Food Safety: Innovative Analytical Tools for Safety Assessment

Food safety and quality are key objectives for food scientists and industries all over the world. To achieve this goal, several analytical techniques (based on both  destructive detection and nondestructive detection) have been proposed to fit the government regulations.

The book aims to cover all the analytical aspects of the food quality and safety assessment. For this purpose, the volume describes the most relevant techniques employed for the determination of the major food components (e.g. protein, polysaccharides, lipds, vitamins, etc.), with peculiar attention to the recent development in the field. Furthermore, the evaluation of the risk associated with food consumption is performed by exploring the recent advances in the detection of the key food contaminants (e.g. biogenic amines, pesticides, toxins, etc.).

Chapters tackle such subject as:

• GMO Analysis Methods in Food
• Current Analytical Techniques for the Analysis of Food Lipids
• Analytical Methods for the Analysis of Sweeteners in Food
• Analytical Methods for Pesticides Detection in Foodstuffs
• Food and Viral Contamination
• Application of Biosensors to Food Analysis

• Language: English
• Publisher: Wiley
• Release date: Dec 6, 2016
• ISBN: 9781119160571

Book preview

Preface

Food safety and quality are key objectives for food scientists and industries all over the world, both in developed and developing countries. Several different approaches have been proposed to characterize and eventually improve the nutritional value and the safety standards of food products, with innovative and fashionable techniques acting on food production, processing and analysis.

With the aim to provide a detailed overview of recent developments in food science in a multidisciplinary context, from agriculture to chemistry and engineering, and from physics to biology and medicine, we planned a new book series highlighting how the knowledge and research in different fields can be applied to address quality and safety issues.

In this first volume we show the recent developments in the analytical techniques (based on both destructive and nondestructive detection) proposed to fit the government regulations related to food quality. The development of effective analytical routes for the evaluation of food quality and safety is, indeed, a key objective for the food industry, for both safe and valuable production and storage. The distinctive aspect of this project is the evaluation of both the nutritional and contamination elements in foodstuffs, with specific attention given to the efficiency and applicability in practical analyses.

The volume is organized into two parts. The first part is related to the evaluation of the major food components (e.g., protein, polysaccharides, lipds, vitamins, etc.), with particular attention paid to recent developments in the field. In the second part, the risks associated with food consumption are evaluated by exploring recent advances in the detection of the key food contaminants (e.g., pollutants, pesticides, toxins, etc.).

Thanks to the valuable contributions of scientists working in these specialized fields, we present a detailed overview of the available techniques, with key advantages and limitations, highlighting the possibility to select an innovative and effective strategy to achieve the main goal of maximizing the nutritional values of foodstuffs while minimizing the risk of toxicity for consumers.

Umile Gianfranco Spizzirri

Giuseppe Cirillo

Department of Pharmacy, Health and Nutritional Sciences

University of Calabria

September 2016

Chapter 1

Food Analysis: A Brief Overview

Giuseppe Cirillo, Donatella Restuccia, Manuela Curcio, Francesca Iemma and Umile Gianfranco Spizzirri*

Department of Pharmacy, Health and Nutritional Sciences, University of Calabria, Rende (CS), Italy

*Corresponding author: [e-mail]

Abstract

Food products are complex mixtures consisting of naturally occurring compounds with nutritional value and contaminating substances, generally originating from technological processes, agrochemical treatments, or packaging materials. Since the exact content of a food product must be assessed before it can be put on the market, routine and/or specialized analysis protocols have been pointed out for the characterization of all compounds present in food and beverages. The main approaches involve separation methods (e.g., chromatography), spectroscopic and biologically derived protocols. By developing efficient methodology with high reproducibility and low detection limits, high quality and safety standards can be achieved to fit the developed government regulations.

Keywords: Food quality, food analysis, separation methods, molecular recognition

1.1 Introduction

Their nutritional and health-related properties make food and beverages highly important products able to provide humans with different biologically active compounds [1, 2]. In the last few years, production, collection, storage, and distribution of food and beverages have been significantly influenced by technological and scientific developments with considerable advantages for both food quality and safety [3–5].

Food products are complex mixtures consisting of naturally occurring compounds with nutritional value (e.g., lipids, carbohydrates, proteins, vitamins, phenolic compounds, organic acids and aromas) [6] and contaminating substances (e.g., pesticides, polycyclic aromatic hydrocarbons, chlorinated and brominated compounds, veterinary drugs, toxins, mutagenic compounds, metals, and inorganic compounds), generally originating from technological processes, agrochemical treatments, or packaging materials [7–9].

