From fundamentals in calibration to modern methodologies: A tutorial for small molecules quantification in liquid chromatography-mass spectrometry bioanalysis.

Over the last two decades, liquid chromatography coupled to mass-spectrometry (LC‒MS) has become the gold standard to perform qualitative and quantitative analyses of small molecules. When quantitative analysis is developed, an analyst usually refers to international guidelines for analytical method validation. In this context, the design of calibration curves plays a key role in providing accurate results. During recent years and along with instrumental advances, strategies to build calibration curves have dramatically evolved, introducing innovative approaches to improve quantitative precision and throughput. For example, when a labeled standard is available to be spiked directly into the study sample, the concentration of the unlabeled analog can be easily determined using the isotopic pattern deconvolution or the internal calibration approach, eliminating the need for multipoint calibration curves. This tutorial aims to synthetize the advances in LC‒MS quantitative analysis for small molecules in complex matrices, going from fundamental aspects in calibration to modern methodologies and applications. Different work schemes for calibration depending on the sample characteristics (analyte and matrix nature) are distinguished and discussed. Finally, this tutorial outlines the importance of having international guidelines for analytical method validation that agree with the advances in calibration strategies and analytical instrumentation.

The discrimination threshold: A selection criterion for analytical methods based on measurement uncertainty - Application to animal stress studies.

Many biological studies seek to confirm the effect of a treatment on the levels of chemical markers in biological tissues. This requires choosing the analytical method best able to detect the difference between basal levels and those found after treatment. We propose a new approach to calculate a criterion we call the 'discrimination threshold' (DT), and we applied it to an analytical method that we developed to determine cortisol in cattle plasma to detect stress. DT is derived from the measurement uncertainty (MU), and combines the variabilities of both the analytical method and the marker. The uncertainty of the analytical method comes from the method validation study. The marker variability (here cortisol) is modeled from a literature review. The graphical representation of DT allows estimating the applicability of the method. Our analytical method is shown to perform well when the difference in blood cortisol exceeds 18 ng. mL-1.

Derivation of uncertainty functions from validation studies in biological fluids: application to the analysis of caffeine and its major metabolites in human plasma samples.

Procedures for estimating the measurement uncertainty (MU) of the concentration of a given analyte in a sample are of major concern for analytical chemists. Unfortunately, it is still unclear how and why MU should be assessed. While several possibilities exist, an appropriate approach consists in using method validation data for the evaluation of MU. This was demonstrated by a validation study achieved in the framework of a clinical study related to caffeine in sports medicine, where the results were used for the evaluation of MU. After validation of the method developed using ultra-high pressure liquid chromatography-mass spectrometry for caffeine and its three main metabolites, accuracy profiles were built for each analyte. The first important conclusion is that the developed method was valid for all compounds and met the given specifications for the application (fit for purpose). Relevant estimates of combined standard uncertainty were computed to obtain uncertainty functions, which allow obtaining values of MU as a function of the concentration of the analyte. The great advantage of both uncertainty function and uncertainty profile is the development of a continuous model that enables easy calculation of the standard, expanded and relative expanded uncertainty at any concentration within the validation domain. In fact, the expanded uncertainty interval is assumed to contain 95% of all possible measurements, regardless of the concentration. Finally, the uncertainty function enables the determination of the lowest limit of quantification by selecting adequate acceptance limits, with the limit of quantification being defined as the point where the relative uncertainty equals the acceptance limit threshold. It has to be noted that further discussions remain mandatory to establish which criteria should be applied to define an adequate decision threshold, and the proposal afforded in this work may open new avenues in this direction.

New approach for the assessment of cluster diets.

