Usefulness of capability indices in the framework of analytical methods validation

Analytical methods capability evaluation can be a useful methodology to assess the fitness of purpose of these methods for their future routine application. However, care on how to compute the capability indices have to be made. Indeed, the commonly used formulas to compute capability indices such as Cpk, will highly overestimate the true capability of the methods. Especially during methods validation or transfer, there are only few experiments performed and, using in these situations the commonly applied capability indices to declare a method as valid or as transferable to a receiving laboratory will conduct to inadequate decisions.     

Determination and interpretation of environmental water samples contaminated by uranium mining activities

Interpretation of environmental behavior of uranium is based on several steps of data analysis and statistical inference. First step is sampling and analyzing of uranium in field samples by routine laboratory methods. Such methods have to fulfill multiple requirements like robustness, efficiency, low detection limit and precision. A comparison of different approaches in assigning uncertainty to experimentally obtained analytical data shows that classical error estimation is not significantly inferior to more sophisticated modern techniques like inverse regression or orthogonal regression. A second step is the correlation of analytical data with current state of insight into environmental behavior of uranium. Such a correlation furthers the choice of adequate geochemical models and quality of geochemical data base for subsequent detailed analysis, e.g. by geochemical modeling. An appraisal of the individual steps in this complex analysis is given on the basis of statistical procedures for calibration and an E_H-pH diagram of uranium for atmospheric conditions.     

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.     

Teaching Chemometrics with a Bioprocess: Analytical Methods Comparison Using Bivariate Linear Regression

We present an advanced analytical chemistry laboratory experiment involving chemometrics. Students perform a comparison of two analytical methods by checking several analyte concentrations within a certain range by using least-squares linear regression. They obtain statistical information such as the presence of constant and proportional biases. The exercise is based on the determination of glucose levels using two colorimetric methods (enzymatic and Somogyi—Nelson) in a very simple batch system formed by an infusion of tea, glucose, and a combination of a yeast (Schizosacaromyces pombe) and a bacteria (Acetobacter xylimun), usually named Kombucha. Several samples are collected during a week of laboratory work, and measurements are performed in a subsequent four-hour laboratory class. Although commercial computer software exists for a variety of statistical applications, specific programs for the application of statistics to analytical chemistry are not prevalent. In order to solve this particular problem, a Matlab 5.3 routine is presented.     

Determination and interpretation of environmental water samples contaminated by uranium mining activities

Interpretation of environmental behavior of uranium is based on several steps of data analysis and statistical inference. First step is sampling and analyzing of uranium in field samples by routine laboratory methods. Such methods have to fulfill multiple requirements like robustness, efficiency, low detection limit and precision. A comparison of different approaches in assigning uncertainty to experimentally obtained analytical data shows that classical error estimation is not significantly inferior to more sophisticated modern techniques like inverse regression or orthogonal regression. A second step is the correlation of analytical data with current state of insight into environmental behavior of uranium. Such a correlation furthers the choice of adequate geochemical models and quality of geochemical data base for subsequent detailed analysis, e.g. by geochemical modeling. An appraisal of the individual steps in this complex analysis is given on the basis of statistical procedures for calibration and an E_H-pH diagram of uranium for atmospheric conditions. Received: 30 July 1998 / Revised: 18 November 1998 / Accepted: 26 November 1998     

Protocols for optimizing MS/MS parameters with an ion-trap GC-MS instrument

The product ion spectrum allows us to achieve very selective detection of pesticides and to eliminate the ambiguities caused by more conventional analytical approaches. Owing to the enhanced capabilities of GC/MS/MS for multiresidue pesticides, the development of a GC/MS/MS pesticides library will be useful. The aim of this study was to develop a sensitive and specific analytical method for the identification and quantification of compounds without the need of a time-consuming procedure. Two methods were studied in order to optimize the nonresonant conditions of dissociation of five pesticides (deltamethrin, metalaxyl, myclobutanil, procymidone, pirimicarb). The first permits a systematic investigation of the influence of the qz trapping parameter on sensitivity in precursor ion detection and on the efficiency of collision-induced dissociation (CID). The second is more suited for analytical laboratories and less time-consuming and allows us to reach similar results: the experiments were conducted step by step at a constant stability parameter.     

Validation requirements for chemical methods in quantitative analysis – horses for courses?