The exact composition (in terms of both natural components and contaminants) must be assessed before a foodstuff can be put on the market, and several limitations are imposed by national and international control agencies in order to assure safety and quality, and to avoid frauds [10–12]. For this reason, routine and/or specialized analysis protocols have been pointed out for the characterization of all compounds present in food and beverages [13–15]. Various types of methods, including microbial methods, sensory analysis, biochemical and physicochemical methods, are used in food analysis. Spectroscopic and chromatographic methods have become very popular for separation and identification of food components due to their high reproducibility and low detection limits [16]. Similarly, biologically based assays, including polymerase chain reaction (PCR) techniques, and immunological-based methods, are also used for detection of specific targets in food samples [17].

This chapter focuses on the principal instrumental techniques proposed in food and beverage analysis.

1.2 Chromatographic Techniques in Food Analysis

Separation techniques, such as gas chromatography (GC), liquid chromatography (LC), and capillary electrophoresis (CE), have largely been used for analysis of compounds in food samples [18]. The complexity of food matrices often requires not only extensive sample preparation, but also online coupling techniques, which are used for their superior automation and high-throughput capabilities.

Many detectors with different types of selectivity can be used in gas chromatography. A first classification can be done in terms of detected compounds. Nonselective detectors are able to detect all compounds except the carrier gas, selective detectors respond to a range of compounds with a common physical or chemical property, while specific detectors are able to detect a single chemical compound. Detectors can also be grouped into concentration- and mass-flow-dependent detectors. The signal from a concentration-dependent detector is related to the concentration of solute in the detector, and does not usually destroy the sample, while mass-flow-dependent detectors usually destroy the sample, and the signal is related to the rate at which solute molecules enter the detector.

The coupling of separation techniques in tandem with mass spectrometry (MS) or high-resolution MS (time-of-flight) is a valuable tool for enhancing the selectivity and sensitivity of a detection system, giving precise information on the identity of compounds [19].

Nowadays, gas chromatography coupled to mass spectrometry (GC-MS, GC-MS/MS) with electron impact ionization is a routine technique for analysis of nonpolar, semipolar, volatile and semivolatile food compounds such as polycyclic aromatic hydrocarbons, pesticides and dioxins [20, 21].

In contrast, for polar and nonvolatile substances, LC is the technique of choice, with increased application over the last few years [22]. Detection by liquid chromatography can be carried out in different ways, highly affecting its applicability to food analysis. Ultraviolet (UV) detection has been mostly used as well as mass spectrometers or refractive index (RI) detectors [23, 24]. Mass spectrometers are sensitive and universal detectors, but they are expensive; moreover, both UV and MS detectors suffer from non-uniform responses due to differences in absorptivity and ionization efficiencies as a function of chemical structure, respectively. Most types of MS can be used to analyze food components, including triple quadrupole, quadrupole time-of-flight, LTQ Orbitrap, ion trap, and magnetic sector mass spectrometers. Others, such as RI detectors, provide a more universal response but only for moderately high concentrations. Moreover, RI detectors are relatively insensitive and incompatible with gradient elution and difficult to stabilize. In recent years, LC coupled to an evaporative light-scattering detector (ELSD) has represented a useful alternative [25]. The use of an ELSD approach for spectrophotometric derivatization (i.e., insertion of chromophoric groups) is feasible and therefore the drawbacks of derivatization (e.g., dependence on experimental parameters, incompleteness of derivatization reaction, use of salt-laden mobile phases, prolonged analysis time, additional cost for derivatization system and reagents) can be eliminated [26]. LC coupled to ELSD was successfully proposed to determine lipids and biogenic amines in different food matrices [27]. Within the past decade, the introduction of ultra-high pressure liquid chromatography (UHPLC) and rapid-scan and sensitive MS instruments has resulted in a seismic shift away from traditional chromatographic techniques towards multiclass, multiresidue methods with short injection cycle times and minimal sample preparation [28]. Comprehensive methods for some of the more important contaminant groups in residue analysis have been developed for UHPLC-MS/MS, including anthelmintics, β-agonists, steroids, quinolones and others. Expected future developments include the possibility to analyze larger numbers of classes of compounds; the use of ever higher temperatures and pressures to create more effective separation methods; and further reduction in sample preparation via online solid-phase extraction and other techniques, to increase the speed of analysis beyond current standards.