Dietary risk assessment is a major public health concern, positioned in the context of establishing overall food safety policy. It requires some understanding of population food choices although geographical location and social-cultural environment are variable. Several years ago, a cluster analysis based on FAO consumption data, ranging from 1990 to 1994, was at the origin of the 13, so called, GEMS/Food cluster diets. This analysis required the initial identification of 19 food markers based on geographical and cultural differences. This paper proposes a new modelling of FAO food consumption database in order to define new cluster diets based on updated consumption data from 2002 to 2007 and better adapted statistical methods. Two statistical methods were combined to extract, consumption systems that generate a substructure from the initial food consumption database and then by deriving a clustering of countries according to their consumption system profiles. The clustering resulted in 17 cluster diets composed of 2 up to 30 countries. The few discrepancies between these new clusters and former ones may be due to more recent data, and to the fact that the new approach is based on another mathematical modelling which does not require any initial identification of food markers.

The OBELIX project: early life exposure to endocrine disruptors and obesity.

The hypothesis of whether early life exposure (both pre- and early postnatal) to endocrine-disrupting chemicals (EDCs) may be a risk factor for obesity and related metabolic diseases later in life will be tested in the European research project OBELIX (OBesogenic Endocrine disrupting chemicals: LInking prenatal eXposure to the development of obesity later in life). OBELIX is a 4-y project that started in May 2009 and which has the following 5 main objectives: 1) to assess early life exposure in humans to major classes of EDCs identified as potential inducers of obesity (ie, dioxin-like compounds, non-dioxin-like polychlorinated biphenyls, organochlorine pesticides, brominated flame retardants, phthalates, and perfluorinated compounds) by using mother-child cohorts from 4 European regions with different food-contaminant exposure patterns; 2) to relate early life exposure to EDCs with clinical markers, novel biomarkers, and health-effect data related to obesity; 3) to perform hazard characterization of early life exposure to EDCs for the development of obesity later in life by using a mouse model; 4) to determine mechanisms of action of obesogenic EDCs on developmental programming with in vivo and in vitro genomics and epigenetic analyses; and 5) to perform risk assessments of prenatal exposure to obesogenic EDCs in food by integrating maternal exposure through food-contaminant exposure and health-effect data in children and hazard data in animal studies.

Uncertainty in intake due to portion size estimation in 24-hour recalls varies between food groups.

Portion size estimation is expected to be one of the largest sources of uncertainty in dietary assessment of the individual. Therefore, we demonstrated a method to quantify uncertainty due to portion size estimation in the usual intake distributions of vegetables, fruit, bread, protein, and potassium. Dutch participants of the European Food Consumption Validation study completed 2 nonconsecutive 24-h recall interviews. In short, the uncertainty analysis consists of Monte Carlo simulations drawing values for portion size from lognormal uncertainty distributions. The uncertainty of the usual intake distribution and accompanying parameters (IQR and the shrinkage factor) were estimated. For the food groups, portion size uncertainty had the greatest effect for vegetables and the least for fruit: the relative 95% uncertainty interval (UI) of the IQR of the usual intake distribution was 0.61-1.35 for vegetables, 0.77-1.24 for bread, and 0.99-1.10 for fruit. For protein and potassium, the resulting relative width of the UI of the IQR for portion size uncertainty are similar: 0.88-1.14 for protein and 0.86-1.14 for potassium. Furthermore, a sensitivity analysis illustrated the importance of the specified uncertainty distributions. The examples show that uncertainty in portion sizes may be more important for some foods such as vegetables. This may reflect differential quantification errors by food groups that deserve further consideration. In conclusion, the presented methodology allows the important quantification of portion size uncertainty and extensions to include other sources of uncertainty is straightforward.

Extraction of food consumption systems by nonnegative matrix factorization (NMF) for the assessment of food choices.

In Western countries where food supply is satisfactory, consumers organize their diets around a large combination of foods. It is the purpose of this article to examine how recent nonnegative matrix factorization (NMF) techniques can be applied to food consumption data to understand these combinations. Such data are nonnegative by nature and of high dimension. The NMF model provides a representation of consumption data through latent vectors with nonnegative coefficients, that we call consumption systems (CS), in a small number. As the NMF approach may encourage sparsity of the data representation produced, the resulting CS are easily interpretable. Beyond the illustration of its properties we provide through a simple simulation result, the NMF method is applied to data issued from a French consumption survey. The numerical results thus obtained are displayed and thoroughly discussed. A clustering based on the k-means method is also achieved in the resulting latent consumption space, to recover food consumption patterns easily usable for nutritionists.

