Quality Assurance in Mycotoxin Analysis

Mycotoxin analysis in food and biological fluids is receiving more and more concern, in view also of increasing involvement by the European Union regarding legislation. Basically all the analytical steps regarding mycotoxin analysis have to be performed according to accurate criteria which are strictly connected to the quality of results in terms of reliability. The only rationale for reducing this difficulty is to apply quality assurance principles. Quality assurance principles define, in fact, the rules to be observed for performing this analysis with a degree of uncertainty that is as low as may be possible. In particular sampling techniques, if carried out improperly, give rise to uncertainty concerning the representativeness of samples that is so critical as to induce a dramatic source of errors in the final analysis. Therefore it seems appropriate to plan training courses for personnel on the various side-effects related to the available sampling and subsampling techniques depending on the commodity. Other contributions to the overall error derive from improper methodologies used by technicians in the pre-treatment step of the samples (incorrect use of glassware, standard solutions, etc.), and finally from the operations involved in the whole analytical procedure. In addition, the use of reference materials and certified reference materials together with the utilization of validated methods of analysis will be dealt with as concrete procedures for obtaining the certainty of final results of good quality. This aspect takes on a relevant outcome if applied to official control activities from authorized bodies acting at a national level.     

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.     

Method validation and reference materials

For implementation of food and feed legislation, there is a strong need for development and harmonisation of reliable, validated and if possible, robust and simple analytical methods. In addition, precise methods used for measuring the exposure of humans to certain types of food contaminants and residues (natural, man-made or produced during technological treatment) such as, e.g. mycotoxins, acrylamide, pesticides and allergens have to be available, in order to compare results derived from monitoring studies. Methods should be validated (in-house or in a collaborative trial) according to harmonised protocols and good laboratory practice must be in place in order to be compliant with internationally harmonised standards. The way in which this is implemented depends strongly on the analyte, interference within the food matrix and other requirements that need to be met. Food and feed certified reference materials, when matrix matched and containing the appropriate concentration of the certified substance, are an extremely useful tool in validation of measurements.Presented at BERM-9—Ninth International Symposium on Biological and Environmental Reference Materials, 15–19 June 2003, Berlin, Germany.     

Measurement uncertainty of food carotenoid determination

For consistent interpretation of an analytical method result it is necessary to evaluate the confidence that can be placed in it, in the form of a measurement uncertainty estimate. The Guide to the expression of Uncertainty in Measurement issued by ISO establishes rules for evaluating and expressing uncertainty. Carotenoid determination in food is a complex analytical process involving several mass transfer steps (extraction, evaporation, saponification, etc.), making difficult the application of these guidelines. The ISO guide was interpreted for analytical chemistry by EURACHEM, which includes the possibility of using intra- and interlaboratory information. Measurement uncertainty was estimated based on laboratory validation data, including precision and method performance studies, and also, based on laboratory participation in proficiency tests. These methods of uncertainty estimation were applied to analytical results of different food matrices of fruits and vegetables. Measurement uncertainty of food carotenoid determination was 10–30% of the composition value in the great majority of cases. Higher values were found for measurements near instrumental quantification limits (e.g. 75% for β-cryptoxanthin, and 99% for lutein, in pear) or when sample chromatograms presented interferences with the analyte peak (e.g. 44% for α-carotene in orange). Lower relative expanded measurement uncertainty values (3–13%) were obtained for food matrices/analytes not requiring the saponification step. Based on these results, the saponification step should be avoided if food carotenoids are not present in the ester form. Food carotenoid content should be expressed taking into account the measurement uncertainty; therefore the maximum number of significant figures of a result should be 2.     

Data processing pipelines for comprehensive profiling of proteomics samples by label-free LC-MS for biomarker discovery

Label-free quantitative LC-MS profiling of complex body fluids has become an important analytical tool for biomarker and biological knowledge discovery in the past decade. Accurate processing, statistical analysis and validation of acquired data diversified by the different types of mass spectrometers, mass spectrometer parameter settings and applied sample preparation steps are essential to answer complex life science research questions and understand the molecular mechanism of disease onset and developments. This review provides insight into the main modules of label-free data processing pipelines with statistical analysis and validation and discusses recent developments. Special emphasis is devoted to quality control methods, performance assessment of complete workflows and algorithms of individual modules. Finally, the review discusses the current state and trends in high throughput data processing and analysis solutions for users with little bioinformatics knowledge.     