The use of supercritical fluids is another useful technology attracting increased interest from researchers in the food sector. Carbon dioxide is the most commonly used supercritical fluid, because it is nontoxic, nonexplosive, and the experimental conditions required are easily achievable, since the critical temperature and pressure are, respectively, 31 °C and 73 bar [29]. Supercritical fluid chromatography (SFC) was initially performed with pure CO2 as the mobile phase, but nowadays SFC is very often carried out under subcritical conditions because CO2 is modified with an organic modifier or additive in order to increase the solubility of polar compounds [30]. In contrast to LC, SFC allows the use of higher flow rates with lower pressure falls through the column, leading to greater efficiency in short analysis times and reduced consumption of organic solvents. This implies sharper peaks, improved resolutions and faster methods due to the shorter times for column equilibration. Moreover, it offers the possibility of analyzing thermally labile and polar compounds which cannot be analyzed by GC without derivatization. Traditionally, SFC applications have been focused on lipid compounds, which could be due to the high solubility of these analytes in supercritical CO2, while more recent studies have shown their suitability for analyzing more polar compounds such as amino acids or carbohydrates [31].

Miniaturized separation techniques, such as electromigration methods (capillary electrophoresis, CE, and capillary electrochromatography, CEC), are alternative methodologies to GC and LC with a big potential regarding analysis time and costs, and offering different advantages, e.g., minor quantities of solvents, stationary phases and samples, easier coupling with MS, shorter analysis time, etc. [32]. Among these techniques, CEC has found a special place due to the combined advantages coming from CE and LC. On the one hand, the interaction between the analytes and the stationary phase inside the capillary column provides a high selectivity, while the presence of the electroosmotic flow reduces the solute dispersion in the column, highly increasing the efficiency. Different authors have reviewed the overall application of CEC [33, 34] for the analysis of certain compounds, such as nucleosides and nucleotides in food materials [35], proteins and peptides [36], natural/bioactive compounds [37] or phytochemicals [38].

1.3 Spectroscopic Methods

The spectroscopic methods used for food analysis include ultraviolet-visible (UV-Vis) spectroscopy, fluorescence spectroscopy, Raman spectroscopy and infrared spectroscopy (IR), X-ray spectroscopy, and nuclear magnetic resonance (NMR), such as electron spin resonance. The underlying mechanism at the basis of their application in routine analysis is described below.

UV/visible spectroscopy is a very simple physicochemical method with respect to experimental setup, developed in the middle of the 1900s. Recently it has been used for qualitative and quantitative analysis of food components such as carotenoids and related compounds [39], while a multivariate screening methodology based on UV-visible and multivariate classification was proposed for testing adulteration in sauces with the banned Sudan I dye [40].

Fluorescence spectroscopy is a highly developed and noninvasive technique that enables the online measurement of substrate and product concentrations or the identification of characteristic process states. The application of fluorescence spectroscopy, especially 2D fluoresence, is becoming more and more interesting for the analysis of fluorescent proteins and biological molecules (e.g., aminoacids, vitamins, and coenzymes) in foodstuffs [41].

Raman spectroscopy is another powerful technique for molecular analysis of foodstuffs since a fingerprint spectrum can be obtained for a target molecule. In this way, food components, additives, processes and changes during shelf life, adulterations and numerous contaminants, such as microorganisms, chemicals and toxins, can be determined [42]. Major advantages of this technique are its ability to provide information about concentration, structure, and interaction of biochemical molecules within intact cells and tissues nondestructively. In addition, it does not require homogenization, extraction, the use of dyes or any other labeling agent, or any pretreatment of samples [43].

Infrared spectroscopy embraces a number of techniques allowing the analysis of different types of samples (e.g., liquid, solids, pastes), determining specific applications such as attenuated total reflection (ATR, ATR-MIR), Fourier transform (FT-MIR, FT-NIR), transmittance (T), transflectance or reflectance (R), and diffuse reflectance (DF) [44]. For a long time, infrared (IR) spectroscopy was not considered to be a method for fundamental research in food science [45]. More recently, the possibility to generate spectra containing hundreds of variables (absorbance intensities measured at each wavenumber or wavelength) resulting in the production of large data sets, allow the extension of this technique to food analysis, and methods and techniques based in IR spectroscopy are now used for the routine analysis of several foods for chemical properties such as moisture, fat and protein [46]. Vibrational spectroscopy in general and IR spectroscopy in particular presents an unique opportunity to interrogate or analyze the food matrix as a whole, on a chemical and biochemical level, as the fingerprints contain information from all components of the sample.