 Although the validation process necessary to ensure that an analytical method is fit for purpose is universal, the emphasis placed on different aspects of that process will vary according to the end use for which the analytical procedure is designed. It therefore becomes difficult to produce a standard method validation protocol which will be totally applicable to all analytical methods. It is probable that far more than 30% of the methods in routine laboratory use have not been validated to an appropriate level to suit the problem at hand. This situation needs to change and a practical assessment of the degree to which a method requires to be validated is the first step to a reliable and cost effective analytical industry. Received: 22 September 1997 · Accepted: 28 November 1997     

Development of metrology for pH measurement in Thailand

It has not been long that metrology is well accepted as an important part in analytical chemistry since it helps the chemists to receive the best measurement and accurate results with traceability. The National Institute of Metrology Thailand (NIMT), which is a public agency under the supervision of the Ministry of Science and Technology, not only focuses on physical standards but also provides and maintains standards in chemical field. pH measurement is one of the most widely used in the laboratories including industries and medical area in Thailand. The chemical laboratory starts working on the project with the objective of disseminating an accurate result in routine pH measurement. In 2002, the laboratory provided a service in calibration of pH meter and organized the first local interlaboratory comparison program (NIMT–C-ILC-1: pH buffer) in pH measurement. There were three buffer solution samples in the range of acid, neutral, and base. A total of 44 laboratories participated in this program. The NIMT chemical laboratory also participated in the proficiency testing program that was conducted by PSB Corporation Testing Group in Singapore. In 2003, NIMT started research in preparation of secondary buffers by using highly accurate pH meters with glass electrode systems. The laboratory produced three secondary buffers, which were pH 4.01, 6.86, and 9.18 with uncertainty 0.020 pH at 25°C. The competence of the laboratory was shown by the measurement results of the pilot study (APMP.QM-P06), which was organized by the APMP electrochemical analysis working group (EAWG/TCQM) in 2005. The title of this study was “pH determination of two phosphate buffers by Harned cell method and glass electrode method”. NIMT aims to achieve for establishment of the primary method for pH measurement in the near future. Presented at -- “BERM-10” -- April 2006, Charleston, SC, USA     

Polycyclic aromatic hydrocarbons in edible fats and oils: occurrence and analytical methods

This review deals with analytical methods for polycyclic aromatic hydrocarbon (PAH) determination in oils and fats. The data reported in the introduction deal with PAH dietary intake from this group of food and contamination levels recently found by some authors in different vegetable oils, stressing the importance of establishing a method suitable for routine analyses. Traditional sample preparation relies on tedious, time-consuming procedures. They generally consist of an extraction step (liquid-liquid partition, caffeine complexation, saponification) followed by one or more purification procedures (column chromatography, thin-layer chromatography, solid-phase extraction). The analytical determination is usually carried out by HPLC and spectrofluorometric detection, or through high-resolution capillary GC coupled to flame ionisation detection or mass spectrometry. LC is a valid alternative to the traditional sample preparation, and off-line LC-LC allows performing an accurate PAH analysis in less than 2 h. Also supercritical fluid extraction, allowing performing both extraction and clean-up in one combined step, is a promising technique. Hyphenated techniques such as LC-GC and LC-LC-GC seem to be very promising. A completely on-line method for alkylated PAH determination in oils or lipidic extracts contaminated with mineral oil involves a two-dimensional LC-step with intermediate eluent evaporation and GC transfer through a vaporiser/overflow interface.     

Developing multiple high-throughput GLP methods for an investigational drug candidate in various matrices within a 96-well plate

In early pharmaceutical product development, an investigational drug candidate is typically dosed to various species for toxicological and pharmacokinetic studies. Most of these studies require multiple analytical methods that have to be validated with good laboratory practice (GLP) prior to the application in regulated studies. Usually, these analytical methods are developed in either a serial or parallel approach. For either approach, the development of multiple analytical methods takes tremendous work from scientists and instruments, and thus is not cost-effective. In this respect, a new strategy has been developed for simultaneous GLP method development using liquid chromatographic separation and tandem mass spectrometric detection. This high-throughput approach allows system suitability, carryover, calibration curve, accuracy, precision, matrix effect and selectivity to be evaluated in one 96-well plate. The strategy has been successfully implemented for multiple investigational drug candidates at Abbott Laboratories. The methods developed with this strategy are accurate, precise, selective, robust and matrix-independent. As an example, ABT-279 was used to demonstrate the feasibility of this strategy.     