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     

Hidden contributors to uncertainty and accuracy of results of residue analysis

Accurate analytical results with known uncertainty are required for the safety assessment of pesticides and testing the conformity of marketed food and feed with the maximum residue limits. The available information on various sources of errors was examined with special emphasis to those which may remain unaccounted for based on the current practice of many laboratories. The method validation typically covers the steps of the pesticide residue determination from the extraction of spiked samples to the instrumental determination, which contribute to only 10–40% of total variance of results. Though the variability of sampling, sample size reduction and sample processing may amount to the 60–90% of total variance, it generally remains unnoticed leading to wrong decisions. Another important source of gross error is the mismatch of the residues analysed and those included in the relevant residue definition. Procedures which may be applied for eliminating or reducing the errors are discussed.     

Uncertainty in chemical analysis and validation of the analytical method: acid value determination in oils

Quantifying uncertainty in chemical analysis, according to EURACHEM document (1995), is based on known relationships between parameters of the analytical procedure and corresponding results of the analysis. This deterministic concept is different from the cybernetic approach to analytical method validation, where the whole analytical procedure is a "black box". In the latter case, analytical results only are the basis for statistical characterization of the method without any direct relationship with intermediate measurement results like weighings, volumes, instrument readings, or other parameters like molecular masses. This difference requires the harmonization of parameters to be validated and to be included in the uncertainty calculation. As an example, results of the uncertainty calculation and validation are discussed for a new method of acid value determination in oils by pH measurement without titration.     

The application of the measurement uncertainty concept to in-process control in pharmaceutical development

 Any analytical data is used to provide information about a sample. The "possible error" of the measurement can be of extreme importance in order to have complete information. The measurement uncertainty concept is a way to achieve quantitative information about this "possible error" using an estimation procedure. On the basis of the analytical result, the chemist makes a decision on the next step of the development process. If the uncertainty is unknown, the information is not complete; therefore this decision might be impossible. The major problem for the in-process control (IPC) procedure is that not only the repeatability but also the intermediate precision (which expresses the variations within laboratories related to different days, different analysts, different equipment, etc.) has to be good enough to make a decision. Unfortunately, the statistical information achieved from one single analytical run only gives information about the repeatability. This paper shows that the estimation of the measurement uncertainty for IPC is a way to solve the problem and gives the necessary information about the quality of the procedure. An example demonstrates that an estimate of uncertainty based on the standard deviations of an analytical method gives a value similar to one based on the standard deviations obtained from a control chart. Therefore, the estimation is both a very useful and also a very cost-effective tool. Though measurement uncertainty cannot replace validation in general, it is a viable alternative to validation for all methods that will never be used routinely. Received: 24 May 1996 Accepted: 10 August 1996     

The Existing Methods and Novel Approaches in Mycotoxins’ Detection

Mycotoxins represent a wide range of secondary, naturally occurring and practically unavoidable fungal metabolites. They contaminate various agricultural commodities like cereals, maize, peanuts, fruits, and feed at any stage in pre- or post-harvest conditions. Consumption of mycotoxin-contaminated food and feed can cause acute or chronic toxicity in human and animals. The risk that is posed to public health have prompted the need to develop methods of analysis and detection of mycotoxins in food products. Mycotoxins wide range of structural diversity, high chemical stability, and low concentrations in tested samples require robust, effective, and comprehensible detection methods. This review summarizes current methods, such as chromatographic and immunochemical techniques, as well as novel, alternative approaches like biosensors, electronic noses, or molecularly imprinted polymers that have been successfully applied in detection and identification of various mycotoxins in food commodities. In order to highlight the significance of sampling and sample treatment in the analytical process, these steps have been comprehensively described.     