X-ray, also called roentgen ray, is electromagnetic radiation with the wavelength range of 0.01–10 nm. The photon energy of an X-ray is in the range of 0.1–120 keV, which leads to strong penetrability. X-ray, similar to other electromagnetic waves, can show the following phenomena: reflection, refraction, scattering, interference, diffraction, polarization and absorption. Usually, X-rays whose photon energy is up to about 10 keV (10–0.10 nm wavelength) are classified as soft X-rays, and those of 10–120 keV (0.10–0.01 nm wavelength) are hard X-rays, due to their penetrating abilities. As hard X-rays pollute food, only the soft XRI technique is used in food inspection. X-ray has been employed for the evaluation of frozen products [47], in fruit-storage control [48], and fungal infection in wheat, namely Aspergillus niger, A. glaucus group, and Penicillium spp. [49]. Applications of X-ray in food manufacture were also reported [50], with the development of image-processing methods based on an X-ray instrument for the control of eye formation of cheese throughout the ripening period [51]. Foreign objects whose density is similar to that of water cannot be easily recognized by the X-ray technique [52].

The noninvasive, nondestructive nature of NMR relaxometry and magnetic resonance imaging (MRI) and the fact that both qualitative and quantitative data on physical and chemical properties of a wide range of samples can be gathered, have made them popular in food-related applications [53]. NMR techniques are applied in food science research and industrial processes to assess the product quality [54]. Changes in micro-cellular structure, diffusion of polymers and investigation of heat and mass transfer within the materials are performed by NMR/MRI measurements [55]. In addition, low-field NMR relaxometry and MRI have been used in the analysis of water content, mobility and distribution [56] as well as measurement of fat content and solid fat ratio and protein content [57].

Electron spin resonance (ESR) spectroscopy is a suitable tool useful in the detection of paramagnetic ion and free radicals with superior sensitivity limit and reduced acquisition time in respect to other analytical methods. ESR analysis has been approved as one of the standard reference methods for the detection of irradiated food containing bone, cellulose and crystalline sugar [58]. Furthermore, ESR technique was employed for the characterization of the antioxidant properties of sulfite and thiols in beer [59], determination of antocyanin in refined sugar [60], and sugar content in peony roots.

1.4 Biologically-Based Methodologies in Food Analysis

In food analyses, enzyme-linked immunosorbent assays (ELISAs), protein-based immunoassays lateral flow strip/protein strip tests, realtime polymerase chain reaction (RTPCR), and flow cytometry are widely explored techniques to assess food authenticity and detect biological contamination [17].

ELISA is an immunological technique involving the use of a selected enzyme to detect the presence of a specific antibody or antigen in a food sample in both a qualitative or quantitative format [61].

In food analysis, two variants of ELISAs have been widely explored, namely the indirect and sandwich ELISA. In the first protocol, two antibodies are employed, one of which is specific to the antigen to be detected, while the second is coupled to the enzyme and causes the production of a signal by a chromogenic or fluorogenic substrate [62]. In the sandwich ELISA protocol, the antigen is bound between two antibodies: one acting as capture and the other as detection antibody [63]. ELISA protocols are widely used for the detection of allergens, toxins, and antibiotic contamination with high specificity [64, 65].

Protein immunoassays are based on the molecular recognition of antigens by antibodies to form a stable complex [66–68]. Although the high specificity of the underlying mechanism is the basis of molecular recognition, widely adopted for the detection of allergens [69], this technique cannot discriminate among phylogenetically related species and suffers from false-negative results due to protein denaturation at high temperatures [70]. A direct upgrade of this technique is the lateral flow immunoassays, suitable for qualitative, semiquantitative and to some extent quantitative monitoring of pathogens, drugs, hormones, toxins and metabolites in foodstuffs [71]. Furthermore, the introduction of flow cytometric bead-based technology confers new opportunities for immunoassay protocols [72]. According to the literature, this technology allows (i) evaluation of multiple analytes in a single sample; (ii) utilization of minimal sample volumes; (iii) high reproducibility; (iv) direct comparison with already developed assays; and (v) a more rapid evaluation of multiple samples in a single platform.

