery of an analyte in an assay is the detector response obtained from an amount of the analyte added to and extracted from the biological matrix, compared to the detector response obtained for the true concentration of the pure authentic standard.  Recovery pertains to the extraction efficiency of an analytical method within the limits of variability.  Recovery of the analyte need not be 100%, but the extent of recovery of an analyte and of the internal standard should be consistent, precise, and reproducible.  Recovery experiments should be performed by comparing the analytical results for extracted samples at three concentrations (low, medium, and high) with unextracted standards that represent 100% recovery.
被分析物在分析中的回收率是指从生物基质中萃取出的添加的被分析物的量的检测响应与纯的可靠的标准的实际浓度获得的检测响应的比值。回收率与分析方法可变限度的萃取效率有关。被分析物回收率不一定是100%,但是被分析物和内标的回收程度和应始终如一,精密的病友重现性。回收率试验由三个浓度(低、中、高)萃取样品的分析结果和代表100%回收的未萃取标准比较来进行。
C.  Calibration/Standard Curve 校准/标准曲线
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A calibration (standard) curve is the relationship between instrument response and known concentrations of the analyte.  A calibration curve should be generated for each analyte in the sample.  A sufficient number of standards should be used to adequately define the relationship between concentration and response.  A calibration curve should be prepared in the same biological matrix as the samples in the intended study by spiking the matrix with known concentrations of the analyte.  The number of standards used in constructing a calibration curve will be a function of the anticipated range of analytical values and the nature of the analyte/response relationship.  Concentrations of standards should be chosen on the basis of the concentration range expected in a particular study.  A calibration curve should consist of a blank sample (matrix sample processed without internal standard), a zero sample (matrix sample processed with internal standard), and six to eight non-zero samples covering the expected range, including LLOQ.
校准(标准)曲线是仪器响应和已知被分析物浓度之间的相互关系。校准曲线应由每个样品中的被分析物获得。应使用足够数量的标准来适当定义浓度和响应的相互关系。校准曲线应应使用与预期研究样品相同的生物基质并添加含已知浓度被分析物的基质来制备。构建校准曲线使用的标准数量是预期分析值范围和被分析物类型/响应的相互关系的函数。标准的浓度应基于特定研究的预期浓度范围来选择。校准曲线应由空白(基质样品不添加内部标准品处理),零样品(用内标处理基质样品)和6-8个涵盖预期范围的非零样品(包括LLOQ)组成

        1.      Lower Limit of Quantification (LLOQ) 定量下限
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The lowest standard on the calibration curve should be accepted as the limit of quantification if the following conditions are met:
如果符合以下条件,校准曲线上最低标准应视为定量限
                The analyte response at the LLOQ should be at least 5 times the response compared to blank response.
                Analyte peak (response) should be identifiable, discrete, and reproducible with a precision of 20% and accuracy of 80-120%.
在LLOQ上的被分析物响应至少是空白响应的5倍。
被分析物峰(响应)应可鉴别,完全分离并有重现性(精密度20%和准确度80-120%)
        2.      Calibration Curve/Standard Curve/Concentration-Response 校准曲线/标准曲线/浓度-响应

        The simplest model that adequately describes the concentration-response relationship should be used.  Selection of weighting and use of a complex regression equation should be justified.  The following conditions should be met in developing a calibration curve:
应使用最简单的模型适当地描述浓度-响应相互关系。权重选择和使用复杂的回归方程应合理。在绘制校准曲线时应满足以下条件
                20% deviation of the LLOQ from nominal concentration LLOQ处与标示浓度20%以内的偏离
                15% deviation of standards other than LLOQ from nominal concentration 除LLOQ以外其他点与标示浓度的偏离在15%以内。
        At least four out of six non-zero standards should meet the above criteria, including the LLOQ and the calibration standard at the highest concentration.  Excluding the standards should not change the model used.
6个非0标准中至少有4个符合上述标准,包括LLOQ和最高浓度校准标准。删除标准点应不会改变模型
D.  Stability稳定性
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Drug stability in a biological fluid is a function of the storage conditions, the chemical properties of the drug, the matrix, and the container system.  The stability of an analyte in a particular matrix and container system is relevant only to that matrix and container system and should not be extrapolated to other matrices and container systems.  Stability procedures should evaluate the stability of the analytes during sample collection and handling, after long-term (frozen at the intended storage temperature) and short-term (bench top, room temperature) storage, and after going through freeze and thaw cycles and the analytical process.  Conditions used in stability experiments should reflect situations likely to be encountered during actual sample handling and analysis.  The procedure should also include an evaluation of analyte stability in stock solution.
