Elsevier

Analytica Chimica Acta

Volume 909, 25 February 2016, Pages 9-23
Analytica Chimica Acta

Tutorial
Chromatographic fingerprinting: An innovative approach for food 'identitation' and food authentication – A tutorial

https://doi.org/10.1016/j.aca.2015.12.042Get rights and content

Highlights

  • Chemical approaches for food authentication, i.e., chemical markers, component profiling and instrumental fingerprinting, have been described.

  • Difference between chromatographic signals and data are clarified.

  • The food authentication framework using chromatographic fingerprinting has been properly established and discussed.

  • A new term, named food ‘identitation’, has been proposed as a prior and necessary stage in order to close the life cycle of the food authentication process.

  • Right analytical applications of chromatographic fingerprinting on food ‘identitation’ and food authentication have been reviewed.

Abstract

Fingerprinting methods describe a variety of analytical methods that provide analytical signals related to the composition of foodstuffs in a non-selective way such as by collecting a spectrum or a chromatogram. Mathematical processing of the information in such fingerprints may allow the characterisation and/or authentication of foodstuffs. In this context, the particular meaning of 'fingerprinting', in conjunction with 'profiling', is different from the original meanings used in metabolomics. This fact has produced some confusion with the use of these terms in analytical papers. Researchers coming from the metabolomic field could use 'profiling' or 'fingerprinting' on a different way to researchers who are devoted to food science. The arrival of an eclectic discipline, named 'foodomics' has not been enough to allay this terminological problem, since the authors keep on using the terms with both meanings. Thus, a first goal of this tutorial is to clarify the difference between both terms. In addition, the chemical approaches for food authentication, i.e., chemical markers, component profiling and instrumental fingerprinting, have been described. A new term, designated as 'food identitation', has been introduced in order to complete the life cycle of the chemical-based food authentication process. Chromatographic fingerprinting has been explained in detail and some strategies which could be applied has been clarified and discussed. Particularly, the strategies for chromatographic signals acquisition and chromatographic data handling are unified in a single framework. Finally, an overview about the applications of chromatographic (GC and LC) fingerprints in food authentication using different chemometric techniques has been included.

Introduction

Fingerprinting is described as a variety of analytical techniques or methods which can inform about the composition of some foods in a non-selective way with the main aim of characterizing or authenticating the food. However this term, together with the term 'profiling', comes from the metabolomic terminology. The concept of 'metabolite profiling' was firstly proposed by Horning and Horning, in 1971, in relation to the determination of metabolites using mass spectrometry (MS) [1], [2]. The uses of the term 'metabolomic fingerprinting' is more recent and it may have been introduced by Fiehn at the end of the twentieth century [3] from the idea of infrared fingerprint.

The metabolome of a biological system refers to the complement of all low molecular weight metabolites in that system (typically <1500 Da). Metabolomics refers to the quantitative analysis of the metabolome, or a fraction of this, by the use of advanced analytical techniques. However, it also involves sampling, sample preparation, chemical analysis and data analysis. Whilst the measurement and quantification of individual or small numbers of metabolites is well established in biochemistry, metabolomics differs from more analyses in the number of classes of metabolites being detected, the range of analytical techniques being employed and the need for advanced signal processing and bioinformatics tools [4], [5].

Generally, the metabolomics approaches can be generally grouped as 'profiling (targeted)' or 'fingerprinting (untargeted)' strategies [6]. While 'profiling' involves the analysis of a group of related metabolites, which are in most cases identified and quantified, 'fingerprinting' is based on the determination of as many metabolites as possible without necessarily identifying or quantifying the compounds present. The 'metabolomics (targeted) profiling' provides direct functional information, and the data can be integrated into metabolic models, whereas the 'metabolomics (untargeted) fingerprinting' provides a pattern that is used to classify samples based on provenance of either their biological relevance or origin by using a fingerprinting technology that is rapid but does not necessarily give specific metabolite information. According to these conceptions, 'metabolite profiling' is the analysis of a given set of known metabolites, for instance, a set of amino and organic acids, whereas 'metabolic fingerprinting' is an unspecific analysis of a test material, e.g., a set of mass peaks obtained by mass spectrometry.

However, a double distinction can also be applied for each case. The 'metabolite profiling' does not need to be quantitative, but often is at least semiquantitative, hence it is not possible to optimize the method for all the known metabolites. However, the 'metabolite targeted analysis' is a quantitative analysis of metabolites participating in a specific part of the metabolism in a sample associated to specific pathways, for example, a particular enzyme system that would be directly affected by abiotic or biotic perturbation [7], [8]. In a similar way, 'metabolite untargeted analysis' is currently differentiated from fingerprinting analysis. The 'metabolite untargeted analysis' is a first approximation to the complexity of the unknown metabolites in a test material and it should be seen as a first step before more concrete approaches as profiling analysis. This strategy, known as top-down strategy, avoids the need for a prior specific hypothesis on a particular set of metabolites and, instead, analyses the global metabolite profile [9]. The term 'fingerprinting' remain faithful to the initial mean, and in this approach the intention is not to identify each observed metabolite, but to compare patterns or 'fingerprints' of metabolites that change in response to disease, environmental or genetic alterations.