Validation of alternative methods for the analysis of drinking water and their application to Escherichia coli.

In Europe, the Drinking Water Directive of the European Commission indicates which methods (most of which are CEN/ISO-standardized methods) should be used for the analysis of microbiological parameters (European Commission, Environment, Council Directive 98/83/EC of 3 November 1998). According to the Directive, alternative methods "may be used, providing it can be demonstrated that the results obtained are at least as reliable as those produced by the methods specified." The prerequisite for the routine use of any alternative method is to provide evidence that this method performs equivalently to the corresponding reference method. In this respect, the ISO 16140 standard (ISO, ISO 16140. Microbiology of Food and Animal Feeding Stuffs-Protocol for the Validation of Alternative Methods, 2003) represents a key issue in generating such a procedure based on an interlaboratory study. A new statistical tool, called the accuracy profile, has been developed to better interpret the data. The study presented here is based upon the enumeration of Escherichia coli bacteria in water. The reference method may require up to 72 h to provide a confirmed result. The aim of this publication is to present data for an alternative method by which results can be obtained in 18 h (Colilert-18/Quanti-Tray) based upon defined substrate technology (DST). The accuracy profile is a statistical and graphical decision-making tool and consists of simultaneously combining, in a single graphic, ß expectation tolerance intervals (ß-ETIs) and acceptability limits (λ). The study presents the validation criteria calculated at the three levels of contamination used in the trial for a ß equal to 80% and a λ equal to ±0.3 and combines the accuracy profiles of Escherichia coli for a λ of ±0.3 log10 unit/100 ml, a λ of ±0.4 log10 unit/100 ml, and a ß of 80% or 90%. Several interesting conclusions can be drawn from these data. The accuracy profile method has been applied to the validation of the Colilert-18/Quanti-Tray method against reference method ISO 9308-1 (ISO, ISO 9308-1. Water Quality--Detection and Enumeration of Escherichia coli and Coliform Bacteria. Part 1. Membrane Filtration Method, 2000), using a ß of 80% and a λ of 0.4; the alternative method can be validated between 1.00 and 2.05 log10 units/100 ml, equivalent to 10 to 112 CFU/100 ml.

Construction of measurement uncertainty profiles for quantitative analysis of genetically modified organisms based on interlaboratory validation data.

A method is presented for estimating the size of uncertainty associated with the measurement of products derived from genetically modified organisms (GMOs). The method is based on the uncertainty profile, which is an extension, for the estimation of uncertainty, of a recent graphical statistical tool called an accuracy profile that was developed for the validation of quantitative analytical methods. The application of uncertainty profiles as an aid to decision making and assessment of fitness for purpose is also presented. Results of the measurement of the quantity of GMOs in flour by PCR-based methods collected through a number of interlaboratory studies followed the log-normal distribution. Uncertainty profiles built using the results generally give an expected range for measurement results of 50-200% of reference concentrations for materials that contain at least 1% GMO. This range is consistent with European Network of GM Laboratories and the European Union (EU) Community Reference Laboratory validation criteria and can be used as a fitness for purpose criterion for measurement methods. The effect on the enforcement of EU labeling regulations is that, in general, an individual analytical result needs to be < 0.45% to demonstrate compliance, and > 1.8% to demonstrate noncompliance with a labeling threshold of 0.9%.

Interpretation of interlaboratory trials based on accuracy profiles.

