Analytical and Sample Preparation Techniques for the Determination of Food Colorants in Food Matrices

Color additives are widely used by the food industry to enhance the appearance, as well as the nutritional properties of a food product. However, some of these substances may pose a potential risk to human health, especially if they are consumed excessively and are regulated, giving great importance to their determination. Several matrix-dependent methods have been developed and applied to determine food colorants, by employing different analytical techniques along with appropriate sample preparation protocols. Major techniques applied for their determination are chromatography with spectophotometricdetectors and spectrophotometry, while sample preparation procedures greatly depend on the food matrix. In this review these methods are presented, covering the advancements of existing methodologies applied over the last decade.


Introduction
Codex Alimentarius gives a definition for food additives as "any substance that its intentional addition of which to a food aiming for a technological (including organoleptic) purpose in the manufacture, processing, preparation treatment, packing, packaging, transport or holding of such food results, or may be reasonably expected to result, in it or its by-products becoming a component of the food or otherwise affecting the characteristics of such foods" [1,2]. Carocho et al. highlighted that the definition given by the Codex Alimentarius does not include the term contaminants or substances added to food for maintaining or improving nutritional qualities [2].
In food technology, food colorants, of several types, are chemical substances that are added to food matrices, to enhance or sustain the sensory characteristics of the food product, which may be affected or lost during processing or storage, and in order to retain the desired color appearance [3][4][5]. These are classified based on several criteria: firstly, based on their origin in nature, nature-identical or, if synthetic, whether they are organic or inorganic. Another classification could be based on their solubility (e.g., soluble or insoluble) or covering ability (e.g., transparent or opaque), though an overlap may exist among one or more of these classifications. The most common and widely used classification is based on the distinction between soluble and insoluble color additives (colorants or pigments), which can be further categorized as natural or synthetic [4].
In addition, as described by Martins et al., there were several food additives that had been used extensively in the past but are no longer allowed, due to existing evidence of their side effects, toxicity in the medium-and long-term, as well as a high frequency of potential health incidents [6]. It is also as they bound to DNA [22]. Many other studies have shown the chemical property of synthetic colorants, namely, Tartrazine [23], azorubine [17,24,25], Allura Red [17,26,27] Sunset Yellow, Quinoline Yellow [17], and Patent Blue [28], to bind to human serum albumin (HSA). Masone and Chanforan compared binding affinities of artificial colorants to human serum albumin (HSA), exhibiting more affinity to HSA than to their natural equivalents' colorants and interacting with its functions. The results supported the hypothesis of their potential risk to human health [17]. Finally, there are dyes, which are rather inexpensive, and which have been used in the food industry, such as Sudan I-IV, which are classified as both a toxic and carcinogenic [24][25][26][27][28][29][30][31]. In Figure 1, basic structures of colorants used in the food industry some of them with toxicological concern (Sudan I-IV) are presented. in vitro experiments, and they concluded there was an in vitro toxic effect on human lymphocytes as they bound to DNA [22]. Many other studies have shown the chemical property of synthetic colorants, namely, Tartrazine [23], azorubine [17,24,25], Allura Red [17,26,27] Sunset Yellow, Quinoline Yellow [17], and Patent Blue [28], to bind to human serum albumin (HSA). Masone and Chanforan compared binding affinities of artificial colorants to human serum albumin (HSA), exhibiting more affinity to HSA than to their natural equivalents' colorants and interacting with its functions. The results supported the hypothesis of their potential risk to human health [17]. Finally, there are dyes, which are rather inexpensive, and which have been used in the food industry, such as Sudan I-IV, which are classified as both a toxic and carcinogenic [24][25][26][27][28][29][30][31]. In Figure 1, basic structures of colorants used in the food industry some of them with toxicological concern (Sudan I-IV) are presented. The main regulatory authorities, EFSA in Europe and the US Food and Drug Administration (FDA) in the United States, are responsible for the evaluation and assessment of food products to enhance and promote health safety [2,4,5]. The European Union, set a re-evaluation program of food additives, including food colorants, to be performed by EFSA by 2020, based on the EU Regulation 257/2010. This re-evaluation program was set in order to assess the safety of all authorized food additives in the European Union before 20 January 2009 [32].
However, despite the existence of different regulatory frameworks, the overall approach follows similar steps, which are based on well-established risk assessment procedures [33].
Authorization for the use of food colorants in the production of food products is subject to a number of toxicity tests, in order to define and evaluate acute, sub-chronic and chronic toxicity, The main regulatory authorities, EFSA in Europe and the US Food and Drug Administration (FDA) in the United States, are responsible for the evaluation and assessment of food products to enhance and promote health safety [2,4,5]. The European Union, set a re-evaluation program of food additives, including food colorants, to be performed by EFSA by 2020, based on the EU Regulation 257/2010. This re-evaluation program was set in order to assess the safety of all authorized food additives in the European Union before 20 January 2009 [32].
The regulatory framework in Europe, in brief, contains the authorization procedure in Regulation  [4,5,10,33].
However, despite the existence of different regulatory frameworks, the overall approach follows similar steps, which are based on well-established risk assessment procedures [33].