Towards smaller and faster gas chromatography-mass spectrometry systems for field chemical detection

Gas chromatography-mass spectrometry (GC-MS) is already an important laboratory method, but new sampling techniques and column heating approaches will expand and improve its usefulness for detection and identification of unknown chemicals in field settings. In order to demonstrate commercially-available technical advances for both sampling and column heating, we used solid phase microextraction (SPME) sampling of both water and air systems, followed by immediate analysis with a resistively heated analytical column and mass spectrometric detection. High-concern compounds ranging from 140 to 466 amu were analyzed to show the applicability of these techniques to emergency situations impacting public health. A field portable (about 35 kg) GC-MS system was used for analysis of water samples with a resistively heated analytical column externally mounted as a retrofit using the air bath oven of the original instrument design to heat transfer lines. The system used to analyze air samples included a laboratory mass spectrometer with a dedicated resistive column heating arrangement (no legacy air bath column oven). The combined sampling and analysis time was less than 10 min for both air and water sample types. By combining dedicated resistive column heating with smaller mass spectrometry systems designed specificallyfor use in the field, substantially smaller high performance field-portable instrumentation will be possible.     

Method development approaches for capillary ion electrophoresis

Capillary ion electrophoresis (CIE) is a capillary electrophoretic technique optimized for rapid determination of low-molecular-mass inorganic and organic ions. CIE predominantly employs indirect UV detection since the majority of the analytes lack specific chromophores. Described are three methods for detection and electrolyte optimization. The first method discussed approaches for optimizing sensitivity, selectivity and peak confirmation using a chromate electrolyte and selected detection wavelengths. Peak confirmation is aided by using both direct detection of analytes. The second and third methods involve an unattended electrolyte development approach for instruments that only provide fresh electrolyte on the injection side of the capillary. The electrolyte composition is changed in both the injection side vial and in capillary before each sample injection while leaving the receiving side electrolyte vial constant at the initial electrolyte composition. In one mode, the concentration of the electroosmotic flow (EOF) modifier used to induce anodic flow is varied while keeping the background electrolyte composition constant. In a second experiment, the background electrolyte co-ion is sequentially changed from high mobility to low mobility while keeping the EOF modifier concentration constant. The end effect is to achieve a broad range of controlled peak symmetry for analytes in a simple matrix. The results are compared to separations obtained when the injection side and receiving side electrolytes are manually matched.     

Review of validation and reporting of non-targeted fingerprinting approaches for food authentication

Food fingerprinting approaches are expected to become a very potent tool in authentication processes aiming at a comprehensive characterization of complex food matrices. By non-targeted spectrometric or spectroscopic chemical analysis with a subsequent (multivariate) statistical evaluation of acquired data, food matrices can be investigated in terms of their geographical origin, species variety or possible adulterations. Although many successful research projects have already demonstrated the feasibility of non-targeted fingerprinting approaches, their uptake and implementation into routine analysis and food surveillance is still limited. In many proof-of-principle studies, the prediction ability of only one data set was explored, measured within a limited period of time using one instrument within one laboratory. Thorough validation strategies that guarantee reliability of the respective data basis and that allow conclusion on the applicability of the respective approaches for its fit-for-purpose have not yet been proposed. Within this review, critical steps of the fingerprinting workflow were explored to develop a generic scheme for multivariate model validation. As a result, a proposed scheme for “good practice” shall guide users through validation and reporting of non-targeted fingerprinting results. Furthermore, food fingerprinting studies were selected by a systematic search approach and reviewed with regard to (a) transparency of data processing and (b) validity of study results. Subsequently, the studies were inspected for measures of statistical model validation, analytical method validation and quality assurance measures. In this context, issues and recommendations were found that might be considered as an actual starting point for developing validation standards of non-targeted metabolomics approaches for food authentication in the future. Hence, this review intends to contribute to the harmonization and standardization of food fingerprinting, both required as a prior condition for the authentication of food in routine analysis and official control.     

Characterization of a suite of ginkgo-containing standard reference materials

A suite of three ginkgo-containing dietary supplement Standard Reference Materials (SRMs) has been issued by the National Institute of Standards and Technology (NIST) with certified values for flavonoid aglycones, ginkgolides, bilobalide, and selected toxic trace elements. The materials represent a range of matrices (i.e., plant, extract, and finished product) that provide different analytical challenges. The constituents have been determined by at least two independent analytical methods with measurements performed by NIST and at least one collaborating laboratory. The methods utilized different extractions, chromatographic separations, modes of detection, and approaches to quantitation. The SRMs are primarily intended for method validation and for use as control materials to support the analysis of dietary supplements and related botanical materials. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Multi-residue method for the analysis of 101 pesticides and their degradates in food and water samples by liquid chromatography/time-of-flight mass spectrometry

