Guideline on bioanalytical method validation

在创新药物研发过程中,生物基质（如血清、血浆、血液、尿液、唾液）中的药物浓度测定是一个重要的方面。其数据可用于支持新活性物质的应用和仿制药及已授权药品的变更申请。动物的毒代动力学研究和临床试验,包括生物等效性研究的结果为原料药或产品的安全性和有效性提供关键性的数据支持。因此,应用经过充分验证并记录到一个满意标准的生物分析方法以得到可靠的结果,这是非常重要的。2012年2月1日,欧洲药品管理局（EMA）开始实施最新的《生物样品分析方法验证指南》（Guideline on bioanalytical method validation）,本指南适用于动物的毒代动力学研究和所有阶段的临床试验中获得的生物样品中的药物浓度的生物分析方法的验证。本文摘录其方法学验证部分,抛砖引玉,供相关研究人员参考。     

Method validation in the bioanalytical laboratory

Bioanalytical methods, based on a variety of physico-chemical and biological techniques such as chromatography, immunoassay and mass spectrometry, must be validated prior to and during use to engender confidence in the results generated. The fundamental criteria for assessing the reliability and overall performance of a bioanalytical method are: the evaluation of drug and analyte stability, selectivity, limits of quantification and detection, accuracy, precision, linearity and recovery. The extent to which a method is validated is dependent on its prospective use, the number of samples to be assayed and the use to which the data are put.

Specific analytical techniques may require additional validation such as antibody-binding characteristics, peak purity determination, evaluation of matrix effects or structural confirmation of the analyte. Ideally each assay should be cross-validated with a method utilizing a highly specific detector such as a mass spectrometer. Once in use, the performance of the method should be monitored using quality control standards. If a method is set up in another laboratory, the performance of the assay should be monitored with quality control standards sent from the originating laboratory.     

Key elements of bioanalytical method validation for macromolecules

The Third American Association of Pharmaceutical Scientists/US Food and Drug Administration (FDA) Bioanalytical Workshop, which was held May 1 and 2, 2006, in Arlington, VA, addressed bioanalytical assays that are being used for the quantification of therapeutic candidates in support of pharmacokinetic evaluations. One of the main goals of this workshop was to discuss best practices used in bioanalysis regardless of the size of the therapeutic candidates. Since the last bioanalytical workshop, technological advancements in the field and in the statistical understanding of the validation issues have generated a variety of interpretations to clarify and understand the practicality of using the current FDA guidance for assaying macromolecular therapeutics. This article addresses some of the key elements that are essential to the validation of macromolecular therapeutics using ligand binding assays. Because of the nature of ligand binding assays, attempts have been made within the scientific community to use statistical approaches to interpret the acceptance criteria that are aligned with the prestudy validation and in-study validation (sample analysis) processes. We discuss, among other topics, using the total error criterion or confidence interval approaches for acceptance of assays and using anchor calibrators to fit the nonlinear regression models.     

Cost-effective and business-beneficial computer validation for bioanalytical laboratories

Computerized system validation is often viewed as a burden and a waste of time to meet regulatory requirements. This article presents a different approach by looking at validation in a bioanalytical laboratory from the business benefits that computer validation can bring. Ask yourself the question, have you ever bought a computerized system that did not meet your initial expectations? This article will look at understanding the process to be automated, the paper to be eliminated and the records to be signed to meet the requirements of the GLP or GCP and Part 11 regulations. This paper will only consider commercial nonconfigurable and configurable software such as plate readers and LC-MS/MS data systems rather than LIMS or custom applications. Two streamlined life cycle models are presented. The first one consists of a single document for validation of nonconfigurable software. The second is for configurable software and is a five-stage model that avoids the need to write functional and design specifications. Both models are aimed at managing the risk each type of software poses whist reducing the amount of documented evidence required for validation.     

Key elements of bioanalytical method validation for small molecules

Method validation is a process that demonstrates that a method will successfully meet or exceed the minimum standards recommended in the Food and Drug Administration (FDA) guidance for accuracy, precision, selectivity, sensitivity, reproducibility, and stability. This article discusses the validation of bioanalytical methods for small molecules with emphasis on chromatographic techniques. We present current thinking on validation requirements as described in the current FDA Guidance and subsequent 2006 Bioanalytical Methods Validation Workshop white paper.     

