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Chemical Analysis of Food: Techniques and Applications
Chemical Analysis of Food: Techniques and Applications
Chemical Analysis of Food: Techniques and Applications
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Chemical Analysis of Food: Techniques and Applications

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Chemical Analysis of Food: Techniques and Applications reviews new technology and challenges in food analysis from multiple perspectives: a review of novel technologies being used in food analysis, an in-depth analysis of several specific approaches, and an examination of the most innovative applications and future trends. This book won a 2012 PROSE Award Honorable Mention in Chemistry and Physics from the Association of American Publishers.

The book is structured in two parts: the first describes the role of the latest developments in analytical and bio-analytical techniques and the second reviews the most innovative applications and issues in food analysis. Each chapter is written by experts on the subject and is extensively referenced in order to serve as an effective resource for more detailed information. The techniques discussed range from the non-invasive and non-destructive, such as infrared spectroscopy and ultrasound, to emerging areas such as nanotechnology, biosensors and electronic noses and tongues. Important tools for problem-solving in chemical and biological analysis are discussed in detail.

  • Winner of a PROSE Award 2012, Book: Honorable Mention in Physical Sciences and Mathematics - Chemistry and Physics from the American Association of Publishers
  • Provides researchers with a single source for up-to-date information in food analysis
  • Single go-to reference for emerging techniques and technologies
  • Over 20 renowned international contributors
  • Broad coverage of many important techniques makes this reference useful for a range of food scientists
LanguageEnglish
Release dateSep 1, 2012
ISBN9780123848635
Chemical Analysis of Food: Techniques and Applications

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    Chemical Analysis of Food - Yolanda Picó

    book.

    Preface

    Food products are analyzed for a variety of reasons—e.g., compliance with legal and labeling requirements, assessment of product quality, determination of nutritive value, detection of adulterations, research and development, etc. Food analysis is an area in continuous evolution, which is especially impelled by the increasing demand of the consumers for food safety and quality, the concern of food authorities to ensure safe food of the highest nutritional quality, and the effort of producers and industry to meet these demands. It is also particularly complex because it integrates and applies principles of biology, chemistry, microbiology, biochemistry, nutrition, and engineering to characterize new ingredients and food products, detect the food processing techniques used, and ensure the safety and nutritional value of the food supply. The progress of food science and its concepts have driven change of classic analytical methods (titrimetric or gravimetric analysis) to instrumental and biochemical ones (chromatography, biosensors, spectroscopy) because of the new quantitative and qualitative information provided. In this context, in addition to the many excellent comprehensive descriptions of historical and already well-established classical methods, this book addresses the most recent advances in analytical and bioanalytical techniques and their application in innovative and emerging areas within food science.

    Chemical analysis of foods presents what is new or challenging within this subject through multiple topics: reviewing novel technologies increasingly applied to food analysis; describing and analyzing in depth several specific approaches, and providing a picture of the most pioneering applications with an insight into future trends. The purpose of this book is to offer an updated and high-quality original contribution on new developments in food analysis and its emerging applications.

    The book contains twenty-three chapters written by experts on the subject and is structured in two parts: the first one describes the role of the latest developments in analytical and bioanalytical techniques, and the second one deals with the most innovative applications and issues in food analysis. The two first introductory chapters about sampling and sample preparion and data analysis and chemometrics are followed by a review of the most recently applied techniques in process (on-line) control and in laboratories for the analysis of major or minor compounds of food. These techniques ranged from the non-invasive and non-destructive ones, such as infrared spectroscopy, magnetic resonance, and ultrasounds, to emerging areas as nanotechnology, biosensors, and electronic noses and tongues, including those already well established in food analysis, such as chromatographic and electrophoretic techniques. These chapters also include two important tools for solving problems in chemical and biological analysis: mass spectrometry and molecular-based techniques.

    The second part of the book looks at the areas of food authenticity, safety, and traceability. Important and innovative issues, such as fraudulent practices, biological active components, flavors and odors, novel foods including those modified genetically, dietary supplements, food proteomics, metal speciation and radionuclides, are covered.

    This book attempts to fill a void in information on recently developed analytical techniques for professionals, students, and academics in food analysis by offering information on modern instrumentation, techniques, and applications. It is hoped that it will be helpful to learn more on chemical analysis of food and of particular interest to those involved in food research and development, as well as food product characterization and analysis. It is also intended to serve as general reference for post-graduate students, which are not exposed to many of the emerging technologies and applications in food analysis, as well as a practical reference guide for a wide range of experts: biologists, biochemists, microbiologists, food chemists, toxicologists, chemists, agronomists, hygienists, and everybody who needs to use analytical techniques for evaluating food quality and safety. The techniques and applications discussed in this book are not only emerging now but they also will be in the future critical for continued assurance of an affordable, safe, and available food supply.

    I would like to thank the authors that have agreed to participate in this initiative for their insight and stimulating chapters and for the time and effort devoted to them. They provide the perfect blend of knowledge and skills that went into authoring this book. I would also really like to thank Prof. Damià Barceló for providing me with the opportunity to become the editor of this book as well as to the project managers and all the staff from Elsevier for offering excellent support and advice. Finally and foremost, I hope that the book lives up to the expectations of the readers. You are the ones who will make the book an integral part of food analysis.