Validation is a very active field in analytical chemistry as illustrated by the numerous publications addressing this topic. According to most definitions, method validation is required to confirm the fitness-for-purpose of a particular analytical method. When considering interlaboratory validation, organization and calculation of parameters that describe method performance follow well-recognized standards. But the strategy used to verify whether the method is fit for a particular purpose is still under discussion. This paper presents a method for assessing the fitness-for-purpose of analytical methods based on results produced during interlaboratory trials. It is based on the construction of accuracy profiles, which can be used as a graphical decision support tool, and demonstrates how accuracy profiles, initially developed for in-house validations, can be extended to interlaboratory studies. These interlaboratory accuracy profiles use measurements collected under reproducibility conditions to give an interval where an expected proportion of future measurements will be located. This interval can be compared to an acceptability interval defined by the end-user to simply decide whether a method is fit-for-purpose or not. Several examples of application illustrate how data can be interpreted to draw conclusions about produced accuracy profiles and fitness-for-purpose. Hence, the accuracy profile could be used as a harmonized procedure to assess the performance of analytical methods that undergo interlaboratory validation.

Validation of an alternative method for counting Enterobacteriaceae in foods based on accuracy profile.

Several years ago, microbiologists developed a harmonized standard for the validation of alternative methods against the reference method, namely International Organization for Standardization (ISO) 16140:2003. Since then a new and better statistical approach known as the accuracy profile concept has been proposed for interpretation of data collected during a validation study. This approach defines acceptability limits that are used to verify that an alternative analytical method can produce results acceptable for a defined analytical objective. This paper demonstrates how the accuracy profile approach can be efficiently applied to any data set collected according to ISO guidelines and how to interpret the final calculations to decide if a method is valid or not. The procedure was applied to data obtained in the validation of a commercial kit for the enumeration of Enterobacteriaceae in foods. A comparison of several statistical outputs derived from application of the accuracy profile demonstrated that this simple numerical method can be applied despite the non-normal distribution of bacterial colony counts.

Determination of complex polysaccharides by HPAE-PAD in foods: validation using accuracy profile.

The increasing supplementation of foods with carbohydrates substitutes and the growing regulatory requirements for controlling these products, turn into the necessary development and validation of accurate analytical control techniques. This paper presents the simultaneous validation of two close analytical procedures for the determination of sucralose and fructooligosaccharides (FOS) in fruit juices using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAE-PAD). This study consisted in applying the accuracy profile procedure with a three-level validation experimental design. Decision criteria, namely acceptability limits (+/-10%) and proportion of result contained in the calculated tolerance intervals (80%), were decided on a consensus basis with end-users, whereas no official references were available. In conclusion, the proposed analytical procedures were validated over the selected validation domains for fruit juices and came out on very capable techniques. Validation strategy was purposely oriented towards the ease of use in routine and the liability of the methods rather than extreme performances. This objective is consistent with this of contract laboratories which need to reach a known level of guarantee for the results which they produce. In that respect, accuracy profile represents a very convenient tool to ascertain such a goal.

Application of the ANOVA-PCA method to stability studies of reference materials.

Near infrared spectroscopy (NIRS) is an analytical technique that can be very useful for stability studies in particular because of its non destructive analytical capability. However, the spectral interpretation and treatment of this kind of multivariate data remains difficult without the use of chemometrics. In this article, a recent chemometrics method, analysis of variance--principal component analysis (ANOVA-PCA), was used for NIRS stability studies of sunflower and bread wheat external reference materials (ERM). It provided a practical tool for the study of the significance of various storage conditions according to an experimental design. Thus, the effect of the temperature, the nature of the atmosphere in the packaging and the storage duration were tested. ANOVA-PCA highlighted the influence of temperature and storage duration on the stability of the sunflower materials. For the bread wheat materials, the storage conditions did not have a significant effect on stability. Consequently, by applying ANOVA-PCA to near infrared spectral data, the sunflower materials were found to be considered stable for the time length of the study, i.e. 18 months stored in a cold room, while the bread wheat materials were found to be considered stable for the time length of the study, i.e. 12 months under the same conditions.

Validation of analytical methods based on accuracy profiles.