Another emerging method for food analysis is RTPCR, the method of choice for food analysis to detect and differentiate between phylogenetically related species and to check the adulteration or the authenticity of food products [73], due to its rapid and highly sensitive identification capabilities [74]. Compared to conventional PCR, which is a cyclic process doubling the target sequences after each cycle and involving denaturation, annealing, and extension steps, in RT-PCR no post-processing is required to analyze the amplification process, as it monitors the increasing copy number of amplicon in real time after each cycle [17]. In this technique, several fluorescent dyes, with emission ranges between 487 and 560 nm, have been developed for the quantitative estimation of PCR amplicons, as the total fluorescence intensity changes in direct proportion to the amount of DNA in the sample. RTPCR-based protocols have been developed for the detection of allergens, genetically modified organisms, as well as bacterial and viral contamination [75].

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Chapter 2

Recent Analytical Methods for the Analysis of Sweeteners in Food: A Regulatory Perspective

Romina Shah* and Lowri S. de Jager

U.S. Food and Drug Administration, Center for Food Safety and Applied Nutrition, College Park, Maryland, USA

*Corresponding author: [e-mail]

Abstract

Non-nutritive or low calorie sweeteners are commonly used worldwide in the food industry, often in combination in order to limit undesirable tastes. The list of allowable sweeteners varies among countries and it is important for regulatory agencies and food safety laboratories to monitor these highly consumed products to ensure compliance with worldwide regulations. Current analytical methods for confirmation and quantification of sweeteners must allow for confirmation of analyte identity in order to be compatible with today’s standards. Various methods for the determination of non-nutritive sweeteners have been reported in the literature. The most common multi-sweetener methods involve high performance liquid chromatography (HPLC) with different types of detection. The modern technique of HPLC-MS/MS is the current method of choice for the determination and confirmation of sweeteners in foods. In addition to multi-sweetener analyses there is also a need for single sweetener analytical methods in certain circumstances.

Keywords: Non-nutritive sweeteners, foods, LC-MS/MS

2.1 Introduction

Non-nutritive sweeteners are commonly used in foods as alternatives to sugar to provide a sweet taste with little or no calories [1]. They are an important class of food additives which are added to foods to cause a technical effect such as sweetening [2]. Sweeteners are grouped into two main categories, bulk and intense sweeteners. Bulk sweeteners, such as sugar alcohols, provide texture and preservative effects to low calorie foods, with equivalent or less sweetening strength relative to sucrose. Sugar alcohols have been given quantum satis, meaning that they are harmless enough to have no specific quantity restriction [3].

Intense sweeteners have sweetening capacities greater than sucrose with varying potencies. These compounds can be synthetic, semi-synthetic or natural. The majority are synthetic compounds, including aspartame (ASP), sucralose (SCL), saccharin (SAC), cyclamate (CYC), acesulfame-potassium (ACS-K), alitame (ALI), neotame (NEO) and dulcin (DUL). Neohesperidine dihydrochalcone (NHDC) is a semi-synthetic sweetener, while stevioside (STV) and rebaudioside (REB) A are natural sweeteners [4]. The list of allowable sweeteners varies among nations worldwide [5]. For example, CYC and NHDC are not approved for use as food additives by the US Food and Drug Administration (FDA) but are authorized in the European Union (EU) [6].

The oldest sweetener on the market, SAC is approved for use in nearly 90 countries. It has a sweetening strength about 450 times that of sucrose and exhibits high water solubility and storage stability [7]. In the 1980s, its consumption was linked with bladder cancer in rats and as such was prohibited in Canada [8]. Despite its bitter metallic aftertaste it is approved for use in many foods and beverages [9]. Unlike SAC, DUL does not have a bitter aftertaste and has a sweetening capacity about 250 times that of sucrose. However, DUL has not gained widespread use due to concerns over its toxicity [7]. It is not approved for use in the USA.

Discovered in 1967, ACS-K exhibits good storage stability [9]. It is 200 times sweeter than sucrose and its use is associated with a slight bitter aftertaste at high concentrations [8]. ACS-K is widely used and approved in 90 countries with few health problems linked with its use [9]. It has very good water solubility and is stable at high cooking and baking temperatures [7].

In contrast, ASP is the most controversial artificial sweetener regarding its health effects. There have been reports about adverse neurological effects and cancer in rats. It is 180 times sweeter than sucrose and thus only small quantities are added to foods to achieve the desired sweetness. Since ASP is not heat-stable it degrades in liquids during prolonged storage [8]. Therefore, it cannot be used in baking or cooking and beverage products with ASP have expiry dates for acceptable consumption [9]. It has been approved for use by the US FDA and the EU. Phenylalanine is a metabolite of ASP, which cannot be metabolized by people with phenylketonuria, a rare genetic disorder. Excessive intake of phenylalanine has been linked to brain damage [7]. As a result, all products containing ASP must be labeled to indicate the presence of a phenylalanine source [8].