    PART I

    Analytical Techniques

    Chapter 1 Basics and Advances in Sampling and Sample Preparation

    Chapter 2 Data Analysis and Chemometrics

    Chapter 3 Near-Infrared, Mid-Infrared, and Raman Spectroscopy

    Chapter 4 Nuclear Magnetic Resonance

    Chapter 5 Low-Intensity Ultrasounds

    Chapter 6 The Applications of Nanotechnology

    Chapter 7 Microfluidic Devices

    Chapter 8 Electronic Noses and Tongues

    Chapter 9 Mass Spectrometry

    Chapter 10 Liquid Chromatography

    Chapter 11 Gas Chromatography

    Chapter 12 Electrophoresis

    Chapter 13 Molecular Techniques

    Chapter 1

    Basics and Advances in Sampling and Sample Preparation

    L. Ramos

    Department of Instrumental Analysis and Environmental Chemistry, IQOG-CSIC, Juan de la Cierva 3, Madrid, Spain

    Outline

    1.1. Introduction

    1.2. Types of Samples and the Analytical Procedure

    1.3. Trends in Sample Preparation for Food Analysis

    1.4. Conclusions

    Acknowledgments

    1.1 Introduction

    The first problem faced when dealing with food science is probably the statement of the concept of food. A number of possible definitions for this concept can be found in the specialized literature. Some of them focus on its composition (typically, carbohydrates, fats, protein and water), others in the way used by humans to seek food items (which, in most cultures, has nowadays changed from hunting and gathering to farming, ranching, and fishing). In other cases, definitions focus on the nature of the matter itself and/or the expected benefices associated to its consumption. Finally, one should recognize that, above definitions, the concept food is also highly cultural dependent. Items considered food may be sourced from water, minerals, plants, animals, or other categories such as fungus, fermented, elaborated, and processed products. Taking into consideration some of these viewpoints, food could be defined as any substance or product, liquid or solid, natural, elaborated, or processed that, because of their characteristics, applications, components, preparation, and conservation state, is eaten or drunk by humans as nourishment and enjoyment.

    Whatever the definition adopted, it is a general consensus that, almost without exception, food is a complex heterogeneous mixture of a relatively wide range of chemical substances. Also, it is agreed that the two key aspects regarding food are its chemical composition and its physical properties. The reason is that these feature categories determine the nutritional value of the considered food item and its sanitary state, as well as its acceptation by consumers and functional activity. This explains why both food analysis and legislation focus on these two aspects.

    Foodstuffs are analyzed for a number of divergent reasons. Governmental and official agencies watch over the accomplishment of legal, labeling, and authenticity requirements. This includes early detection of possible adulterations and fraudulent practices that could result in economic losses or consumers damage. Food analysis is also of primary importance for the food industry, which assesses the quality of the original raw materials and its maintenance through the complete processing, transportation, and conservation process. Scientific researchers are involved in the constant update of the methodologies used to control all the above-mentioned aspects as well as in the development of new analytical procedures that allow the lowering of the allowed maximum residue levels (MRLs) of toxic components and the inclusion of new ones in current legislation, the detailed characterization of food items, and the development of new foodstuffs with added value. Finally, in recent years, there has been an increasing concern by consumers regarding the quality of food. This has partially been motivated by the different scandals originated by food contamination with toxicants and/or forbidden products but, also and more important, by the nowadays accepted relationship between diet and health and the increasing demand of foodstuffs with added nutritional properties. The latter frequently results in the development and addition of new ingredients, whose effect on the original food item at short and long time should also be tested.

    It is evident from previous considerations that food analysis is an extremely wide field in constant evolution involving analysis and chemical determinations of very different nature and with widely divergent goals. These differences translate also to the methods in use for food analysis. As shown in Fig. 1.1, these methods range from subjective (e.g., organoleptic determinations) to objective procedures based on physical, chemical, microscopic, and microbiologic determinations. Other approaches based on, for example, biological determinations and personal questionnaires are also used. This volume reviews the current state-of-the-art and last developments regarding chemical methods and will pay special attention to those based on the use of modern instrumental analytical techniques that, in many instances, have only recently started to be applied in this dynamic research field.

    FIGURE 1.1 Different types of methods applied for food analysis.

    1.2 Types of Samples and the Analytical Procedure

    Food analysis demands chemical determinations at very different levels and for different purposes. As previously indicated, for conventional foods, chemical analysis and controls are applied from independent ingredients and raw materials to the processed products and end-products and, when required, to all intermediate items to guarantee food quality. These types of determinations become especially relevant during the development and implementation of new processing and conservation procedures, or when developing new formula and products.

    As in any other analytical process, the chemical analysis of foodstuffs involves a number of equally relevant steps with a profound effect on the validity of the data generated (Fig. 1.2).

    FIGURE 1.2 Steps in the analytical process.

    Although in some cases on-site determination is possible, most samples have to be transported to the laboratory for chemical analysis. Thereby, in many instances, the first issue to consider is how many samples (or subsamples) should be taken, of which size and from where to guarantee the representativeness of the subsamples. Whether random or purposeful, significant consideration needs to be given to the sampling protocol in order to obtain at the end of the analytical process data meaningful and interpretable. Sampling is a complex process that firstly depends on the nature of the matrix to be sampled (solid or liquid), its size (as a whole or as subsamples), and the goal of the analysis (e.g., determination of main components or trace analysis), just to mention a few parameters. In some cases, the procedure and minimum amount of sample necessary to develop a particular analysis is clearly stated in current legislations [see, e.g., (90/642/EEC, 1993) and (2002/63/EC, 2002) for the determination of pesticides residues in products of plant and animal origin]. In other cases, protocols similar to those set in legal texts can be followed or alternative procedures can be adopted as far as they guarantee the representativeness of the sampling process. In-depth discussion on this complex matter is out of the scope of this chapter. Therefore, the reader is referred to texts of a more specialized nature for a detailed discussion on this topic [see, e.g., Curren et al., (2002); Woodget and Cooper, 1987].

