Ruggedness testing in analytical chemistry

Very high standards of accuracy and precision are currently expected from analytical methods. This is particularly true for pharmaceutical applications, due not only to the potential toxicity of drugs but also to the strict controls of regulatory bodies. This paper deals with the validation of methods by testing ruggedness to changes in the analytical method conditions. As a ruggedness test examines a number of variables simultaneously and hence a large experimental response surface, the design has to be carefully chosen. Advice is offered on the test design, including the selection of factors and the levels at which to test them. Some results are shown for high-performance liquid chromatography methods.     

化学分析方法验证和确认的应用研究

分析方法的验证/确认是实验室引进新方法时必做的工作,也是实验室技术工作的重点和难点之一。对实验室采用分析方法的验证和确认工作进行归纳总结,详述了分析方法选择性、测量范围、线性范围、检出限和定量限、精密度、准确度的验证/确认方法及结果判定方式。适用于实验室引进的标准方法(包括标准变更)和非标准方法(实验室设计/制定的方法、超出预定范围使用的标准方法、扩充和修改过的标准方法)的验证和确认。     

The NIH analytical methods and reference materials program for dietary supplements

Quality of botanical products is a great uncertainty that consumers, clinicians, regulators, and researchers face. Definitions of quality abound, and include specifications for sanitation, adventitious agents (pesticides, metals, weeds), and content of natural chemicals. Because dietary supplements (DS) are often complex mixtures, they pose analytical challenges and method validation may be difficult. In response to product quality concerns and the need for validated and publicly available methods for DS analysis, the US Congress directed the Office of Dietary Supplements (ODS) at the National Institutes of Health (NIH) to accelerate an ongoing methods validation process, and the Dietary Supplements Methods and Reference Materials Program was created. The program was constructed from stakeholder input and incorporates several federal procurement and granting mechanisms in a coordinated and interlocking framework. The framework facilitates validation of analytical methods, analytical standards, and reference materials.     

Different approaches to legal requirements on validation of test methods for official control of foodstuffs and of veterinary drug residues and element contaminants in the EC

 In order to ensure food consumer protection as well as to avoid barriers to trade and unnecessary duplications of laboratory tests and to gain mutual recognition of results of analyses, the quality of laboratories and test results has to be guaranteed. For this purpose, the EC Council and the Commission have introducedprovisions – on measures for quality assurance for official laboratories concerning the analyses of foodstuffs on the one hand and animals and fresh meat on the other, – on the validation of test methods to obtain results of sufficient accuracy. This article deals with legal requirements in the European Union on basic principles of laboratory quality assurance for official notification to the EC Commission and on method validation concerning official laboratories. Widespread discussions and activities on measurement uncertainty are in progress, and the European validation standards for official purposes may serve as a basis for world-wide efforts on quality harmonization of analytical results. Although much time has already been spent, definitions and requirements have to be revised and further additions have to be made.     

Comparison of quantitative methods for analysis of polyphasic pharmaceuticals

Adequate very sensitive quantification methods are needed for the development and are also now required for the monitoring of undesirable solid form(s) as routine tests. The pre-requisite for quantitation are selectivity, sensitivity and most important the purity of standards and their proper storage, what is a challenge for metastable forms. Several analytical techniques are available such as X-ray diffraction, spectroscopy, thermal analysis and microcalorimetry. The different steps of the validation of the analytical methods and problems to be solved are discussed. Examples illustrate the different techniques and compare their possible advantages and limits. The relative standard deviation of measurements should allow for checking the homogenization procedure of mixtures for calibration. The validation should be carried out following ICH guidelines for validation of analytical methods. Comparison of different techniques in adequate concentration range add confidence in the analytical results.     