Due to all this and since there is no single analytical method, different terms are often used in the field of metabolomics, different authors have given different definitions along the bibliography and there are a lot of definitions in bibliography, as well as misuses. For this, these four terms are often used interchangeably; also some of them have been mixed up such us 'untargeted metabolomic profiling' as synonym of 'global profiling' [10], or 'targeted metabolomic fingerprinting' which could lead to misunderstanding. Indeed, in reviewing the literature over the past 40 years, it is evident that these various disciplines of metabolite analysis are related via an evolution of methods and technology.

The amount of data provided by the different metabolomics approaches, mainly from untargeted and fingerprinting strategies, is of great complexity, and correct treatment of those data is of the utmost importance [11], [12]. There are also classifications of metabolomics based on the specific objective of the analysis and data manipulation. For instance, Cevallos et al. [13] classified metabolomics as targeted and untargeted but also propose an interesting sorting as discriminative, informative, and/or predictive. Discriminative analyses aim to find differences between sample populations without necessarily creating statistical models or evaluating possible pathways that may elucidate such differences. Discrimination is usually achieved by the use of multivariate data analysis. In contrast, informative metabolomic analyses focus on the identification and quantification of targeted or untargeted metabolites to obtain sample intrinsic information. Informative metabolomics is used in the development and continuous update of metabolite databases. Finally, some metabolomics reports have been predictive. In this case, statistical models based on metabolite profile and abundance are created to predict a variable that is difficult to quantify by other means.

In general, in the strategy of fingerprinting, a chemical picture is made by direct analysis of crude sample extracts, typically by pyrolysis mass spectrometry (Py-MS) [14], direct infusion mass spectrometry (DIMS) [15], ambient mass spectrometry (Ambient MS), also designated as direct ionisation mass spectrometry [16], or nuclear magnetic resonance spectrometry (NMR) [17]. Both techniques, MS and NMR, either generates a metabolite profile, in which the signals are assigned to specific metabolites, or a metabolite fingerprint, in which the analysis is based on the distribution of intensities in the corresponding spectra rather than the assignment of the signals. However, the metabolites detected by profiling must be recognized consistently and should be also quantified. For this, profiling requires, in general, a separation of the compounds and it is typically done by gas or liquid chromatography (GC, LC) or by capillary electrophoresis (CE) prior to the spectrometric detection by, e.g., UV, Fluorescence, NMR, or MS [2], [18], [19]. Therefore, this coupling tremendously expands the capability of the chemical analysis of highly complex biological samples.

Planning an efficient strategy for metabolome analyses requires consideration of what kind of information is needed, what kind of chemistry is expected the concentration range and what are the analytical facilities available. The selection of different analytical approaches (including the analytical techniques) will depend on the topic of the study and choosing a suitable analytical strategy requires a clear formulation of the problem to which we want some answers.

This tutorial aims at presenting the use of the fingerprinting methodology, applied on chromatography as a technique able of yielding proper fingerprints, independently of the coupled detector, and as suitable technique for food authentication. This tutorial involves several topics, which are: (i) the discussion about the meanings, in the metabolomics and food authentication fields, of the related terms 'profiling', 'fingerprinting', and both targeted and untargeted analysis; (ii) a proposal of new terms related to food authentication as 'food identitation'; and (iii) a critical overview about the applications of chromatographic (GC and LC) fingerprints analytical methods in food authentication using different chemometric techniques. These applications have been tabled according to several items as chromatographic method, chemical fraction, analytical aim, and chemometric target: similarity analysis, exploratory and clustering analysis, signal resolution, classification and regression on food properties.

Section snippets

Food authentication: targets and approaches. Current perspectives

Food authentication involves the confirmation of the stated specifications as true. It could include many aspects as the identification and/or quantification of characteristic components, adulterants and/or contaminants, and the verification of the differentiated quality requirements, among them the botanical or geographical origin and the manufacturing or processing procedure [20], [21], [22]. The authenticity is bound to the truthfulness and, therefore, food is considered authentic (or

Chromatographic fingerprinting for food authentication: framework

The essential aim of the chromatography is the separation of chemical species and it is not the achieving of instrumental signals related to the chemical composition of the material test in study. This last key role is played by the detector which is the true analytical measurement instrument and is defined as a device that measures the change in the composition of the eluent by measuring physical or chemical properties [46]. So, only the combination of both chromatographic and detection

Applications of chromatographic fingerprinting on food 'identitation' and food authentication

In this section, it is shown an overview of the published articles about right applications of the fingerprinting methodology for both food 'identitation' and food authentication. Therefore, the articles which state the use of chromatographic fingerprints but effectively they are applying profiling methodology, are not included.