Analytical Techniques in the Use of Natural Food Colorants Determinations
The available bibliography concerning the methods of analysis for the natural colorants is limited, compared to that for the synthetic ones, and it is exclusively oriented to their determination in the different naturally deriving products.
All the relative information concerning analytical methods for natural colorants, including tested matrices, analytical technology, type of detection and settings, analytical columns if used, elution parameters, mobile phases, injection volumes, and analytical figures of merit (LOD, LOQ), have been reviewed and are summarized in Table 1.
It can be concluded from Table 1 that evaluation of methods' performance criteria was not within the aims of the above-mentioned reports, as they were focusing in activity, bioavailability, processing impact, and adulteration. Thus, no analytical figures of merit are reported in these papers.
From Table 1 and Figure 2 it could be perceived that the predominant technique is HPLC combined with spectrophotometric (UV-Vis) or Diode Array (DAD) detectors, followed by HPLC by MS/MS. Spectrophotometric UV-Vis methods seem also to be preferred by the researchers in this field as they show low instrument cost and do not involve expert skill. However, it should be considered that the individual features of the spectra obtained for single colors are highly dependent on the pH-adjustment of the solution or the mobile phase, using proper acid or alkali. The pH adjustment certainly affects maximum absorption wavelength, where shifts and intensities based on the different pH can be observed. Although sample preparation is much less demanding in comparison to the LC methods, these techniques present a significant disadvantage, which is the lack of ability to analyze simultaneously a bigger number of food colorants. It can be concluded from Table 1 that evaluation of methods' performance criteria was not within the aims of the above-mentioned reports, as they were focusing in activity, bioavailability, processing impact, and adulteration. Thus, no analytical figures of merit are reported in these papers.
From Table 1 and Figure 2 it could be perceived that the predominant technique is HPLC combined with spectrophotometric (UV-Vis) or Diode Array (DAD) detectors, followed by HPLC by MS/MS. Spectrophotometric UV-Vis methods seem also to be preferred by the researchers in this field as they show low instrument cost and do not involve expert skill. However, it should be considered that the individual features of the spectra obtained for single colors are highly dependent on the pHadjustment of the solution or the mobile phase, using proper acid or alkali. The pH adjustment certainly affects maximum absorption wavelength, where shifts and intensities based on the different pH can be observed. Although sample preparation is much less demanding in comparison to the LC methods, these techniques present a significant disadvantage, which is the lack of ability to analyze simultaneously a bigger number of food colorants.

Sample Preparation for Natural Colorant Analysis
Several sample preparation protocols are reported in the literature by applying various techniques. The applied protocol is strongly dependent by the type and nature of the food sample. Below in Table 2, a short description of the sample preparation protocols is given, along with their application for the clean-up of food samples, for the quantification of natural food colorants. A hydrolysis step with a deprotonation step (ethanol, HCl solution) is being reported depending on the food matrix, including dilution methods and SFE with supercritical CO2.   Ref. Anthocyaninderived extracts

Acacia decurrens
Willd. Bark Spectrophotometric analysis UV-Vis 400-800 nm n/a n/a n/a n/a [35] -Tulipa gesneriana L. Spectrophotometric analysis UV-Vis 765 nm n/a n/a n/a n/a n/a [ Spectrophotometric analysis UV-Vis λ = 536 nm n/a n/a n/a n/a n/a [41] Betalains Rivina humilis L. fruits, juice Spectrophotometric analysis UV-Vis λ = 535 nm n/a n/a n/a n/a n/a [42]  n/a n/a [46] Crocetin, Crocin Grape seed, monascus, gardenia, and red radish Spectrophotometric analysis UV-Vis 438 nm; 462 nm n/a n/a n/a n/a n/a [47] Monascus red pigments