A comprehensive multi-residue method for the chromatographic separation and accurate mass identification of 101 pesticides and their degradation products using liquid chromatography/time-of-flight mass spectrometry (LC/TOF-MS) is reported here. Several classes of compounds belonging to different chemical families (triazines, organophosphorous, carbamates, phenylureas, neonicotinoids, etc.) were carefully chosen to cover a wide range of applications in the environmental field. Excellent chromatographic separation was achieved by the use of narrow accurate mass windows (0.05 Da) in a 30 min interval. Accurate mass measurements were always below 2 ppm error for all the pesticides studied. A table compiling the accurate masses for 101 compounds together with the accurate mass of several fragment ions is included. At least the accurate mass for one main fragment ion for each pesticide was obtained to achieve the minimum of identification points according to the 2002/657/EC European Decision, thus fulfilling the EU point system requirement for identification of contaminants in samples. The method was validated with vegetable samples. Calibration curves were linear and covered two orders of magnitude (from 5 to 500 microg/L) for most of the compounds studied. Instrument detection limits (LODs) ranged from 0.04 to 150 microg/kg in green-pepper samples. The methodology was successfully applied to the analysis of vegetable and water samples containing pesticides and their degradation products. This paper serves as a guide for those working in the analytical field of pesticides, as well as a powerful tool for finding non-targets and unknowns in environmental samples that have not been previously included in any of the routine target multi-residue methods.     

A mussel tissue certified reference material for multiple phycotoxins. Part 2: liquid chromatography-mass spectrometry, sample extraction and quantitation procedures

A freeze-dried mussel tissue certified reference material (CRM-FDMT1) containing multiple groups of shellfish toxins has been prepared. Toxin groups present in the material include okadaic acid and the dinophysistoxins, azaspiracids, yessotoxins, pectenotoxins, spirolides and domoic acid. In this work, analytical methods have been examined for the characterisation of the candidate CRM. A comprehensive extraction procedure was developed, which gave good recovery (>98%) for all lipophilic toxins studied. A fast liquid chromatography–mass spectrometry (LC-MS) method was developed that separates the major toxins according to the MS ionisation mode of optimum sensitivity. Matrix effects associated with analysis of these extracts using the developed LC-MS method were assessed. Standard addition and matrix-matched calibration procedures were evaluated to compensate for matrix effects. The methods and approaches will be used for the precise characterisation of the homogeneity and stability of the various toxins in CRM-FDMT1 and for the accurate assignment of certified values. The developed methods also have excellent potential for application in routine regulatory monitoring of shellfish toxins.     

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。     

Quantitative Determination of Copper: Combining Project-Based Laboratories with Single-Analyte Detection

A multiweek experiment is presented for use in undergraduate instrumental analysis courses. The experiment combines project-based laboratories and single-analyte detection to provide students with experience in method development and validation, and to give them a more realistic experience in the analytical laboratory. Working together as a team, students develop methods for the detection of an analyte (i.e., copper) in water samples using at least two spectroscopic instruments (e.g., ICP-AES, AA, UV-vis, fluorescence). Student teams are given only topical information about their projects, and must research and plan the analyses, learn the instrumental methods to be used, obtain figures of merit (e.g., detection limits) from Beers law plots, analyze commercial water samples, and produce a standard operating protocol for one of their methods, which will be validated by another team during a subsequent laboratory. Goals of this approach include promoting teamwork and building student confidence in approaching and operating unfamiliar instrumentation. Even more importantly, students are placed in the position of being scientists and having to make decisions and recommendations. Each step of the analytical process must be carefully considered, and its significance assessed as there are no recipes to follow as they develop their methods and make comparisons between different techniques for the determination of a single analyte.     

Applying non-parametric statistical methods to the classical measurements of inclusion complex binding constants

A study on using non-parametric statistical methods was carried out to calculate the binding constant of an inclusion complex and to estimate its associated uncertainty. First, a correct evaluation of the stoichiometry was carried out in order to ensure an accurate determination of the binding constant. For this purpose, the modified Benesi-Hildelbrand method had been previously applied. Then, four statistical methods (three non-parametric methods: two bootstrap approaches, the jackknife method and a parametric one: Fieller's theorem) were employed in order to compute the binding constant. The results obtained from applying these methods and the combination of the methods: jackknife after bootstrap and bootstrap after jackknife were compared. The best results in terms of accuracy were obtained from the application of a bootstrap method: the resampling residuals approach. These procedures were applied to the inclusion complex 2-hydroxil-propyl-beta-cyclodextrin-2,4-dichloro-phenoxyacetic, which shows photochemically-induced fluorescence.