Nuclear forensics—metrological basis for legal defensibility

The admissibility of nuclear forensics measurements and opinions derived from them in US Federal and State courts are based on criteria established by the US Supreme Court in the case of Daubert v. Merrell Dow and the 2000 Amendment of Rule 702 of the Federal Rules of Evidence. These criteria are being addressed by new efforts that include the development of certified reference materials (CRMs) to provide the basis for analytical method development, optimization, calibration, validation, quality control, testing, readiness, and declaration of measurement uncertainties. Quality data is crucial for all stages of the program, from R&D, and database development, to actual casework. Weakness at any point in the program can propagate to reduce the confidence of final conclusions. The new certified reference materials will provide the necessary means to demonstrate a high level of metrological rigor for nuclear forensics evidence and will form a foundation for legally defensible nuclear chemical analysis. The CRMs will allow scientists to devise validated analytical methods, which can be corroborated by independent analytical laboratories. CRMs are required for ISO accreditation of many different analytical techniques which may be employed in the analysis of interdicted nuclear materials.     

Cold vapour atomic fluorescence spectrometry and gas chromatography-pyrolysis-atomic fluorescence spectrometry for routine determination of total and organometallic mercury in food samples

Two procedures have been investigated for the quantification of the different forms of mercury in food. A two-stage procedure has been developed to determine firstly total inorganic and organometallic species, and then the full separation of all organomercury species. The procedure involves solubilisation of the samples using alkaline extractions or enzymolysis, followed by the extraction of organic mercury in an organic solvent, preferably a mixture of dichloromethane and hexane (3:2). For the total organic mercury determination, the organic extract is analysed for "total" mercury after nitric acid/peroxide digestion, evaporation of the solvent and detection by cold vapour-atomic fluorescence spectrometry. Full organomercury speciation requires a clean-up step before analysis of the final extract in dichloromethane by gas chromatography coupled to a pyrolyser and an atomic fluorescence detector (GC-pyro-AFS). A detection limit of 6 ng l-1, and reproducibility of 2% was achieved for the CV-AFS method; GC-pyro-AFS yielded 200 ng l-1 and 5% for detection limit and coefficient of variation, respectively. Both procedures were validated with the use of various certified reference materials over a wide range of mercury concentrations, and by spiking experiments. The validated methods were tested successfully on a wide range of commercially available food samples.     

Quality criteria of the isotope dilution method for the determination of polycyclic aromatic hydrocarbons, petroleum-derived hydrocarbons and phenol in leachate of large-scale lysimeter experiments

A comprehensive analytical procedure for polycyclic aromatic hydrocarbons, petroleum-derived hydrocarbons and phenol using the isotope dilution method and employing high-resolution gas chromatography and mass spectrometry was developed and validated for leachates from source-term experiments and the different sampling sites of lysimeters. The use of glassware and other materials is efficiently limited to minimize the risk of contamination. The relative standard deviation elaborated allows precise reliable measurements. Limit of detection and recovery data are useful to judge the quality of each single measurement.     

Natural toxins: risks,regulations and the analytical situation in Europe

Natural toxins in food and feed are considered important food safety issues of growing concern, in particular mycotoxins, phycotoxins and plant toxins. Most scientific developments have occurred in the past few decades in the area of mycotoxins. Formal health risk assessments have been carried out by the Joint Expert Committee on Food Additives of the World Health Organization and the Food and Agriculture Organization. Limits and regulations for mycotoxins in food and feed have been established in many countries, including practically all European countries. An array of (formally validated) analytical methods and (certified) reference materials have become available. Several European research projects, funded by the European Commission and supported by the European Standardization Committee, have significantly contributed to this development. Quantitative methods of analysis for mycotoxins often make use of immunoaffinity cleanup with liquid chromatographic or gas chromatographic separation techniques in combination with various types of detectors, including mass spectroscopy. For screening purposes (bio)sensor-based techniques are among the promising newcomers. For the phycotoxins the situation is less advanced. Formal risk assessments by authoritative international bodies have not been carried out. Methods of analysis, formally validated according to internationally harmonized protocols, are scarce and animal testing still plays a key role in official methodology. The development of the analytical methodology is partly hampered by the limited availability of certain reliable calibrants and reference materials, although this situation is gradually improving. New regulations in the European Union have increased the pressure to develop and validate chemical methods of analysis. Joint efforts in the European context are now directed towards significantly improving this situation, and techniques such as liquid chromatography–mass spectroscopy offer promise in this respect. Both the working group on biotoxins of the European Standardization Committee and the network of National Reference Laboratories for Marine Biotoxins have taken up responsibilities here. The plant toxins are a category of natural toxins, where the situation is the least developed with respect to regulations, validated methods of analysis and reference materials. Yet, their occurrence in a wide range of consumable plant species demands the attention of the analytical community.     