    Samples should remain unaltered during transportation and storage until the moment of the analysis. As a rule of thumb, samples must be stored for the shortest possible time. When applicable, stabilization procedures that, for example, retard biological action, hydrolysis of chemical compounds, and complexes, and reduce the volatilization of components and adsorption effects, should be adopted.

    Once in the laboratory, samples are typically subjected to a number of operations and manipulations before instrumental analysis of the target compounds. These several treatments are grouped under the generic name of sample preparation. The number and nature of these operations and treatments typically depend on the nature and anticipated concentration level of the target compounds, and also on those of the potential matrix interfering components and on the selectivity and sensitivity of the analytical technique selected for final separation and/or detection. Sample preparation would include from the labeling and mechanical processing and homogenization of the received samples, to any type of gravimetric or volumetric measurement carried out. Sample preparation also includes all treatments conducted to decompose the matrix structure in order to perform the fractionation, isolation, and enrichment of the target analytes. Treatments developed to make the tested analyte(s) compatible with the detector (e.g., change of phase and derivatization reactions) and to enhance the sensitivity of the detector are also considered part of the sample preparation protocol.

    Table 1.1 presents a simplified overview on food components and food contaminants typically considered for chemical analysis. In most instances, these analytes are also the subject of routine controls. Target compounds include from metals and organometallic species to volatile components. The latter include flavor and fragrances, but also off-flavors that can create problems with unacceptable food products. Many main and minor components with nutritional or added functional value, such as lipids, proteins, carbohydrates, vitamins, and antioxidants, are also analyzed for legal, quality, or research reasons. In addition, food additives, residues, and a large variety of contaminants of different origin and nature are nowadays matter of continuous monitoring and control to ensure the accomplishment of current legislations. The increasing social pressure for safe foods contributes to support the constant research efforts carried out to improve the accuracy and sensitivity of the analytical methodologies used to determine these particular compounds.

    TABLE 1.1 Overview of the Typical Food Components

    Except for the few cases in which direct determination is feasible (e.g., spectroscopy determination of main food components in combination with chemometrics, see Chapter 2; control process by low intensity ultrasounds, see Chapter 5; use of sensors, see Chapter 7), the determination of the analytes mentioned in Table 1.1 requires some type of sample preparation before instrumental analysis, almost irrespective of the technique selected for final separation-plus-detection. In the simplest case, this consists of the usually quantitative (i.e., exhaustive and nonselective) extraction of the compound(s) of interest from the matrix in which they are entrapped, a fractionation or clean-up step to isolate them from other coextracted materials, and a final concentration of the purified extracts to ensure analyte(s) accurate detection. As in other application areas, in food analysis, the several analytical steps involved in such procedures are most frequently carried out off-line, which make them tedious and time consuming. In general, the complexity of the procedures increases as the concentration of the target compound decreases and so the possibility of loss and contamination of the analyte due to the continual manual manipulation of the extracts. In recent years, much effort has been devoted to eliminating these drawbacks. This has led to the development of faster and more powerful and/or versatile extraction techniques, often incorporated from other research areas, such as environmental and molecular analysis (see e.g., Chapters 6, 7 and 13). These include, for example, automated purge-and-trap (P&T), solid-phase microextraction (SPME), and stir-bar-sorptive extraction (SBSE) for the analysis of volatile components (Table 1.2); a number of solvent-based microextraction techniques especially adapted for the determination of semi- and nonvolatile analytes in liquid sample; other techniques suitable for the treatment of viscous and (semi-) solid samples, such as matrix solid-phase dispersion (MSPD), widely used enhanced fluid/solvent extraction techniques, such as supercritical fluid extraction (SFE), pressurized liquid extraction (PLE), subcritical water extraction (SWE), and microwave-assisted extraction (MAE) and ultrasound-assisted extraction (USE); and also microfluidic devices, DNA arrays, real-time PCR, and other molecular techniques. The latter approaches will be the matter of subsequent chapters within this volume. Thereby, in this chapter, the last trends in the use of some of the modern analytical techniques previously mentioned for food analysis will be revised through selected representative application examples.

    TABLE 1.2 Overview of Selected Analytical Techniques in Use for Food Analysis

    1.3 Trends in Sample Preparation for Food Analysis

    Every single physico-chemical treatment carried out to isolate the analytes from other matrix components that could interfere during their instrumental determination and/or to increase their concentration in the extract subjected to analysis is considered a step of the sample preparation protocol. According to this consideration, one can conclude that most of conventional and official sample preparation methods (AOAC, 1990; Nollet, 1996) in use for food analysis are long, laborious, and highly manipulative multistep procedures prone to loss, degradation, and/or contamination of the target analytes. Therefore, in this field, sample preparation is a key part of the analytical process with a profound effect on (i) the time required to complete the analytical process, (ii) the cost of the determination in terms of solvents and sorbents consumption, and (iii) the validity of the final result.

    Again as in other application areas, sample treatment is considered the bottleneck of the analytical methodologies in use for food analysis. It is estimated that 60–80% of the work activity and operating costs in the analytical laboratories is spent in preparing samples for introduction into the analytical system selected for instrument determination. It is also estimated that this part of the analytical process is responsible for more than 50% of the error associated to the final reported data. These figures explain the efforts carried out during the last decades to develop analytical approaches that represent a faster, more automated, cost-effective, and greener alternative to the previously mentioned traditional protocols.