生物流体(尿囊液?)药物稳定性是贮存条件,药物化学特性,基质和容器系统的函数。特定基质或容器系统中的分析物的稳定性仅与该基质和容器系统有关,不能外推至其他基质和容器中。稳定性程序应评估取样和样品处理时的,在长期(预期贮存温度下冻存)和短期(台式,室温)贮存和经过冰冻和解冻过程后和分析过程中的分析物的稳定性。稳定性试验的条件应考虑在实际样品处理和分析中的偶然情况。该程序也应包括分析物在储备液中的稳定性的评估。
All stability determinations should use a set of samples prepared from a freshly made stock solution of the analyte in the appropriate analyte-free, interference-free biological matrix.  Stock solutions of the analyte for stability evaluation should be prepared in an appropriate solvent at known concentrations.
所有稳定性测定应使用一系列的由新制备的适当的无分析物,无干扰的生物基质储备溶液制备的样品。用于稳定性评估的分析物储备液应使用适当的已知浓度的溶剂制备。
          1.  Freeze and Thaw Stability 冰冻和解冻稳定性
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2010/7/5 8:20:55 Last edit by myreebok
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1.  Freeze and Thaw Stability 冰冻和解冻稳定性

          Analyte stability should be determined after three freeze and thaw cycles.  At least three aliquots at each of the low and high concentrations should be stored at the intended storage temperature for 24 hours and thawed unassisted at room temperature.  When completely thawed, the samples should be refrozen for 12 to 24 hours under the same conditions.  The freeze–thaw cycle should be repeated two more times, then analyzed on the third cycle.  If an analyte is unstable at the intended storage temperature, the stability sample should be frozen at -70 C during the three freeze and thaw cycles.
分析物稳定性应在三个冻存-解冻周期后测定。至少低和高浓度各三个等分(溶液)应在预期的贮存温度下贮存24小时并在没有外部辅助的情况下在室温下解冻。当完全解冻后,样品应在相同的条件下复冻12到24小时。冰冻-解冻周期应重复2次以上,然后再第三个周期分析。如果分析物在预期贮存温度下不稳定,稳定性样品在冰冻-解冻周期中应在-70℃冻存。
          2.  Short-Term Temperature Stability 短期温度稳定性
          Three aliquots of each of the low and high concentrations should be thawed at room temperature and kept at this temperature from 4 to 24 hours (based on the expected duration that samples will be maintained at room temperature in the intended study) and  analyzed.
低和高浓度各三个等分(溶液)应在室温下解冻并在室温下保持4-24小时(根据样品在预期研究时在室温中保存的预期持续时间)
          3.  Long-Term Stability 长期稳定性
          The storage time in a long-term stability evaluation should exceed the time between the date of first sample collection and the date of last sample analysis.  Long-term stability should be determined by storing at least three aliquots of each of the low and high concentrations under the same conditions as the study samples.  The volume of samples should be sufficient for analysis on three separate occasions.  The concentrations of all the stability samples should be compared to the mean of back-calculated values for the standards at the appropriate concentrations from the first day of long-term stability testing.
长期稳定性试验的贮存时间评估应超过第一个样品取样和最后一个样品分析期间的时间。长期稳定性试验应由贮存在与研究样品一样的条件下的至少低和高浓度各三个等分(溶液)测定。样品量应足够进行三次独立分析。所有稳定性样品的浓度应与从长期稳定性试验第一天的标准品适当浓度的倒退计算值的平均值相比较。
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4.  Stock Solution Stability 储备液稳定性
          The stability of stock solutions of drug and the internal standard should be evaluated at room temperature for at least 6 hours.  If the stock solutions are refrigerated or frozen for the relevant period, the stability should be documented.  After completion of the desired storage time, the stability should be tested by comparing the instrument response with that of freshly prepared solutions.
药物和内标的储备液稳定性应在室温下至少6小时评估。如果储备液是在相关时期冷藏或冷冻的,稳定性应记录。在目标贮存时间到达后,稳定性应由与新制备溶液的仪器响应值比较的方法测定。
        5.  Post-Preparative Stability 后制备稳定性
        The stability of processed samples, including the resident time in the autosampler, should be determined.  The stability of the drug and the internal standard should be assessed over the anticipated run time for the batch size in validation samples by determining concentrations on the basis of original calibration standards.
处理后样品的稳定性,包括自动进样器的驻留时间,应确定。药物和内标的稳定性应用超过验证样品预期批运行时间由依据原始校准标准测定浓度来评估
        Although the traditional approach of comparing analytical results for stored samples with those for freshly prepared samples has been referred to in this guidance, other statistical approaches based on confidence limits for evaluation of an analytes stability in a biological matrix can be used.  SOPs should clearly describe the statistical method and rules used.  Additional validation may include investigation of samples from dosed subjects.