Validation is a very living field in analytical chemistry as illustrated by the numerous publications addressing this topic. But, there is some ambiguity in this concept and the abundant vocabulary often does not help the analytical chemist. This paper presents a new method based on the fitness-for-purpose approach of the validation. It consists in building a graphical decision-making tool, called the accuracy profile. Using measurements collected under reproducibility or intermediate precision condition, it allows computing an interval where a known proportion of future measurements will be located. When comparing this interval to an acceptability interval defined by the result end-user it is possible to simply decide whether a method is valid or not. The fundamentals of this method are presented starting from an accepted definition of validation. An example of application illustrates how validation can be experimentally organized and conclusion made.

Experimental evaluation of different precision criteria applicable to microbiological counting methods.

The dispersion of microbiological counting measurements, when repeating the analysis on the same material both within a laboratory (repeatability) and between laboratories (reproducibility) can be characterized by the organization of interlaboratory studies, where several sets of identical test materials are sent to several laboratories. Using the example of data generated by an interlaboratory study on enumeration of Listeria monocytogenes in foods by the standardized reference method (colony-count technique), 2 types of robust estimators of reproducibility standard deviations, based on the median, were examined, in comparison with the classical estimators, based on the mean. Experimental evaluation indicated that the 3 approaches gave consistent results for most of the combinations. The usual log10 transformation of the enumeration results was also questioned before these calculations were conducted.

Quantification of the 35S promoter in DNA extracts from genetically modified organisms using real-time polymerase chain reaction and specificity assessment on various genetically modified organisms, part I: operating procedure.

A highly sensitive quantitative real-time assay targeted on the 35S promoter of a commercial genetically modified organism (GMO) was characterized (sF/sR primers) and developed for an ABI Prism 7700 Sequence Detection System and TaqMan chemistry. The specificity assessment and performance criteria of sF/sR assay were compared to other P35S-targeted published assays. sF/sR primers amplified a 79 base pair DNA sequence located in a part of P35S that is highly conserved among many caulimovirus strains, i.e., this consensus part of CaMV P35S is likely to be present in many GM events. According to the experimental conditions, the absolute limit of detection for Bt176 corn was estimated between 0.2 and 2 copies of equivalent genome (CEG). The limit of quantification was reached below 0.1% Bt176 content. A Cauliflower Mosaic Virus control (CaMV) qualitative assay targeted on the ORF III of the viral genome was also used as a control (primers 3F/3R) to assess the presence of CaMV in plant-derived products. The specificity of this test was assessed on various CaMV strains, including the Figwort Mosaic Virus (FMV) and solanaceous CaMV strains. Considering the performance of sF/sR quantification test, the highly conserved sequence, and the small size of the amplicon, this assay was tested in a collaborative study in order to be proposed as an international standard.

Quantitation of 35S promoter in maize DNA extracts from genetically modified organisms using real-time polymerase chain reaction, part 2: interlaboratory study.

The European Committee for Standardization (CEN) and the European Network of GMO Working Laboratories have proposed development of a modular strategy for stepwise validation of complex analytical techniques. When applied to the quantitation of genetically modified organisms (GMOs) in food products, the instrumental quantitation step of the technique is separately validated from the DNA extraction step to better control the sources of uncertainty and facilitate the validation of GMO-specific polymerase chain reaction (PCR) tests. This paper presents the results of an interlaboratory study on the quantitation step of the method standardized by CEN for the detection of a regulatory element commonly inserted in GMO maize-based foods. This is focused on the quantitation of P35S promoter through using the quantitative real-time PCR (QRT-PCR). Fifteen French laboratories participated in the interlaboratory study of the P35S quantitation operating procedure on DNA extract samples using either the thermal cycler ABI Prism 7700 (Applied Biosystems, Foster City, CA) or Light Cycler (Roche Diagnostics, Indianapolis, IN). Attention was focused on DNA extract samples used to calibrate the method and unknown extract samples. Data were processed according to the recommendations of ISO 5725 standard. Performance criteria, obtained using the robust algorithm, were compared to the classic data processing after rejection of outliers by the Cochran and Grubbs tests. Two laboratories were detected as outliers by the Grubbs test. The robust precision criteria gave values between the classical values estimated before and after rejection of the outliers. Using the robust method, the relative expanded uncertainty by the quantitation method is about 20% for a 1% Bt176 content, whereas it can reach 40% for a 0.1% Bt176. The performances of the quantitation assay are relevant to the application of the European regulation, which has an accepted tolerance interval of about +/-50%. These data were fitted to a power model (r2 = 0.96). Thanks to this model, it is possible to propose an estimation of uncertainty of the QRT-PCR quantitation step and an uncertainty budget depending on the analytical conditions.