    Solid-phase microextraction (SPME) is a miniaturized technique that fulfills most of these requirements. In SPME, the analyte(s) is(are) adsorbed onto a fused-silica fiber coated with an appropriate sorbent layer by simple exposure of the fiber for a preselected time to the headspace (HS) of the sample or by direct immersion in a liquid sample. Since its introduction in 1990 by Pawliszyn’s group (Arthur and Pawliszyn, 1990) as a (virtually) solvent-free preconcentration technique, SPME has profusely been used in many application fields including food analysis. Here, its primary use has been the preconcentration of volatile analytes from liquid, semi-solid, and solid samples, for which it has been demonstrated to be a simple, rather selective, and relatively fast (under nonequilibrium conditions) technique. SPME has been used for different application studies such as lipids oxidation and protein degradation during storage of soup powder (Raitioa et al., 2011), and the evaluation of the traceability of grapes origin (Rocha et al., 2007). In this latter work, a fused SPME silica fiber coated with Carbowax-divinylbenzene was used in the HS-mode to establish the monoterpenoid profile of Vitis vinifera L. cv. ‘Fernao-Pires’ white grape. The use of HS-SPME coupled with comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (GC × GC-ToF MS) allowed determining 56 monoterpenoids in grapes. Among them, 20 were reported for the first time in this fruit. A typical example of the results obtained is shown in Fig. 1.3. The authors concluded that, as monoterpenoids are secondary metabolites whose synthesis is encoded by variety-related genes, the terpenoid profile may be used as a way to trace grape varietal origin.

    FIGURE 1.3 GC × GC contour plot corresponding to ions m / z 93, 121 and 136. Bands or clusters formed by structurally related compounds are highlighted.

    Recently, stir-bar-sorptive extraction (SBSE) has been found to be advantageous as compared with conventional extraction techniques like simultaneous distillation–extraction (SDE) or direct HS, and more modern sample preparation techniques, such as SPME, for the determination of unknown taints in food (Ridgway et al., 2010). SDE uses larger volumes of solvent than SBSE, which provides improved detectability as compared with HS and SPME and also minimizes the potential for contamination from external laboratory sources. In general, SBSE provided better results than these established techniques, although the optimized method was not feasible for the determination of methyl methacrylate and hexanal. Other examples of the use of SBSE and a discussion of the advantages and limitations of this technique as compared with SPME, SPE, and other conventional sample preparation techniques can be found in Olariu et al. (2010).

    Liquid–liquid extraction (LLE) is the technique of choice in most official methods. However, some of these procedures are frequently revisited in an attempt to expand their application field by incorporating new target compounds into the analysis (Mol et al., 2007). The straightforward nature of most LLE methods would suggest that their adaptation for implementing some of the newly developed solvent microextraction techniques is a relatively easy goal, attainable by simple scaling down of the original procedures. Depending on the application, practice can be slightly more complicated. However, the high sensitivity provided by many modern instrumental techniques and the increased use of these miniaturized techniques in food analysis demonstrate the feasibility of the approach [see, e.g., Asensio-Ramos et al., (2011)].

    Single-drop microextraction (SDME) was the first solvent-based microextraction technique introduced and has up to now been one of the most profusely used for food analysis. Typical applications involving SDME are presented in Table 1.3.

    TABLE 1.3 Selected Applications of Solvent Microextraction Techniques

    SDME can be used as a two-phase system, as in the direct-immersion (DI) and drop-to-drop microextraction (DDME) approaches, or as a three-phase system, as in the HS mode or in the more recently introduced liquid–liquid–liquid microextraction (LLLME). In its simplest configuration, a single microdrop of a water-insoluble solvent suspended at the tip of a GC syringe is either immersed in an aqueous sample (DI mode) or exposed to the HS of a sample contained in a vial. Strategies such as stirring, heating, and/or salting out the solution, and derivatization of the target compounds are frequently used to speed up the extraction process. Once the extraction time is completed, the drop is withdrawn into the syringe and the enriched solvent is transferred to the system selected for instrumental analysis without any additional treatment. HS has been used for preconcentration of volatile analytes or derivatives. Meanwhile, the two-phase approaches are particularly suitable for the analysis of less volatile and relatively polar compounds in pristine samples.

    DDME is a modification of the DI-SDME procedure that has been used for the fast, inexpensive clean-up and quantitative preconcentration of different analytes from aqueous solutions with minimum sample consumption. In a representative application, Shrivas and Wu (2007) used DDME with chloroform (0.5 μL) for the rapid determination of caffeine in one drop of beverages and foods, i.e., 7 μL. The extraction took only 5 min and was carried at room temperature and without salt addition. The optimized DDME combined with gas chromatography–mass spectrometry (GC–MS) method exhibited good linearity between 0.05 and 5.0 μg/mL with correlation coefficient of 0.980, recoveries above 97%, a relative standard deviation (RSD) of 4.4%, and a limit of detection (LOD) of 4.0 ng/mL. DDME avoided the main shortcomings of conventional methods of caffeine extraction, like large amount of organic solvent and sample consumption and long sample pretreatment process. The authors proposed the optimized DDME-based procedure as a simple, fast, and feasible diagnosis tool for caffeine in food and beverages.

    Application of SDME to the analysis of polar compounds required a modification that resulted in a three-phase SDME system named LLLME. In this approach, the deionized polar analytes were preconcentrated from the aqueous sample in a few microliters of organic phase and subsequently back-extracted in an aqueous microdrop that acted as receiving phase. Up to now, the technique has mainly been used for the analysis of aqueous samples and biological fluids. To the best of our knowledge, only one study has reported on its application to food analysis. The study (Zhu et al., 2010) proposed the combined use of LLLME with capillary electrophoresis (CE) for the on-line purification and preconcentration of adenine from green tea extracts.