A derivative of ASP, NEO is an odorless, white crystalline powder. It is safer for consumption by people with phenylketonuria because the 3,3-dimethyl group in its structure blocks the breakdown to phenylalanine [10]. NEO is 7000–13000 times sweeter than sucrose, with a taste very similar to sucrose. Its use is not associated with any bitter aftertaste and it has extensive shelf-life stability in dry conditions. It is also very stable in aqueous solutions in the neutral and acidic pH ranges [7]. In addition, NEO is heat stable and thus can be used in cooking and baking. It is approved for use in the USA, Australia, New Zealand and the EU.

The dipeptide sweetener ALI has a sweetening capacity 2000 times greater than sucrose. Due to the presence of an amide moiety in its structure, ALI is relatively heat stable [7]. It has no aftertaste and is characterized by a clean, sweet flavor. It is approved for use as a sweetener in Australia and Mexico but not in the USA or EU [7].

Discovered in the 1960s, NHDC has a sweetening strength ~1500 times greater than sucrose. Industrially, it is produced by hydrogenation of a flavonoid (neohesperidin) found in citrus fruits. NHDC is known to have menthol-licorice-like aftertastes and antioxidant properties [8]. It exhibits good stability in aqueous solutions [7].

Sucralose is thermally stable and contains three chlorine atoms in its structure, making it an organochloride. It is about 600 times sweeter than sucrose and can be used during cooking and baking [9]. It is approved for use by the US FDA in a variety of foods and beverages. There is some concern about its safety due to the fact that other organochlorides such as dioxins and pesticides are linked with toxic and carcinogenic effects [8]. However, human and animal studies have shown SCL to be safe for human consumption [9].

Steviol glycosides are natural components in the extract of Stevia rebaudiana Bertoni, a plant native to Paraguay [11]. Stevia has been used for years in Japan, Korea, China, Brazil, and Paraguay as a food additive or as a household sweetener [12]. Steviol glycosides under certain conditions are considered Generally Recognized as Safe (GRAS) by the FDA and are approved in the EU. Stevia produces several diterpene glycosides, the most abundant being STV and REB A [13]. Five other steviol glycosides have been identified as minor components of the stevia leaf, including Reb C, D, F, dulcoside A, and rubusoside. The steviol glycosides have similar structures: a steviol aglycone is connected at C-4 and C-13 to mono, di, or trisaccharides consisting of glucose and/or rhamnose residues [14, 15]. Steviolbioside and Reb B are thought to be hydrolysis products of STV and Reb A formed during the extraction process of the glycosides from the plant [16]. The distribution of steviol glycosides in plant extracts can vary greatly depending on the extraction and purification process [17]. One issue preventing the wide use of stevia as an artificial sweetener is the presence of a bitter aftertaste in some extracts. REB A has been reported to have the least bitterness of the major steviol glycosides [18]. The sweetening power of the steviol glycosides also differ, with REB A being 400 times sweeter than sucrose while STV is about 300 times sweeter [16, 19].

Sweeteners are often used in combination to enhance sweetness and limit undesirable aftertastes [7]. A classic example is the blend of SAC-CYC formulated in a 1:10 ratio. The bitter aftertaste of SAC is masked by CYC and due to an additive effect the sweetening power of the mixture is greater. Food products containing sweeteners are heavily promoted as beneficial for the treatment of obesity and management of diabetes [7]. Sweeteners can be found in a large number of food products including the following: tabletop sweeteners, carbonated and non-carbonated beverages, baked goods, preserves and confectionery (icings, frostings, and syrups), alcoholic drinks, candies and dairy products such as yogurt and ice cream [20].

There is considerable controversy surrounding the adverse health effects of non-nutritive sweeteners. Consumers worldwide have reported side effects linked to sweetener consumption, including mood and behavioral changes, skin irritations, headaches, allergies, respiratory difficulties, and cancer [7]. As such, it is important to monitor and control the concentration of sweeteners in foods to ensure compliance with different country-specific regulations. The EU limits the amount of sweeteners added to food and sets a maximum usable dose (MUD) for specific food commodities [20]. In order to ensure that products are in compliance with regulations, it is necessary to have reliable, robust and quantitative methods for the simultaneous determination of several commonly used sweeteners in a single analysis.