Sample Preparation for Natural Colorant Analysis
Several sample preparation protocols are reported in the literature by applying various techniques. The applied protocol is strongly dependent by the type and nature of the food sample. Below in Table 2, a short description of the sample preparation protocols is given, along with their application for the clean-up of food samples, for the quantification of natural food colorants. A hydrolysis step with a deprotonation step (ethanol, HCl solution) is being reported depending on the food matrix, including dilution methods and SFE with supercritical CO 2 . Table 2. Sample preparation techniques for the analysis of natural food colorants in food products.

Food Colorant
Extraction/Sample Preparation Ref.

Analytical Techniques in the Use of Synthetic Food Colorants Determinations
The need to determine synthetic colorants in food matrices originating from their known toxicity, renders the analytical task even more challenging as food matrices are ordinarily very complex. Various analytical techniques are used to determine synthetic food colorants in food samples, including spectrophotometry, thin layer chromatography, capillary electrophoresis, high performance liquid chromatography and mass spectrometry (MS).
Certain chemical properties and characteristics of the substances/colorants that influence their separation, such as hydrophilicity/hydrophobicity, existence of acidic or alkaline groups should to be taken into account. Using a Reversed Phase (RP) liquid chromatography separation, more polar compounds are eluting first followed by the less polar. However, their chromatographic separation is normally performed at neutral pH (ca. 7), and thus, any presence of acidic or alkaline groups could affect the elution sequence.
Ordinarily, organic solvents such as methanol, acetonitrile, or their mixture are used for analysis by HPLC. The addition of acetonitrile improves significantly chromatographic peaks' shape (i.e., asymmetry). Nevertheless, the addition of an inorganic electrolyte as a chemical modifier to the mobile phase can be considered as important in order to advance the separation of all the ionizable species [12,28,37,49].
Food colorants are compounds that absorb exceedingly in the visible region. Thus, spectrophotometry is sufficient and appropriate for their quantitative analysis. It is generally preferred as a quite straightforward technique, with respective low instrumental cost (i.e., compared to MS/MS). However, in several cases, its main drawback is the lack of specificity, as in case of mixtures of absorbing species. A solution to overcome the problem of specificity is the application of mass spectrometry (MS). In this case, all spectral interventions or interferences, presented on UV-Vis/DAD detectors, are overpassed. High analytical sensitivity could succeed, even in more difficult food matrices, though after proper clean-up. In addition, tandem MS technique could provide structural information based on the molecular mass/ion and the respective fragmentation pattern. Regarding the ionization mode, in most cases, for synthetic colorants, the electro spray ionization (ESI) is preferred because synthetic food colorants are polar molecules, and their ionization efficiency depends on the existence of matrix interferences, present in sample or in the mobile phase. In general, negative mode (ESI-) is more effective, though in other non-regulated substances (i.e., Sudan I-IV) the positive ionization is preferred. During the MS/MS analysis, chemical modifiers (i.e., HCOONH 4 or CH 3 COONH 4 ) are added to the mobile phases, in order to improve and facilitate the better ionization of each target analyte.
Capillary electrophoresis follows in frequency of use the HPLC-DAD/UV-Vis or MS/MS techniques, applied for the quantification of food colorants. These methods present good separation of both small and large molecules, using high voltages. Other reported techniques are FIA (Flow Injection Analysis) and TLC (Thin Layer Chromatography). These could be considered as relatively simple analytical techniques, even for quantification, though in certain cases they could lack specificity and could be affected by matrix interferences.
For synthetic food colorants, all the respective references containing details about the tested matrices, analytical techniques, detection and settings, analytical columns if used, elution, mobile phases, injection volumes, and figures of merit (LOD, LOQ) are presented below in Table 3.
As it could be extrapolated from Table 3, a significant number of LC-MS, LC-MS/MS or LC-UV/Vis methods are available, which are dedicated to simultaneous detection of either a significant or limited number of artificial colorants (whether authorized or delisted), even including illegal Sudan-type dyes. In addition, to Table 3, Figure 3 gives the percentage distribution of the analytical techniques, regarding the analysis of synthetic food colorants. It could be easily concluded that HPLC/U(H)PLC is the most frequently applied technique, followed by capillary electrophoresis and enzyme-linked immunosorbent assay (ELISA) as well as other residual methods. In the case of ELISA, it needs to be highlighted that it cannot be applied for a group of substances/food colorants but only for standalone substances, for which the monoclonal antibodies have been developed. As it could be extrapolated from Table 3, a significant number of LC-MS, LC-MS/MS or LC-UV/Vis methods are available, which are dedicated to simultaneous detection of either a significant or limited number of artificial colorants (whether authorized or delisted), even including illegal Sudan-type dyes. In addition, to Table 3, Figure 3 gives the percentage distribution of the analytical techniques, regarding the analysis of synthetic food colorants. It could be easily concluded that HPLC/U(H)PLC is the most frequently applied technique, followed by capillary electrophoresis and enzyme-linked immunosorbent assay (ELISA) as well as other residual methods. In the case of ELISA, it needs to be highlighted that it cannot be applied for a group of substances/food colorants but only for standalone substances, for which the monoclonal antibodies have been developed.
The applied analytical techniques are followed by proper detection approaches. In this framework, simple detector UV-Vis/DAD is mostly applied, followed by MS/MS detectors, UV-Vis spectrometry, and electrochemical detection. The UV-Vis/DAD detection wavelengths depend on the analyte color (i.e., blue, yellow, red) set in any case in the maximum absorbance.
Regarding the MS, listed and EU-approved food colorants could be analyzed in the negative ionization, while for other substances (i.e., Sudan I-IV) positive ionization is applied. From observation among the available methods of analysis (Table 3 and Figure 3), it could be concluded that traditional TLC methods require a significant sample preparation step and a timeconsuming analytical procedure. On the other hand, the HPLC methods need longer analysis time, compared to the respective LC-MS/MS methods, in order to obtain good separation for the same number of analytes [87][88][89].