    Hollow fiber-protected two-phase liquid microextraction (HF(2)LPME) was introduced by He and Lee (1997) with the name of liquid-phase microextraction. In its simplest version, the technique involves a small-diameter microporous polypropylene tube (the hollow fiber), typically sealed at one end, to contain the organic extracting solvent. The open end of the hollow fiber is attached to a syringe needle used to fill the fiber with the organic solvent. Once filled, the fiber is immersed in the vial containing the investigated aqueous sample to allow analyte migration through its walls. After a preselected extraction time, the solvent is withdrawn with the syringe and transferred to the instrument selected for analyte determination, typically gas chromatography (GC). The hollow fiber can be considered to act as a membrane. Consequently, this technique is more appropriate for the analysis of dirty aqueous samples than SDME. Due to the higher stability of the solvent, contained in the hollow, it also allows higher stirring rates than SDME. On the contrary, HF-LPME typically used to involve larger extractant volumes (Table 1.3) and longer extraction times than SDME (20–60 min vs. 5–15 min with SDME). In its three-phase format (HF(3)LPME), the analytes preconcentrated in the water-immiscible organic solvent used to fill the pores of the hollow fiber polymer are subsequently extracted to an aqueous acceptor phase that is placed in the lumen of the fiber. The HF(3)LPME technique is typically used to extract water-soluble analytes from aqueous matrices and, because the final acceptor solution is aqueous, liquid chromatography (LC) and CE are usually preferred for final instrumental determination of the tested analytes. During the last few years, a number of studies have demonstrated the feasibility of the techniques for the determination of analytes of very different nature in food and beverages. Applications include the analysis of micropollutants in alcoholic drinks (Plaza Bolaños et al., 2008), orange juice (Barahona et al., 2010), and other beverages (Xiong and Hu, 2008); phenolic compounds in fruit juices (Saraji and Mousavi, 2010), antibiotics in bovine milk (Shariati et al., 2009), and metallic (Abulhassani et al., 2010) and organometallic (Ghasemi et al., 2011) species in complex foodstuffs.

    In dispersive liquid–liquid microextraction (DLLME), the investigated aqueous sample (up to 10 mL) is extracted with a small amount of a water-immiscible extraction solvent (typically 10–50 μL) dissolved in 0.5–2 mL of a water-soluble solvent. The technique can be considered a modification of a miniaturized LLE in which extraction is favored by the formation of small microdrops of the water-immiscible solvent by fast injection of the mixture of organic solvents into the water with a syringe. The enriched organic phase is then separated from the aqueous sample by centrifugation or freezing (depending on its density) and directly subjected to instrumental analysis, typically by GC. Application to polar analytes requires previous pH adjustment and/or in situ derivatization, which can be accomplished by direct addition of the derivatization agent to the sample or by dispersion together with the extraction solvent. Since its introduction in 2006 (Rezaee et al., 2006), this miniaturized and green, but highly manipulative technique, has profusely been used in different application areas. In food analysis, the DLLME has been demonstrated to be a valuable alternative to large-scale conventional procedures for the determination of relatively abundant food components, such a cholesterol (Daneshfar et al., 2009), and also for the analysis of trace organic (Cunha et al., 2009; Liu et al., 2011b) and inorganic (Wen et al., 2011) contaminants and other illegal substances (Liu et al., 2011a).

    Several recent studies have reported on the use of ionic liquid as extractant in DLLME, a trend also observed on SDME and HF(2/3)LPME (Table 1.3). These examples demonstrate that room-temperature ionic liquids are a valuable alternative to classical organic volatile solvents for the extraction of both organic and inorganic compounds that, apart from greening the analytical process, efficiently contribute to reduce the exposure of the analyst to toxic solvents. Ionic liquids can directly be applied to aqueous samples. The analysis of solid matrices is only possible after extraction of the target analytes from the matrix and dilution of the extract in water. Ravelo-Pérez et al. (2009) used this approach for the determination of eight pesticides belonging to classes different from bananas. In this method, the homogenized fruit sample (1 g) was extracted with acetonitrile and, after evaporation and reconstitution of the extract in 10 mL of water, the target compounds were preconcentrated by DLLME using [HMIM][PF6] (88 mg) as extractant and methanol (714 μL) as disperser solvent. The ionic liquid was recovered after centrifugation at 4000 rpm (20 min), diluted in acetonitrile, and analyzed without any further treatment by LC-DAD. Figure 1.4 shows the typical chromatograms obtained for (A) a spiked and (B) a nonspiked banana. Acceptable mean recoveries in the 53–97% range, with RSD values lower than 9%, and LODs (0.32–4.7 μg/kg) below the MRLs set in current legislations were obtained in all instances. These analytical figures of merit would prove the validity of the optimized method for the intended determination, although the observed severe matrix effect made the use of matrix-matched calibration mandatory.

    FIGURE 1.4 LC-DAD chromatograms of (a) a spiked and (b) nonspiked banana sample after matrix extraction and DLLME with [HMIM][PF 6 ] as extractant. Peak identification: (1) thiophanate-methyl, (2) carbofuran, (3) carbaryl, (4) tebuconazole, (5) iprodione, (6) oxyfluorfen, (7) hexythiazox, and (8) fenazaquin.

    Solid-phase extraction (SPE) is the most widely used technique for the treatment of aqueous samples and extracts in laboratories. A large variety of sorbents, ranging from classical sorbents, such as silica, florisil, and C8 or C18, to modern cross-linked polymers are nowadays commercially available in different formats, including conventional SPE cartridges and disks for off-line and on-line analysis as well as 96-well plates. As illustrated in several reviews (Beyer and Biziuk, 2008; Buldinia et al., 2002; Ihnat, 2003; Kinsellaa et al., 2009; Ridgway et al., 2007; Rostagno et al., 2010), all of them have been used for food analysis.