Validation of analytical methods involved in dissolution assays: Acceptance limits and decision methodologies

Dissolution tests are key elements to ensure continuing product quality and performance. The ultimate goal of these tests is to assure consistent product quality within a defined set of specification criteria. Validation of an analytical method aimed at assessing the dissolution profile of products or at verifying pharmacopoeias compliance should demonstrate that this analytical method is able to correctly declare two dissolution profiles as similar or drug products as compliant with respect to their specifications. It is essential to ensure that these analytical methods are fit for their purpose. Method validation is aimed at providing this guarantee. However, even in the ICHQ2 guideline there is no information explaining how to decide whether the method under validation is valid for its final purpose or not. Are the entire validation criterion needed to ensure that a Quality Control (QC) analytical method for dissolution test is valid? What acceptance limits should be set on these criteria? How to decide about method's validity? These are the questions that this work aims at answering. Focus is made to comply with the current implementation of the Quality by Design (QbD) principles in the pharmaceutical industry in order to allow to correctly defining the Analytical Target Profile (ATP) of analytical methods involved in dissolution tests. Analytical method validation is then the natural demonstration that the developed methods are fit for their intended purpose and is not any more the inconsiderate checklist validation approach still generally performed to complete the filing required to obtain product marketing authorization.     

A validation concept for purity determination and assay in the pharmaceutical industry using measurement uncertainty and statistical process control

 The analytical chemists in process development in the pharmaceutical industry have to solve the difficult problem of producing high quality methods for purity determination and assay within a short time without a clear definition of the substance to be analyzed. Therefore the quality management is very difficult. The ideal situation would be that every method is validated before use. This is not possible because this would delay the development process. A process-type quality development approach with an estimation type fast validation (measurement uncertainty) is therefore suggested. The quality management process consists of the estimation of measurement uncertainty for early project status. Statistical process control (SPC) is started directly after measurement uncertainty estimation and a classical validation for the end of the project. By this approach a process is defined that allows a fast and cost-efficient way of supporting the development process with the appropriate quality at the end of the process and provides the transparency needed in the development process. The procedure presented tries to solve the problem of the parallelism between the two development processes (chemical and analytical development) by speeding up the analytical development process initially. Received: 25 March 1997 · Accepted: 17 May 1997     

Optimization and Verification of Droplet Digital PCR Even-Specific Methods for the Quantification of GM Maize DAS1507 and NK603

In recent years, digital polymerase chain reaction (dPCR), a new molecular biology technique, has been gaining in popularity. Among many other applications, this technique can also be used for the detection and quantification of genetically modified organisms (GMOs) in food and feed. It might replace the currently widely used real-time PCR method (qPCR), by overcoming problems related to the PCR inhibition and the requirement of certified reference materials to be used as a calibrant. In theory, validated qPCR methods can be easily transferred to the dPCR platform. However, optimization of the PCR conditions might be necessary. In this study, we report the transfer of two validated qPCR methods for quantification of maize DAS1507 and NK603 events to the droplet dPCR (ddPCR) platform. After some optimization, both methods have been verified according to the guidance of the European Network of GMO Laboratories (ENGL) on analytical method verification (ENGL working group on “Method Verification.” (2011) Verification of Analytical Methods for GMO Testing When Implementing Interlaboratory Validated Methods). Digital PCR methods performed equally or better than the qPCR methods. Optimized ddPCR methods confirm their suitability for GMO determination in food and feed.