SERS-Raman
Radiation of 514.5 nm from an air-cooled argon ion laser was used for SERS excitation n/a n/a n/a n/a LOD = 10 −7 -10 −5 M [83] Sunset yellow beverage, dried bean curd, braised pork ELISA n/a n/a n/a n/a LOD = 25 pg mL −1 [84]  The applied analytical techniques are followed by proper detection approaches. In this framework, simple detector UV-Vis/DAD is mostly applied, followed by MS/MS detectors, UV-Vis spectrometry, and electrochemical detection. The UV-Vis/DAD detection wavelengths depend on the analyte color (i.e., blue, yellow, red) set in any case in the maximum absorbance.
Regarding the MS, listed and EU-approved food colorants could be analyzed in the negative ionization, while for other substances (i.e., Sudan I-IV) positive ionization is applied.
From observation among the available methods of analysis (Table 3 and Figure 3), it could be concluded that traditional TLC methods require a significant sample preparation step and a time-consuming analytical procedure. On the other hand, the HPLC methods need longer analysis time, compared to the respective LC-MS/MS methods, in order to obtain good separation for the same number of analytes [87][88][89].
As reported recently by Periat et al., full-scan screening methods using HR-MS (High Resolution Mass Spectrometry) have proven to be an alternative to triple quadrupole methods as they could maximize the number of control and analyzed target colorants. Main advantages of the HR-MS can be the reduced sample preparation and the combined targeted analysis with untargeted screening of food colorants with high MS resolving power. Quadropole Time-of-Flight (QTOF) used by Li et al. and by Periat et al. for the detection and identification of coloring compounds in spices provided not only mass accuracy but also MS/MS spectra information and thus increased selectivity. A drawback of the approach could be the high cost of the instrumentation [85,86]. As reported by Li et al., HR-MS accurate mass measurements can detect a large number of target analytes, avoiding isobaric interferences in complex samples [89]. A combination of an ESI (or APCI) ionization with an anion trap analyzer linked to a TOF mass analyzer (ESI/APCI-IT-TOF/MS) provides simultaneously multi tandem MS (up to MS 2 ) with respective mass accuracy. Currently, there is an increasing interest on the fragmentation mechanism of synthetic food dyes; use of ESI-IT-TOF/MS n in positive as well as in negative ionization modes [87][88][89] has been increased.