    Current trends in the use of SPE for food analysis agree with those observed in closely related research areas, such as environmental analysis. These include the preference for the so-called universal sorbents, i.e., those able to simultaneously retain polar and nonpolar analytes, in an attempt to increase the number of analytes monitored in a single analysis; the use of highly cross-linked polymers to improve the retention of very polar analytes; and the use of very selective sorbents based on restricted access media (RAM) or molecular imprinted polymers (MIPs) (Turiel and Martín-Esteban, 2010). Food analysis is at present benefited by the development experienced in the last decade in the field of nanotechnologies. In a representative study, López-Feria et al. proposed the use of carbon nanotube-based solid-phase extraction for the control of multiclass pesticides in virgin olive oils (López-Feria et al., 2009). Carboxylated single-walled carbon nanotubes (SWCNs) were preferred to multiwalled carbon nanotubes for the application. Once optimized, the method consisted of the direct elution of the investigated olive oil diluted with n-C6 (1:5,v/v) through an SPE column containing 30 mg of the selected nanotubes. After washing the column with 3 mL of n-C6, the analytes were eluted with 0.5 mL of ethyl acetate. The extract was finally concentrated, reconstituted on methanol, and analyzed by GC–MS. Complete sample preparation was carried out in less than 8 min and the SPE column could be reutilized more than 100 times. The low LODs achieved (in the 1.5 and 3.0 μg/L) allowed the application of the method to control the target pesticides in very restrictive samples, such as the ecological virgin olive oil.

    Probably the most successful development introduced in the last few years in the field of SPE has been the method known as QuEChERS. The acronym applies to quick, easy, cheap, effective, rugged, and safe, which is supposed to describe the main merits of the analytical procedure introduced by Anastassiades et al. (2003) for the determination of pesticides in fruits and vegetables. The method is a multistep procedure based on dispersive solid-phase extraction (dSPE). In its basic scheme for pesticide analysis in fruits and vegetables (Fig. 1.5) (Wilkowska and Biziuk, 2011), the method involves the initial sample treatment with magnesium sulfate to promote water separation from the organic solvent, followed by treatment with primary secondary amine (PSA) to remove polar components, such as organic acids, some sugars, and polar pigments. Other protocols include sample shaking with graphitized carbon black (GCB) to eliminate sterols and pigments such as chlorophyll.

    FIGURE 1.5 Main steps in QuEChERS procedure for determining pesticides in fruits and vegetables.

    The rapid acceptation of this fast and efficient sample preparation protocol promoted its quick adaptation for other types of analysis, including different application such as the analysis of nonpolar microcontaminants (Ramalhosa et al., 2009) and acrylamide in different food items (Mastovska and Lehotay, 2006), drugs in animal tissues (Stubbings and Bigwood, 2009), and blood (Plossl et al., 2006).

    dSPE has also benefited for the development of new materials. Chen et al. (2009) prepared a magnetic molecularly imprinted polymer for the separation of tetracycline antibiotics from egg and tissue samples by dSPE. The satisfactory results obtained with this method as compared with more conventional configurations such as MIP-SPE and MIP-SPME (Table 1.4), together with the simplicity of the operation methodology and the possibility of recovering the magnetic particles with a simple magnet, make this novel approach an interesting alternative for sample preparation.

    TABLE 1.4 Comparison of QuEChERS Method with Magnetic MIP with the Results Obtained by Using MIP-SPE and MIP-SPME for the Determination of Tetracycline Antibiotics

    Matrix solid-phase dispersion (MSPD) is a widely accepted technique for the treatment of liquid, viscous, and (semi-) solid samples. In MSPD, the extraction and (preliminary) clean-up of the target analytes is carried out in a single step and in a column format. The column configuration simultaneously contributes to simplify the analytical process and to avoid the emulsion problems associated to most of the conventional LLE-based procedures. When the sorbent dispersant and the extraction solvent protocol are properly selected, MSPD can yield ready-to-analyze extracts that, in the case of foodstuffs, are usually processed by GC or LC.

    In food analysis, MSPD has mainly been used for the determination of trace organic micropollutants and, in particular, of pesticides (Barker, 2007; Bogialli and Di Corcia, 2007; Gilbert-Lopez et al., 2009; Kristenson et al., 2006). For this type of application, sorbents used for sample dispersion range from classical ones (e.g., alumina, florisil, carbon, or C8) to new materials like multiwalled carbon nanotubes (Guan et al., 2011) or highly selective dedicated sorbents (Yan et al., 2011).

    Most recent trends focus on the miniaturization of the MSPD process (Kristenson et al., 2001; Ramos et al., 2009) and/or the combined use of MSPD with one or several of the previously described novel sample preparation techniques in order to improve the efficiency and/or selectivity of the MSPD process. In an illustrative example, Yan et al. (2011) proposed the use of a new synthesized kind of aniline-naphthol molecularly imprinted microspheres (0.2 g) selective for Sudans as dispersant for miniaturized MSPD of 0.1 g of egg yolk. After washing the MSPD column with 4 mL of methanol:water (1:1, v/v), analytes were quantitatively extracted with 3 mL of acetone:acetic acid (95:5, v/v). The concentrated eluent (1 mL) was used as dispersive solvent for DLLME. The mixture was shaken and ultrasonicated to form a homogeneous cloudy solution. Phase separation was subsequently performed by centrifugation at 4000 rpm for 10 min. The four studied Sudan dyes were simultaneously determined by LC-UV after concentration of the corresponding enriched phase. Figure 1.6 shows a schematic diagram of the complete sample preparation procedure (Yan et al., 2011). The method showed a good linearity for all target analytes in the investigated 0.02–2.0 μg/g range (r² ≥ 0.9990), with recoveries better than 87% and RSDs below 6%.