The application of qNMR for the determination of rosuvastatin in tablet form

In this study, quantative nuclear magnetic resonance (qNMR) method was used to determine the content of rosuvastatin in tablet. Linearity, range, limit of detection (LOD), limit of quantification (LOQ), accuracy, and precision were determined in validation study of rosuvastatin. Furthermore, validation study of rosuvastatin was performed with high performance liquid chromatography (HPLC). Uncertainties of qNMR and HPLC methods were determined using per EURACHEM/CITAC Guide CG 4 (3th edition), quantifying uncertainty in analytical measurement. qNMR and HPLC methods were linear in the ranges of 0.10 - 5.00 mg/mL and 0.001 - 0.0995 mg/mL, respectively and these lineraties indicate very good linearity performance with regression coefficients (R2 value) above > 0.99. Moreover, LOD and LOQ values using qNMR method were observed as 0.25 mg/mL and 0.80 mg/mL, respectively. These values using HPLC method were found as 0.00051 µg/mL and 0.001695 µg/mL, respectively. The strengths and weaknesses of qNMR method and HPLC method were determined with spectral emphasis on the role of identical reference standards in qualitive and quantitative analyses. It was found that qNMR method is simple, efficient, reliable, and accurate method. Moreover, qNMR method is an easy, practical, and useful method for the validation and optimization of rosuvastatin in the tablet.     

Advances in methodology for the validation of methods according to the International Organization for Standardization. Application to the determination of benzoic and sorbic acids in soft drinks by high-performance liquid chromatography

Robust chemometric techniques such as least median of squares regression, H15 Huber estimator and Lenth's method are fundamental tools in the validation of analytical methods since they contribute the strategies needed to estimate efficiently parameters such as robustness, linear range, selectivity, accuracy (trueness and precision) and the capability of detection. In addition, the capability of discrimination defined as a generalisation of the capability of detection for any nominal concentration is evaluated. The new strategy proposed is applied to the validation of a chromatographic method for use in systematic analysis.     

The validation of methods for regulatory purposes in the control of residues

The topic of validation is diversified. This review outlines the validation strategies which can be found in national, international and supranational regulations, compares them with one another and aims to elaborate on the main principles. European regulations and legislation, Codex alimentarius guidelines, the official methods program of the AOAC, and naturally the relevant ISO standards, particularly the ISO 5725 series, are taken into consideration. The objective of every validation is to demonstrate fitness for purpose. This varies of course in its characteristics for the diverse uses. However, all approaches have in common the objective of harmonisation of food control by using effective and reliable methods. To this end, criteria are determined and validation models developed and made compulsory. ISO 5725 is the central basis for validations for quantitative methods with its validation specifications through method collaborative studies. On the contrary, there are no valid uniform international method specifications for qualitative methods. Collaborative studies are in opposition to single-lab-validations with different sources of error. Whereas laboratory errors are predominant in collaborative studies, the single-lab-validation or in-house validation concentrates particularly on time and processing errors (intermediate precision). In new statistical models for in-house validations, the matrix mismatch error is also considered. The validation models presented here are of a general nature and can be used in principle for all analytical methods. Correct and appropriate statistical modelling is very important.     

Using tolerance intervals in pre-study validation of analytical methods to predict in-study results. The fit-for-future-purpose concept

It is recognized that the purpose of validation of analytical methods is to demonstrate that the method is suited for its intended purpose. Validation is not only required by regulatory authorities, but is also a decisive phase before the routine use of the method. For a quantitative analytical method the objective is to quantify the target analytes with a known and suitable accuracy. For that purpose, first, a decision about the validity of the method based on prediction is proposed: a method is declared proper for routine application if it is considered that most of the future results generated will be accurate enough. This can be achieved by using the "beta-expectation tolerance interval" (accuracy profile) as the decision tool to assess the validity of the analytical method. Moreover, the concept of "fit-for-purpose" is also proposed here to select the most relevant response function as calibration curve, i.e. choosing a response function based solely on the predicted results this model will allow to obtain. This paper reports four case studies where the results obtained with quality control samples in routine were compared to predictions made in the validation phase. Predictions made using the "beta-expectation tolerance interval" are shown to be accurate and trustful for decision making. It is therefore suggested that an adequate way to conciliate both the objectives of the analytical method in routine analysis and those of the validation step consists in taking the decision about the validity of the analytical method based on prediction of the future results using the most appropriate response function curve, i.e. the fit-for-future-purpose concept.     

Effect of quality characteristics of single sample preparation steps in the precision and coverage of proteomic studies—A review

Proteomic profiling and biomarker search are analytical tools as many other. Nevertheless, in the proteomic discovery phase considerable sample fractionation is inevitable before readout. Since these procedures are of notable complexity, proteomic tools need in particular analytical quality validation standards as prevail for other analytical methods. With acceptance of the rule of error propagation the values of imprecision and yield of each preparation step determine overall reproducibility and therewith information harvest of a propagated method series. Thereto, we examined recent proteomic reports with reproducibility data and with parallelization, and automation approaches. Based on the data available from literature it is highly probable, that at least a part of current proteomic platforms actually suffer from high technical variance.     