Sample Preparation for the Determination of Synthetic Colorants in Foods
Currently, there is no generally accepted/standard method for synthetic colorant extraction in laboratories. Nevertheless, most extraction procedures follow a common approach, which normally involves firstly the release of desired analytes from their matrices, followed afterwards by removal of extraneous matter/interferences by applying an efficient extraction protocol (i.e., solid-liquid or liquid-liquid extraction) [90].
The applied sample preparation protocols are strongly dependent on the type and nature of the food sample. A short description of the sample preparation protocols, along with their application to the clean-up of food samples, for the analysis of synthetic food colorants is given in Table 4.
Membrane filtration involves the permeation of the analyte through a thin layer of material. Explicitly, in case of beverages, when filtration is involved, a degassing step needs to be done in advance, in order to remove CO 2 [90].
Solid phase extraction (SPE) is one of the most commonly used techniques in determination of food colorants, presenting certain advantages such as simplicity. Polyamide resin used for SPE cleanup retains polar compounds with chemical groups that can be protonated. In acidic pH, during SPE, the colorants are adsorbed to the polyamide stationary phase mainly by Van der Waals interactions. Other hydrophilic substances can mask SPE interaction sites by reducing their binding power for the colorants and consequently reducing the capacity of the cartridges. Some substances, such as amaranth, are strongly retained by SPE cartridges, and the ammonia solution used for elution could be insufficient for its release (low recoveries).   Dispersive solid phase extraction (d-SPE) analysis is a simple sample preparation methodology that is suitable for a wide variety of food and agricultural products, as is also QuEChERS, introduced for pesticides from Anastassiades et al. [91]. In case of synthetic colorants, a modified QuEChERS method has been reported (magnetic-dSPE) using cross-linking magnetic polymer (NH 2 -LDC-MP) containing less hydrophilic amino groups and more lipophilic styrene monomer for cleaning up the synthetic food colorants from wine and soft drinks [53].
Liquid-liquid extraction (LLE) deals with the separation of substances based on their relative solubility in two different immiscible liquids. Common solvents for the extraction of synthetic food colorants from food matrices are water, ethanol, methanol, isopropyl alcohol, ammoniac ethanol, ethyl acetate, ammonia, cyclohexane, and tetra-n-butyl ammonium phosphate. Wu et al. has also reported an extraction method based on Ionic liquid dispersive liquid phase microextraction using the ionic liquid (1-Octyl-3-methylimidazolium tetrafluoroborate ((C 8 MIM)(BF 4 ))) [81].
In the literature, a limited number of protocols exists dealing with other types of extraction methods for synthetic food colorants, such as MAE and Ultrasound Assisted Extraction (UAE). These kinds of extractions require special instrumentation and most probably can be beneficial for a laboratory, as extractions with organic solvents are characterized by consumption of high volumes of solvents, are time consuming, and in some cases have low recoveries [90].

Conclusions
The use of food colorants in the production of foods leads to the need for the development of accurate, precise, sensitive, and selective analytical methods for their analysis and quantification. Certain interest in the impacts of food colorants is being reported worldwide. There is a plethora of analytical research works that deal with the analytical challenge of the analysis and quantification of either natural or synthetic food colorants. The research community gives more attention to the appropriate analysis, in sufficient concentration or mass fraction levels, mostly to synthetic food colorants rather than natural ones.
Analytical methodologies have much more to offer in this direction and, as it could be concluded from synthetic colorants, HPLC is the most frequently used followed by capillary electrophoresis. In terms of detection methods, the simple UV-Vis/DAD is the predominant one followed by tandem MS. The analytical techniques and sample preparation methodologies presented cover the existing methodologies mainly applied during the last decade.
Regarding sample preparation, this is highly sample dependent. It could involve the application of different extraction techniques, such as membrane filtration, liquid-liquid and solid phase extraction techniques, for cleaning-up the highly complex matrix of food products. Sample preparation is of great importance and must be carefully developed, in order to avoid or eliminate existing matrix interferences aiming to the development of simple, selective, and precise methods of extraction.
In the case of simple liquid samples, dilution and injection are preferred, though in other cases such as high protein content foods, specific steps need to be followed for sufficient sample clean-up.