    FIGURE 1.6 Schematic of the MIP/MSPD combined with DLLME proposed for the simultaneous determination of four Sudan dyes in egg yolk. (a) Blending of the sample with the selective MIP (MIM); (b) transfer of the blended sample to the column; (c) completed MSPD column; (d) washing of the MSPD column and elution of the test analytes; (e) eluent to be evaporated, (f) injection of the extractant into the eluent for DLLME; (g) addition of deionized water into the DLLME extractant–dispersant mixture; (h) formation of the emulsion assisted by ultrasounds; (i) emulsion of the ternary mixture; (j) phase separation by centrifugation; and (k) collection of the high-density extractant.

    The main application fields of the techniques based on the use of compressed fluids, namely supercritical fluid extraction (SFE) and pressurized liquid extraction (PLE), so-called subcritical water extraction (SWE) when water is used as extractant, in food analysis are the isolation of relevant natural compounds and of functional products (Mendiola et al., 2007).

    Probably some of the most widely known industrial applications of SFE in food analysis are the extraction of caffeine from coffee and tea and of cholesterol from, e.g., egg. However, the particular features of this technique make it suitable for many other applications. Thereby, SFE with carbon dioxide modified with 35% of methanol and combined off-line with GC–MS has been used for obtaining the amino acid profiles of genetically modified maize and soybean (Bernal et al., 2008). Comparison of these profiles with those obtained for their corresponding isogenic nontransgenic varieties proved that the latter seemed to have higher content of several amino acids.

    The distinguished advantages of SFE for automatic sample treatment and its relatively simple at-line or on-line coupling with different separation-plus-detection instruments have made SFE the technique of choice for a variety of application studies.

    SFE coupled at-line with CE equipped with fluorimetric detection (CE-FD) has been used for the determination of flavin vitamins in food samples (Zougagh and Ríos, 2008). The nonpolar nature of supercritical carbon dioxide was used for the initial elution of the nonpolar interference compounds existing in the matrix; then, the extraction of the studied water-soluble vitamins was achieved by modification of the polarity of the extracting agent with 5% methanol. Extracts were clean enough to allow direct CE-FD analysis. In another interesting study, SFE-LC was used for the determination of air- and light-sensitive food components, such as lycopene (Pól et al., 2004). Here, a single monolithic column was used for trapping and subsequent chromatographic separation of target analytes. The method showed a linear response over the studied range of 0.1–2.5 μg, a good repeatability (RSD, 3.9%), and sensitivity (LOD, 0.5 ng). Complete analysis was done in only 35 min. A typical chromatogram demonstrating the performance of the SFE-LC method proposed for real-life applications is shown in Fig. 1.7.

    FIGURE 1.7 Typical chromatogram obtained for a tomato extract. Peak identification: (1) β-carotene; (2) cis -lycopene; (3) trans -lycopene; and (4) cholesterol (internal standard). Residual carbon dioxide inside the trapping-separation monolithic column showed as a peak eluting at 1.2 min.

    Despite the increasing acceptance of PLE (and SWE) as fast and relatively green techniques for food extraction (Carabias-Martínez et al., 2005; Mendiola et al., 2007), the development of equivalent hyphenated systems with commercial PLE system can still be considered an unachieved goal. The main reason is the relative large volume of extractant used by these PLE devices (typically more than 35 mL), which makes difficult the coupling with both the subsequent clean-up step (if required) and the selected instrumental chromatographic or separation technique. Probably, the most plausible strategy to circumvent this limitation could be the use of a miniaturized PLE setup [see, e.g., Ramos et al. (2007)]. Although these types of devices represent also the best alternative for the PLE treatment of size-limited samples, to the best of our knowledge, no commercially available system of these characteristics is available yet.

    Apart from miniaturization, the main recent highlights concerning PLE are the application of sequential elution protocols during PLE and the preference for the so-called selective PLE. The former approach is usually selected when the study aimed to obtain information as complete as possible regarding sample composition. Sequential PLE has been used in combination with either Fourier transform–ion cyclotron resonance–mass spectrometry (FT–ICR–MS) or capillary electrophoresis–time-of-flight–mass spectrometry (CE–TOF–MS) to study the metabolomics of genetically modified organisms (Leon et al., 2009). Using this sophisticated strategy, the authors found differences in the metabolite levels of three transgenic maize varieties compared with their wild isogenic lines suggesting specific metabolic pathways.

    In selective PLE, sorbent(s) used for purification of the food extracts in conventional sample preparation are packed at the bottom of the extraction cell to perform in-cell purification. In most instances, ready-to-analyze extracts are obtained with concentration as the only required treatment; before final instrumental determination of the target compounds, at least a miniaturized PLE system (Ramos et al., 2007) or a very sensitive detector was used.