Advances in validation, risk and uncertainty assessment of bioanalytical methods

Bioanalytical method validation is a mandatory step to evaluate the ability of developed methods to provide accurate results for their routine application in order to trust the critical decisions that will be made with them. Even if several guidelines exist to help perform bioanalytical method validations, there is still the need to clarify the meaning and interpretation of bioanalytical method validation criteria and methodology. Yet, different interpretations can be made of the validation guidelines as well as for the definitions of the validation criteria. This will lead to diverse experimental designs implemented to try fulfilling these criteria. Finally, different decision methodologies can also be interpreted from these guidelines. Therefore, the risk that a validated bioanalytical method may be unfit for its future purpose will depend on analysts personal interpretation of these guidelines. The objective of this review is thus to discuss and highlight several essential aspects of methods validation, not only restricted to chromatographic ones but also to ligand binding assays owing to their increasing role in biopharmaceutical industries. The points that will be reviewed are the common validation criteria, which are selectivity, standard curve, trueness, precision, accuracy, limits of quantification and range, dilutional integrity and analyte stability. Definitions, methodology, experimental design and decision criteria are reviewed. Two other points closely connected to method validation are also examined: incurred sample reproducibility testing and measurement uncertainty as they are highly linked to bioanalytical results reliability. Their additional implementation is foreseen to strongly reduce the risk of having validated a bioanalytical method unfit for its purpose.     

Aspects of bioanalytical method validation for the quantitative determination of trace elements

Bioanalytical methods are used to quantitatively determine the concentration of drugs, biotransformation products or other specified substances in biological matrices and are often used to provide critical data to pharmacokinetic or bioequivalence studies in support of regulatory submissions. In order to ensure that bioanalytical methods are capable of generating reliable, reproducible data that meet or exceed current regulatory guidance, they are subjected to a rigorous method validation process. At present, regulatory guidance does not necessarily account for nuances specific to trace element determinations. This paper is intended to provide the reader with guidance related to trace element bioanalytical method validation from the authors' perspective for two prevalent and powerful instrumental techniques: inductively coupled plasma-optical emission spectrometry and inductively coupled plasma-MS.     

A new approach to evaluate regression models during validation of bioanalytical assays

The quality of bioanalytical data is highly dependent on using an appropriate regression model for calibration curves. Non-weighted linear regression has traditionally been used but is not necessarily the optimal model. Bioanalytical assays generally benefit from using either data transformation and/or weighting since variance normally increases with concentration. A data set with calibrators ranging from 9 to 10000 ng/mL was used to compare a new approach with the traditional approach for selecting an optimal regression model. The new approach used a combination of relative residuals at each calibration level together with precision and accuracy of independent quality control samples over 4 days to select and justify the best regression model. The results showed that log-log transformation without weighting was the simplest model to fit the calibration data and ensure good predictability for this data set.     

EBF recommendation on the validation of bioanalytical methods for dried blood spots

Over the last few years bioanalysts, pharmacokineticists and clinical investigators have rediscovered the technique of dried blood spots. The revival has provided pharmaceutical R&D a wealth of opportunities to optimize the drug-discovery and development process with respect to animal and patient ethics, new scientific insights and costs savings. On the bioanalytical front, multiple experiments have been performed and a lot of experience has been gained. Nevertheless, the technique still has a number of bioanalytical challenges. The European Bioanalysis Forum discussed the advantages and hurdles of the technique and summarized their current thinking in a recommendation on the validation of bioanalytical methods for dried blood spots, which can be used as a cornerstone for further discussions and experiments.     

Application issues in bioanalytical method validation,sample analysis and data reporting

Although some degree of consensus has been reached concerning the requirements for acceptable method validation, the procedures used to establish them vary significantly between laboratories. Also, issues arising from application of these requirements during validation and subsequent sample analysis need to be addressed. The purpose of this paper is to discuss application issues concerning prerequisites to method validation, and all validation criteria for evaluation of method reliability and overall performance. Other poorly addressed issues such as re-validation, cross-validation, partial sample volume, multicomponent analysis and reporting will also be discussed. Although many issues discussed are of a general nature, the scope of this presentation is primarily to address issues arising from the validation and routine application of chromatographic methods.     

Development and validation of a high-performance liquid chromatographic method for bioanalytical application with rimonabant

A simple and feasible high-performance liquid chromatographic method with UV detection was developed and validated for the quantification of rimonabant in human plasma. The chromatographic separation was carried out in a Hypersil BDS, C₁₈ column (250 mm × 4.6 mm; 5 μm). The mobile phase was a mixture of 10 mM phosphate buffer and acetonitrile (30:70, v/v) at a flow rate of 1.0 ml/min. The UV detection was set at 220 nm. The extraction recovery of rimonabant in plasma at three quality control (QC) samples was ranged from 84.17% to 92.64%. The calibration curve was linear for the concentration range of 20–400 ng/ml with the correlation coefficient (r²) above 0.9921. The method was specific and sensitive with the limit of quantification of 20 ng/ml. The accuracy and precision values obtained from six different sets of QC samples analyzed in separate occasions ranged from 88.13% to 106.48% and 0.13% to 3.61%, respectively. In stability tests, rimonabant in human plasma was stable during storage and assay procedure. The method is very simple, sensitive and economical and the assay was applied to human plasma samples in a clinical (pharmacokinetic) study of rimonabant.     