In addition to multi-sweetener analyses, there is also a need for single sweetener analytical methods such as in the case of CYC. The non-nutritive sweetener CYC was discovered in the 1930s [21]. It is 30–40 times sweeter than sucrose with its efficacy increasing when used in combination with other sweeteners [22]. It is widely used as a sweetening agent in a variety of low-calorie foods and beverages in many countries [21]. However, CYC is banned for commercial use as a food additive by the US FDA (Code of Federal Regulations 21, §189.135) because of research findings that linked its consumption with bladder cancer in rats [23]. Under the ban, CYC cannot be added to or be detectable in food. Since there is an increasing number of foods sold in the USA that are imported from other countries, where CYC is approved for use as a food additive, it is important to have analytical methodology for the detection and confirmation of CYC in foods [22].

2.2 Sample Preparation

Sample preparation/cleanup is the process of isolating target analytes from interferences in food matrices prior to instrumental analysis. This is often the most time-consuming part of the analytical method and is essential to analyte determination. In order to be able to determine whether or not a sample contains sweeteners and authenticate the presence and concentrations of these analytes in various foods, simple to extensive sample cleanup is necessary. Sweeteners are widely used in drinks, candies and yogurts, which are commonly consumed products [24]. Foods are complex matrices due to the considerable differences in their composition, which includes the presence of macromolecules, color additives and preservatives. Furthermore, sweeteners are present in food products at levels that require prepared samples to often be significantly diluted in order to bring the analyte concentrations within the linear range of the method. There are many components in food matrices that have similar polarities to sweeteners, most of which are water soluble, with the exception of DUL and NHDC. Therefore, it is very difficult to isolate sweeteners from food matrix.

There are considerable differences in the concentrations of sweeteners in drinks, possibly due to beverage manufacturing processes that may contribute to these variations. Differences are most likely due to the varying sweetening strengths of these compounds relative to sucrose. Therefore, differing amounts of sweeteners are added to produce the desired sweetening effect [3]. Furthermore, there are significant differences in chemical properties among sweeteners such as solubility and thermal stability [3]. As such, some sweeteners function better in certain food types while others are best suited for use in drinks.

Generally, hard candies, drinks and tabletop sweeteners require minimal sample preparation prior to instrumental analysis. Normally, hard candies and tabletop sweeteners are weighed and dissolved in H2O by the process of shaking and/or vortexing. The samples are then diluted to obtain an analyte concentration within the linear range of the method. This procedure should produce complete dissolution of the candy or tabletop samples, resulting in transparent solutions with no visual insoluble material remaining after shaking. Drink samples are simply diluted with H2O or mobile phase and filtered with sonication of carbonated beverages to remove dissolved gases [25]. Replicate analysis should be performed on all samples and if products are packaged in individual servings (candy, tabletop sweeteners), separate packages should be analyzed.

Liquid-liquid extraction (LLE) is sometimes used as a simple, low-cost method to prepare samples prior to instrumental analyses [5]. LLE involves addition of an organic solvent to the food in liquid form. Sweeteners are then extracted from the liquid aqueous phase into the organic phase [6]. Solid-liquid extraction (SLE) is the process of partitioning target analytes from a solid state into a solvent prior to dilution and filtration. Solid samples can be homogenized, vortexed and centrifuged to separate the supernatant [5, 22, 26].

Yang and Chen [5] used LLE and SLE to extract sweeteners from a water/methanol solution (50:50, v/v). Beverages were degassed when necessary and solid samples were homogenized and extracted. The method was applied to the determination of eight non-nutritive sweeteners in foods. Lim et al. modified the LLE and SLE procedures developed by Yang and Chen to analyze nine artificial sweeteners in Korean foods. Samples analyzed included candies, beverages and yogurts. Sheridan and King [22] applied SLE with homogenization to the analysis of CYC in a wide range of foods, including dried prunes and beans, jarred mangos and peaches, grape tomatoes and strawberry cake. Since CYC is water-soluble the aqueous extract could be centrifuged, filtered and significantly diluted, which limits matrix interferences and MS signal suppression [22]. Scotter et al. also used LLE and SLE for the analysis of CYC in carbonated beverages, fruit juices, milk-based desserts, jams and spreads. Additionally, Carrez I and II solutions (reagents used to precipitate proteins and fats) were prepared and added to the foods under slightly heated conditions for sample clarification [7, 26]. This is followed by centrifugation to separate proteins and fatty material from the water-soluble supernatant in complex matrices such as ice-cream, chocolate syrup and coffee creamers [27]. The supernatant can then be filtered and diluted in preparation for instrumental analysis. Centrifugation without protein separation may be needed to separate solid particles present in some fruit juices [28]. Solvents that are commonly used for extraction are methanol (MeOH), acetonitrile (ACN) and water [28].