尽管本指南中引用了传统的比较贮存样品与新制备样品分析结果的方法,其他根据置信限评估在生物基质中的分析物稳定性统计学方法也可以使用。SOP应明确地描述统计的方法和规则。额外的验证可能包括样品制剂后的调查。
E.  Principles of Bioanalytical Method Validation and Establishment 生物分析方法验证和建立的原则。
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The fundamental parameters to ensure the acceptability of the performance of a bioanalytical method validation are accuracy, precision, selectivity, sensitivity, reproducibility, and stability.
确保生物分析方法验证可接受性的基本参数是准确度,精密度,选择性,灵敏度,重现性和稳定性。
    A specific, detailed description of the bioanalytical method should be written.  This can be in the form of a protocol, study plan, report, and/or SOP.
应起草生物分析方法的明确的,详细的描述。可以以方案,研究计划,报告和/或SOP 的形式。
Each step in the method should be investigated to determine the extent to which environmental, matrix, material, or procedural variables can affect the estimation of analyte in the matrix from the time of collection of the material up to and including the time of  analysis.
应审查方法的每一步骤来确定从收集到物料的时间包括分析时间中环境,基质,物料或程序上变量可以影响基质中分析物定量的范围
    It may be important to consider the variability of the matrix due to the physiological nature of the sample.  In the case of LC-MS-MS-based procedures, appropriate steps should be taken to ensure the lack of matrix effects throughout the application of the method, especially if the nature of the matrix changes from the matrix used during method validation.
由于样品的生理特性,考虑基质的可变性很重要。以基于液质质的程序为例,应采取适当的步骤来确保基质不足影响,特别是如果在验证中使用的基质特性变化。
    A bioanalytical method should be validated for the intended use or application.  All experiments used to make claims or draw conclusions about the validity of the method should be presented in a report (method validation report).
生物分析方法应为预期使用或申报验证。所有用来得到关于方法有效性的主张或结论的试验应形成报告(方法验证报告)
Whenever possible, the same biological matrix as the matrix in the intended samples should be used for validation purposes.  (For tissues of limited availability, such as bone marrow, physiologically appropriate proxy matrices can be substituted.) The stability of the analyte (drug and/or metabolite) in the matrix during the collection process and the sample storage period should be assessed, preferably prior to sample analysis.
在任何可能的时候,为验证目的应使用与预期样品中基质相同的生物基质。(对于组织的有限性,例如骨髓,可以用生理学上有适当代表性的基质来替代)应评估在取样过程和样品贮存期的基质中分析物稳定性(药物和/或代谢物),最好是在样品分析前。
    For compounds with potentially labile metabolites, the stability of analyte in matrix from dosed subjects (or species) should be confirmed.
对于有潜在不稳定代谢物的物质,应确认制剂后基质中分析物稳定性。
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2010/7/5 9:47:04 Last edit by myreebok
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The accuracy, precision, reproducibility, response function, and selectivity of the method for endogenous substances, metabolites, and known degradation products should be established for the biological matrix.  For selectivity, there should be evidence that the substance being quantified is the intended analyte.
对内部物质,代谢物,和已知降解产物,方法的准确度,精密度,重现性,响应函数和选择性应对生物基质确定(个人认为就是对生物基质制订标准)。对选择性,应有凭证证明经定量的物质是目标分析物。
The concentration range over which the analyte will be determined should be defined in the bioanalytical method, based on evaluation of actual standard samples over the range, including their statistical variation.  This defines the standard curve.
应依据涵盖范围的实际标准样品评估,在生物分析方法中定义分析物测定的浓度范围,包括统计学变化。这就定义了标准曲线。
A sufficient number of standards should be used to adequately define the relationship between concentration and response.  The relationship between response and concentration should be demonstrated to be continuous and reproducible.  The number of standards used should be a function of the dynamic range and nature of the concentration-response relationship.  In many cases, six to eight concentrations (excluding blank values) can define the standard curve.  More standard concentrations may be recommended for nonlinear than for linear relationships.
应用足够数量的标准点适当地定义浓度和响应之间的关系。应论证浓度和响应之间的关系的持续性和重现性。使用的标准点数量应是浓度-响应关系动态范围和特性的函数。在许多情况下,6-8个浓度(除空白值)可以定义标准曲线。更多标准点浓度可能推荐为非线性而不是线性关系。
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2010/7/5 10:04:57 Last edit by myreebok