The number of published applications is even limited; only 32 and 26 articles have been found using GC and LC, respectively. Specific chemometrics tools are used with

Concluding remarks

The framework of food authentication, particularly by chromatographic methods, has been described and clarified. As a result, we have detected that, there is not a proper term that describes the step of obtaining the reference fingerprint from genuine foods. For this reason the term 'food identitation' is proposed in order to define this stage and to close the life cycle of the chemical-based food authentication process.

Some reviews have been published in recent years devoted to fingerprinting

Acknowledgement

Authors acknowledge to the programme of R&D strengthening from the University of Granada, which is supported by the Andalusia Regional Government (Consejería de Innovación, Ciencia y Empresa) FQM 232 and, partially, by European Regional Development Funds (ERDF).

Luis Cuadros-Rodríguez, Full Professor of the Department of Analytical Chemistry (University of Granada, Spain), expert in the field of the Chemical Metrology and Qualimetrics (CMQ). He teaches analytical chemistry in Undergraduate and Master's Degrees in Chemistry and Chemical Engineering. His most significant R&D area of interest included the development of quality assurance protocol (calibration, validation, uncertainty estimation, etc.) on analytical process. He has also developed the use

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    Luis Cuadros-Rodríguez, Full Professor of the Department of Analytical Chemistry (University of Granada, Spain), expert in the field of the Chemical Metrology and Qualimetrics (CMQ). He teaches analytical chemistry in Undergraduate and Master's Degrees in Chemistry and Chemical Engineering. His most significant R&D area of interest included the development of quality assurance protocol (calibration, validation, uncertainty estimation, etc.) on analytical process. He has also developed the use of multivariate process optimization by applying statistically designed experiments on analytical methods. His working is currently focused on the analytical control for food quality, particularly on the characterization and authentication of vegetable (olive) oil with the application of chemometrics tools from chromatographic data.

    Cristina Ruiz Samblás, Chemical Engineer since 2007 by University of Granada and University College of London. Master degree in Chemistry, one year later. Ph. D in Analytical Chemistry in 2012 titled “Authentication of vegetable oils by gas chromatography and mass spectrometry. Quantification of olive oil”. During this period, she got two research stays at Rikilt, Institute of Food Safety in The Nederlands and at University of Rome. Postdoctoral stage at University of Granada. Her research experience is devoted to analytical chemistry, specifically, gas chromatography, chemometrics and vegetable oils authentication, mainly olive oil. Also experiences, in the private sector, on accredited food control laboratories, on multiresidue methods of pesticides and developing calibration models of NIR spectroscopy for quality control of olive oil production.

    Lucía Valverde Som studied Chemistry in 2012 from the University of Granada (Spain). She obtained her Master Degree in Advances in Food Quality and Technology, one year later. Currently, she is a Ph.D. student in the department of analytical chemistry at the University of Granada. During this period, she has done research stays at Department of Drug Science and Technology, University of Turin (Italy) with the aim of improve her knowledge in new analytical techniques. Her research interest includes gas chromatography (GC-FID, GC-(IT)MS, GC × GC-(Q)MS) liquid chromatography (UHPLC-(Orbitrap)MS) and chemometrics (similarity analysis and pattern recognition).

    Estefanía Pérez-Castaño holds a MA degree in Technology and Food Quality since 2009 and she received her PhD in Chemistry from the University of Granada (Spain), in 2012. Researcher in Analytical Chemistry (permanent research group “Food and Environmental Analysis”) at the Department of Analytical Chemistry of the University of Granada. Additionally she has taken part in several research projects, research contracts and agreements on analytical quality of the authenticity of the olive oil. Her research expertise focuses on Chemometrics from chromatographic data (chromatographic fingerprints) in order to authenticate olive oil in foodstuffs, including multivariate classification and regression methods with different chemometrics software's. In addition, she is expertise working under GLP and GMP standards.

    Antonio González Casado received the Ph.D. degree in Chemistry from the University of Granada (Spain) in 1997. He is currently Tenured Professor in the Department of Analytical Chemistry at the University of Granada. He is a researcher in the Analysis in Food and Environment Research Group at the University of Granada. His research topics are focused on the development of methods of analysis of vegetable oils using different analytical techniques such as HPLC and GC coupled with mass selective detectors and different chemometrics tools such CA, PDA, PLS, … His current research fields also include the production of certified reference materials of olive oils for quality control. He has co-authored more than 50 papers in international journals.

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