Another technique to prepare solid samples, such as dried fruits, uses a cryogenic grinder. Dried fruits are cut into small pieces and placed into a cryogenic blender. Liquid nitrogen is then poured over the pieces until they are immersed. Once the liquid nitrogen completely evaporates and the pieces are frozen they are blended into a fine powder using an analytical mill. Solvent is then added to a weighed amount of the powder with subsequent vortex mixing, centrifugation, dilution and filtration [28]. This procedure results in a more homogeneous and uniform sample mixture than achieved with normal homogenization because the solid is broken down into very fine particles.

One of the biggest challenges in food analysis is the effect of matrix composition on the performance of the analytical method. In order to determine method accuracy and selectivity, a representative from each food commodity containing no target analytes is fortified with known amounts of sweeteners. The sweeteners chosen for spiking experiments should encompass the range of polarities, including most polar, intermediate and nonpolar compounds. Food products are fortified in triplicate at three different concentrations in accordance with agency guidelines and analyzed alongside an unfortified sample.

Solid-phase extraction (SPE) is a reproducible technique that can be used to isolate sweeteners based on their affinity to a stationary phase. The SPE sorbents are silica- or polymer-based beds that are modified with polar or nonpolar functional groups [29]. There are many types of commercially available SPE cartridges that are packed with C8, C18 and ionexchange sorbent beds [29]. For the isolation of sweeteners from foods, the most successful SPE cartridges have been those with reversed-phase (RP) sorbents such as C8 or C18 [30].

Zygler et al. developed a method for the determination of nine non-nutritive sweeteners using Strata-X polymeric RP 3 mL cartridges packed with 200 mg sorbent bed for the cleanup of beverages, yogurts, and fish products [20]. These SPE cartridges were chosen because extensive testing of different SPE columns, including Chromabond C18ec, Strata-X RP, and Bakerbond Octadecyl, revealed optimal recoveries for all sweeteners were achieved [29].

Scheurer et al. [8] tested several different SPE cartridges and determined that Bakerbond Isolute SDB-1 achieved best recoveries for the extraction of ACS-K, SAC, ASP, CYC, NEO, SCL and NHDC in waste and surface waters. Yogurts represent a much more complex mixture of ingredients than beverages or hard candies, thus requiring a thorough sample cleanup prior to chromatographic analyses [31]. This ensures better long-term performance of the instrument and minimizes ion suppression effects when using mass spectrometric detection.

Shah et al. [32] modified and optimized a previous SPE method for the analysis of yogurts using Macherey-Nagel Chromabond® C18ec 3 mL cartridges packed with 500 mg sorbent bed [29]. Several SPE parameters were tested, including sorbent phase type, cartridge size, sample load volume, and extraction buffer. As previously seen, the most critical factor affecting analyte recoveries was the composition of the extraction buffer [29]. The use of formic acid and N,N- diisopropylethylamine (DIPEA) at pH 4.5 yielded the best recoveries for the sweeteners from yogurts. Compared to triethylamine (TEA), the ion pairing agent DIPEA allows for improved recoveries as it enables a stronger hydrophobic interaction between the sorbent bed and sweeteners [29]. As a result, this enables better retention of the sweeteners on the SPE cartridge, especially ACS-K and CYC. The authors reported that it is imperative to prevent the cartridge from drying out during the course of this SPE procedure.

Yang and Chen [33] developed a SPE method using a Waters Oasis HLB cartridge for the isolation of NEO from beverages, preserved fruits and cake. Dairy and fruit juice beverages were pretreated with MeOH, mixed, centrifuged and loaded on the SPE cartridge. Preserved fruits and cake were homogenized, vortexed, sonicated, centrifuged, and then loaded onto the SPE cartridge. The cartridge was conditioned prior to sample loading and then washed with water followed by MeOH to remove impurities. NEO was eluted with MeOH and concentrated to dryness by vacuum and reconstituted