    1.4 Conclusions

    Foodstuffs are complex mixtures of volatile, inorganic, and organic components at very different concentration levels. Food chemical characterization requires the analysis of all a variety of macromolecules such as proteins, other macronutrients like carbohydrates and lipids, natural bioactive components such as polyphenols, aroma and flavor components, inorganic micronutrients and organometallic compounds, as well as undesirable residues of other small molecules introduced during production, processing, storage and/or transport of food, including contaminants, i.e., plasticizers, pesticides, persistent organic pollutants, veterinary drugs, and toxins. Sample preparation is, in one way or another, required almost in all these types of analyses. Because of the still rather traditional protocols used for many of these determinations, there are many opportunities for improvement and analytical development. The new analytical demands derived from current legislations concerning (and constantly affecting) food routine control and monitoring programs to ensure human health protection also contribute to promote new improvements and developments in the field, with increasing automation probably being one of the main requirements. Finally, as in many other application fields, the large amount of (frequently toxic) wastes generated during sample preparation of foodstuffs demands the development of alternative greener analytical procedure also in this research area.

    Acknowledgments

    Author thanks MICINN for project AGL2009-09733 and CM for program S-2009/AGR-1464 (ANALISYC-II).

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    Chapter 2

    Data Analysis and Chemometrics

    Paolo Oliveri and Michele Forina

    Department of Drug and Food Chemistry and Technology, University of Genoa, Via Brigata Salerno, 13, Genoa, Italy

    Outline

    2.1. Introduction

    2.1.1. From Data to Information

    2.2. From Univariate to Multivariate

    2.2.1. Histograms

    2.2.2. Normality Tests

    2.2.3. ANOVA

    2.2.4. Radar Charts

    2.3. Multivariate Data Analysis

    2.3.1. Principal-Component Analysis

    2.3.2. Signal Pre-Processing

    2.3.3. Supervised Data Analysis and Validation

    2.3.4. Supervised Qualitative Modeling

    2.3.5. Supervised Quantitative Modeling

    2.3.6. Artificial Neural Networks

    2.1 Introduction

    2.1.1 From Data to Information

    Advances in technology and the increasing availability of powerful instrumentation now offer analytical food chemists the possibility for obtaining high amounts of data on each sample analyzed, in a reasonable – often negligible – time frame (Valcárcel and Cárdenas, 2005).

    Often, in fact, a single analysis may provide a considerable number of measured quantities, generally of the same nature. For instance, gas chromatographic (GC) analysis of fatty acid methyl esters allows us to quantify, with a single chromatogram, the fatty acid composition of a vegetable oil sample (American Oil Chemist’s Society, 1998). Spectroscopic techniques as well may supply, with a single and rapid analysis on a sample, multiple data of homogeneous nature: in fact, a spectrum can be considered as a data vector, in which the order of the variables (e.g., absorbances at consecutive wavelengths) has a physical meaning (Oliveri et al., 2011).

    In other cases, a set of samples can be described by a number of heterogeneous chemical and physical parameters at the same time. For example, a global analytical characterization of a tomato sauce may involve the quantification of color and rheological parameters as well as pH and chemical composition and – possibly – a number of sensorial responses (Sharoba et al., 2005). Also in such cases, each sample may be described by a data vector, but without any implication with respect to the order of the variables. Instead, differences in variable magnitude and scale between different variables may affect data analysis if a proper pre-processing approach is not followed.

    The availability of large sets of data does not mean at the immediate time availability of information promptly accessible to the sample analyzed: usually, in fact, a number of steps are required to extract and properly interpret the potential information embodied within the data (Martens and Kohler, 2008).

    A deep understanding of the nature of analytical data is the first basic step for any proper data treatment, because different data types usually require different processing strategies, which closely depend on their nature and origin. For this reason, the data analyst should always have a complete awareness of the problem under study and about the whole analytical process from which data derive – from the sampling to the instrumental analysis. Such knowledge is fundamental: it makes the difference between a chemometrician and a mathematician. A chemometrician is, first of all, a chemist, who is acquainted with his data, and utilizes mathematical methods for the conversion of numerical records into relevant chemical information.

    The analytical food chemist William Sealy Gosset (1876–1937), who worked at the Arthur Guinness & Son brewery of Dublin, can be considered as one of the fathers of chemometrics. In fact, he studied a number of statistical tools and adapted them to better solve actual chemical problems. He had to present his studies using a pseudonym, since his company did not permit him to publish any data. Considering himself as a modest contributor in the field, rather than a statistician, he adopted the pen name Student. His most famous work was on the definition of the probability distribution that is commonly referred to as the Student’s t distribution (Student, 1908).

    The term chemometrics was used for the first time by Svante Wold, in 1972, for identifying the discipline that performs the extraction of useful chemical information from complex experimental systems (Wold, 1972).

    Statistics offers a number of helpful tools that can be used for converting data into information. Univariate methods, which consider one variable at a time, independently of the others, have been and are still extensively used for such purposes. Nonetheless, they usually supply just partial answers to the problems under study, since they underutilize the potential for discovering global information embodied in the data. For instance, they are not able to take into account inter-correlation between variables – a feature that can be very informative, if recognized and properly interpreted.

    Multivariate strategies are able to take into account such an aspect, allowing a more complete interpretation of data structures. However, in spite of their big potential, multivariate methods are generally less used than univariate tools.

    On the other hand, a number of people try multivariate analysis as the last-ditch resort, when nothing seems to provide the desired results, pretending that chemometrics provide valuable information from data that do not contain any informative feature at all.

    Such demeanor is very hazardous especially when complex methods are being used, because there may be the risk of employing chance correlations to develop models with good performances only on appearance – namely, on the same samples used for model building –but with very poor prediction ability on new samples: this is the so-called overfitting. To overcome such a possibility, a proper validation of models is always required. In particular, the more complex the technique applied, the deeper the validation recommended.

    For these reasons, a good understanding of the characteristics of the methods employed for data processing is always advantageous as well.

    In this chapter, an overview of the chemometric techniques most commonly used for data analysis in analytical food chemistry will be presented,

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