Quantifying the measurement uncertainty of results from environmental analytical methods

The Eurachem-CITAC Guide Quantifying Uncertainty in Analytical Measurement was put into practice in a public laboratory devoted to environmental analytical measurements. In doing so due regard was given to the provisions of ISO 17025 and an attempt was made to base the entire estimation of measurement uncertainty on available data from the literature or from previously performed validation studies. Most environmental analytical procedures laid down in national or international standards are the result of cooperative efforts and put into effect as part of a compromise between all parties involved, public and private, that also encompasses environmental standards and statutory limits. Central to many procedures is the focus on the measurement of environmental effects rather than on individual chemical species. In this situation it is particularly important to understand the measurement process well enough to produce a realistic uncertainty statement. Environmental analytical methods will be examined as far as necessary, but reference will also be made to analytical methods in general and to physical measurement methods where appropriate. This paper describes ways and means of quantifying uncertainty for frequently practised methods of environmental analysis. It will be shown that operationally defined measurands are no obstacle to the estimation process as described in the Eurachem/CITAC Guide if it is accepted that the dominating component of uncertainty comes from the actual practice of the method as a reproducibility standard deviation.     

Analytical Method Performance Evaluation (AMPE)--a software tool for analytical method validation

A Windows-based software tool [Analytical Method Performance Evaluation (AMPE)] was developed to support the validation of analytical methods. The software implements standard statistical approaches commonly adopted in validation studies to estimate analytical method performance (limits of detection and quantitation, accuracy, specificity, working range, and linearity of responses) according to ISO 5725. In addition, AMPE proposes the application of innovative and unique approaches for the assessment of analytical method performance. Specifically, AMPE proposes the use of difference-based indexes to quantify the agreement between measurements and reference values, the use of pattern indexes to quantify methods bias with respect to specific external variables, and the application of fuzzy logic to aggregate into synthetic indicators the information collected independently via the different performance statistics traditionally estimated in validation studies. Aggregated measures are particularly useful for methods comparison, when more than one method is available for a specific analysis and it may be of interest to identify the best performing one taking into account, simultaneously, the information available from different performance statistics. Illustrative examples of the type of outputs expected from AMPE-based validation sessions are given. The extensive data handling capabilities and the wide range of statistics supplied in the software package makes AMPE suitable for specific needs that may arise in different validation studies. The installation package, complete with a fully documented help file, is distributed free of charge to interested users along with input files exemplary of the type of entry data required to run validation data analyses.     

Rational experimental design for bioanalytical methods validation illustration using an assay method for total captopril in plasma

Generally, bioanalytical chromatographic methods are validated according to a predefined programme and distinguish a pre-validation phase, a main validation phase and a follow-up validation phase. In this paper, a rational, total performance evaluation programme for chromatographic methods is presented. The design was developed in particular for the pre-validation and main validation phases. The entire experimental design can be performed within six analytical runs. The first run (pre-validation phase) is used to assess the validity of the expected concentration-response relationship (lack of fit, goodness of fit), to assess the specificity of the method and to assess the stability of processed samples in the autosampler for 30 h (benchtop stability). The latter experiment is performed to justify overnight analyses. Following approval of the method after the pre-validation phase, the next five runs (main validation phase) are performed to evaluate method precision and accuracy, recovery, freezing and thawing stability and over-curve control /dilution. The design is nested, i.e., many experimental results are used for the evaluation of several performance characteristics. Analysis of variance (ANOVA) is used for the evaluation of lack of fit and goodness of fit, precision and accuracy, freezing and thawing stability and over-curve control/ dilution. Regression analysis is used to evaluate benchtop stability. For over-curve control/ dilution, additional to ANOVA, also a paired comparison is applied. As a consequence, the recommended design combines the performance of as few independent validation experiments as possible with modern statistical methods, resulting in optimum use of information. A demonstration of the entire validation programme is given for an HPLC method for the determination of total captopril in human plasma.     