New advances in method validation and measurement uncertainty aimed at improving the quality of chemical data.

The implementation of quality systems in analytical laboratories has now, in general, been achieved. While this requirement significantly modified the way that the laboratories were run, it has also improved the quality of the results. The key idea is to use analytical procedures which produce results that fulfil the users' needs and actually help when making decisions. This paper presents the implications of quality systems on the conception and development of an analytical procedure. It introduces the concept of the lifecycle of a method as a model that can be used to organize the selection, development, validation and routine application of a method. It underlines the importance of method validation, and presents a recent approach based on the accuracy profile to illustrate how validation must be fully integrated into the basic design of the method. Thanks to the beta-expectation tolerance interval introduced by Mee (Technometrics (1984) 26(3):251-253), it is possible to unambiguously demonstrate the fitness for purpose of a new method. Remembering that it is also a requirement for accredited laboratories to express the measurement uncertainty, the authors show that uncertainty can be easily related to the trueness and precision of the data collected when building the method accuracy profile.

Bacillus Calmette Guerin triggers the IL-12/IFN-gamma axis by an IRAK-4- and NEMO-dependent, non-cognate interaction between monocytes, NK, and T lymphocytes.

The IL-12/IFN-gamma axis is crucial for protective immunity to Mycobacterium in humans and mice. Our goal was to analyze the relative contribution of various human blood cell subsets and molecules to the production of, or response to IL-12 and IFN-gamma. We designed an assay for the stimulation of whole blood by live M. bovis Bacillus Calmette-Guerin (BCG) alone, or BCG plus IL-12 or IFN-gamma, measuring IFN-gamma and IL-12 levels. We studied patients with a variety of specific inherited immunodeficiencies resulting in a lack of leukocytes, or T, B, and/or NK lymphocytes, or polymorphonuclear cells, or a lack of expression of key molecules such as HLA class II, CD40L, NF-kappaB essential modulator (NEMO), and IL-1 receptor-associated kinase-4 (IRAK-4). Patients with deficiencies in IL-12p40, IL-12 receptor beta1 chain (IL-12Rbeta1), IFN-gammaR1, IFN-gammaR2, and STAT-1 were used as internal controls. We showed that monocytes were probably the main producers of IL-12, and that NK and T cells produced similar amounts of IFN-gamma. NEMO and IRAK-4 were found to be important for IL-12 production and subsequent IFN-gamma production, while a lack of CD40L or HLA class II had no major impact on the IL-12/IFN-gamma axis. The stimulation of whole blood by live BCG thus triggers the IL-12/IFN-gamma axis by an IRAK-4- and NEMO-dependent, non-cognate interaction between monocytes, NK, and T lymphocytes.

Homogeneity check of agricultural and food industries samples using near infrared spectroscopy.

Samples distributed in proficiency testing schemes (PTS) need to be homogeneous in order to be sure that if a laboratory has a result different from the other laboratories, its error can be attributed to its analysis method and not to its sample. This control must be done according to the ISO 13528 draft standard before sending the samples to the laboratories. It can be done by determining homogeneity targets by sub-contracting to accredited laboratories using reference methods, but this engenders logistic and financial problems. That is why a homogeneity check using Near Infrared Spectroscopy (NIR) has been developed for agricultural and food industries samples prepared for PTS at Bipea (Bureau Interprofessionnel d'Etudes Analytiques). To evaluate the homogeneity among samples, this procedure involves a comparison of NIR spectra, the determination of global homogeneity criteria and the use of control charts. The method of control developed and carried out at Bipea allows the rapid and easy monitoring of the performance of the sample preparation.