Development and validation of bioanalytical method for the determination of asulacrine in plasma by liquid chromatography

Asulacrine (9-[(2-methoxy-4-methylsulphonylamino)phenylamino]-N,5-dimethyl-4-acridinecarboxamide), an analogue of the antileukaemia drug amsacrine, has high antitumour activity in mice and has also shown clinical activity. A simple method is described for the quantitation of asulacrine in plasma by liquid chromatography. Chromatographic separation was achieved on a reversed phase C 18 column (250 mm x 4.6mm, particle size 5 microm, Gemini) using isocratic elution (acetonitrile and 0.01 M sodium acetate buffer pH 4.0, 45/55, v/v) at a flow rate of 1 ml/min. Asulacrine and internal standard (the ethylsulphonanilide analogue) were measured using UV detection at 254 nm. The total chromatographic run-time was 8 min with asulacrine and internal standard eluting at approximately 4.7 and approximately 6.5 min, respectively. Limit of quantification was 0.1microg/ml. The linearity range of the method was 0.1-10 microg/ml (r2=0.9995). Mean recoveries from plasma were 100-105%. Intra-batch and inter-batch precision was 7.1 and 7.8%, respectively, and intra-batch and inter-batch accuracy (relative error) was 4.9 and 8.4%, respectively (n=8 in all cases). The bench top, freeze thaw, short-term storage and stock solution stability evaluation indicated no evidence of degradation of asulacrine. The validated method is simple, selective and rapid and can be used for pharmacokinetic studies in mice.     

An analysis of the SFSTP guide on validation of chromatographic bioanalytical methods: progress and limitations

The Société Fran?aise des Sciences et Techniques Pharmaceutiques (SFSTP) published in 1997 a guide on the validation of chromatographic bio-analytical methods, which introduces new concepts in three different areas: stages of the validation, test of acceptability of a method and design of experiments to perform. In 'stages of validation', the SFSTP guide requires two phases to validate a method. The first phase, called 'prevalidation', is intended to (1) identify the model to use for the calibration curve; (2) evaluate the limits of quantitation; and (3) provide good estimates of the precision and bias of the method before designing the 'validation' phase per se. In the 'test of acceptability', the use of the interval hypotheses is envisaged by the SFSTP guide, not on the parameters of bias and precision, but on individual results by mixing mean bias and intermediate precision in a single test. The SFSTP guide also avoids the use of Satterthwaite's df for testing the acceptability. The reasons for those choices are discussed extensively. In 'design of experiments', much effort has been devoted to improving the quality of results by optimally designing and sizing the experiments to perform in validation. The rationale for using near D-optimal designs for the calibration curve is demonstrated and sample sizes are proposed to correctly size the validation experiments.     

A comprehensive method validation strategy for bioanalytical applications in the pharmaceutical industry--1. Experimental considerations

The method validation strategy described consists of four components which are the prevalidation, validation proper, study proper and statistical analyses. These components constitute the platform upon which to evaluate the reliability and reproducibility of a bioanalytical method. Consideration has been given to emulate the study proper conditions to understand the method's limitations and performance expectations. The validation strategy will be presented in two papers. This first paper will describe the overall validation strategy, and the second paper will discuss the statistical analyses and data interpretation.     

Development and validation of a bioanalytical LC-UV method with solid-phase extraction for determination of valproic acid in saliva

A bioanalytical HPLC method with UV detection for the determination of the antiepileptic drug valproic acid in human saliva has been developed and validated. Saliva represents an alternative matrix for therapeutic monitoring of antiepileptic drugs due to the increasing interest in free drug concentration. The proposed method involved solid-phase extraction for sample preparation and yielded very good mean recoveries of 99.4 % and 97.9 % for valproic acid and IS, respectively. The calibration function for valproic acid was linear over the concentration range of 1.0-50.0 μg mL(-1) (R(2) = 0.9989). Within-run and between-run precision and accuracy were studied at four concentrations and RSDs were less than 7.3 and 2.2 %, while accuracy values were higher than 96.8 and 97.5 %, respectively. The described method provides sensitivity, linearity, precision, accuracy and is suitable for analyses of valproic acid in saliva samples.