The effect of the physical matrix on accurate measurements using fixed volume analytical techniques

In order to perform high accuracy analytical measurements most analytical techniques require some form of calibration using standards of the same quantity as that being measured. The highest accuracy calibration standards are those prepared by mass (gravimetrically) as opposed to by volume (volumetrically). The use of gravimetrically prepared standards to calibrate analytical techniques that rely on fixed volume injections can cause systematic errors, even when the analytical technique does not suffer from a chemical matrix interference. The origin of these errors is explained and is demonstrated experimentally for the analysis of sulphate in synthetic seawater samples, and the measurement of the anionic content of particulate matter following extraction with water and wetting agents; where average measurement biases of +2.7 and -3.2%, respectively, were observed. Proposals are offered for methods to overcome this 'physical matrix effect'.     

Improvement of the decision efficiency of the accuracy profile by means of a desirability function for analytical methods validation. Application to a diacetyl-monoxime colorimetric assay used for the determination of urea in transdermal iontophoretic extracts

Validation of analytical methods is a widely used and regulated step for each analytical method. However, the classical approaches to demonstrate the ability to quantify of a method do not necessarily fulfill this objective. For this reason an innovative methodology was recently introduced by using the tolerance interval and accuracy profile, which guarantee that a pre-defined proportion of future measurements obtained with the method will be included within the acceptance limits. Accuracy profile is an effective decision tool to assess the validity of analytical methods. The methodology to build such a profile is detailed here. However, as for any visual tool it has a part of subjectivity. It was then necessary to make the decision process objective in order to quantify the degree of adequacy of an accuracy profile and to allow a thorough comparison between such profiles. To achieve this, we developed a global desirability index based on the three most important validation criteria: the trueness, the precision and the range. The global index allows the classification of the different accuracy profiles obtained according to their respective response functions. A diacetyl-monoxime colorimetric assay for the determination of urea in transdermal iontophoretic extracts was used to illustrate these improvements.     

Analysis of recent pharmaceutical regulatory documents on analytical method validation

All analysts face the same situations as method validation is the process of proving that an analytical method is acceptable for its intended purpose. In order to resolve this problem, the analyst refers to regulatory or guidance documents, and therefore the validity of the analytical methods is dependent on the guidance, terminology and methodology, proposed in these documents. It is therefore of prime importance to have clear definitions of the different validation criteria used to assess this validity. It is also necessary to have methodologies in accordance with these definitions and consequently to use statistical methods which are relevant with these definitions, the objective of the validation and the objective of the analytical method. The main purpose of this paper is to outline the inconsistencies between some definitions of the criteria and the experimental procedures proposed to evaluate those criteria in recent documents dedicated to the validation of analytical methods in the pharmaceutical field, together with the risks and problems when trying to cope with contradictory, and sometimes scientifically irrelevant, requirements and definitions.     

Analytical service: systematic approach to the activity optimization according to accuracy criterion of analysis results

Summary It is taken as an initial conception that the general aim of quantitative analysis is measurement by specific, mainly analytical, methods and instruments. Another important initial position is the necessity to specify the required accuracy of analytical data, taking into account the contribution of analytical error within the error of the whole test result which includes contributions from other sources (sampling, sample storage, transporting, etc.).The approaches to validation of accuracy of field analysis are considered. Taking into account all factors mentioned above, the principles and methods of demonstration of accuracy are stated.The main sources of uncertainty of the really insured accuracy are pointed out. The typical scheme of activity optimization of analytical subdivisions and those systems which use the results on the chemical composition of substances is described. The peculiarities of obtaining basic data and their usage for quality control of field analytical results are discussed.

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Systemoptimierung durch Genauigkeitskriterien analytischer Ergebnisse

     

Some practical examples of method validation in the analytical laboratory

Method validation is a key element in both the elaboration of reference methods and the assessment of a laboratory's competence in producing reliable analytical data. Hence, the scope of the term method validation is wide, especially if one bears in mind that there is or at least should be a close relation between validation, calibration and quality control QA/QC. Moreover, validation should include more than the instrumental step only since the whole cycle from sampling to the final analytical result is important in the assessment of the validity of an analytical result. In this article validation is put in the context of the process of producing chemical information. Two cases are presented in more detail: the development of a European standard for chlorophenols and its validation by a full scale collaborative trial, and the intralaboratory validation of a method for ethylenethiourea using alternative analytical techniques.