Rovina 2017

Accepted Manuscript
Toxicology, extraction and analytical methods for determination of Amaranth in food

and beverage products
Kobun Rovina, Shafiquzzaman Siddiquee, Sharifudin Md Shaarani
PII: S0924-2244(16)30417-4
DOI: 10.1016/j.tifs.2017.05.008
Reference: TIFS 2010
To appear in: Trends in Food Science & Technology
Received Date: 20 September 2016

Revised Date: 6 February 2017
Accepted Date: 14 May 2017
Please cite this article as: Rovina, K., Siddiquee, S., Shaarani, S.M., Toxicology, extraction and
analytical methods for determination of Amaranth in food and beverage products, Trends in Food
Science & Technology (2017), doi: 10.1016/j.tifs.2017.05.008.
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ACCEPTED MANUSCRIPT
1 Toxicology, Extraction and Analytical Methods for Determination of Amaranth in Food
2 and Beverage Products
5 Kobun Rovina1,2, Shafiquzzaman Siddiquee*1, and Sharifudin Md Shaarani2
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9 Biotechnology Research Institute, Universiti Malaysia Sabah, Kota Kinabalu, Sabah,
10 Malaysia
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11 Faculty of Food Science and Nutrition, Universiti Malaysia Sabah, Kota Kinabalu, Sabah,
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12 Malaysia
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16 Corresponding author:
17 *Siddiquee, S
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18 Biotechnology Research Institute, Universiti Malaysia Sabah, Jln UMS, 88400 Kota
19 Kinabalu, Sabah, Malaysia
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20 Email: shafiqpab@ums.edu.my
21 Office phone: 006088320000 ext. 8467
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22 Fax: 006088 320993

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28 ABSTRACT
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30 Amaranth (E 123) is synthetic dyes which commonly used in the industry and scientifically
31 applied as textiles and paper productions as well as for cosmetic and pharmaceutical
32 production. However, higher consumption of Amaranth may lead to hyperactivity and other
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33 disturbed behaviour especially in children. World Health Organization (WHO) with Food and
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34 Agriculture Organization (FAO) has the acceptable daily intake (ADI) of Amaranth between
35 0 to 0.5 mg kg-1. Several extraction and analytical methods have available for quantifying and
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36 identifying the existence level of Amaranth in food products. Herein, we critically discussed
37 those existing analytical methods applied such as high performance liquid chromatography
38 (HPLC), thin layer chromatography

U(TLC), liquid chromatography/tandem mass
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39 spectrometry (LC-MS/MS), spectrophotometric, electrochemical sensor and capillary zone
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40 electrophoresis (CZE). Additionally, a brief description on the uses of different extraction
41 methods such as solid phase extraction (SPE), liquid-liquid extraction (LLE) and membrane
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42 filtration have presented. In this review incorporated valuable information’s that intended as a
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43 guideline or idea in choosing an appropriate method for detection of Amaranth in foodstuffs.
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45 Keywords: Amaranth; synthetic dyes; analytical methods; chromatography; extraction;
46 toxicology
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53 1.0 Introduction
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55 Colour provides the first impression of a food taste, texture and freshness to consumers
56 that will influence their choices on food. Generally, food colorants are categorized into
57 natural and synthetic colours. Natural colours are known as natural-identical that extracted
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58 from plant, fungi or insect. However, natural colours are easily to degrade, more sensitive to
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59 light, temperature, pH and high cost. Prior to that, synthetic colours have been discovered as
60 the most reliable and economical compound because of their unique characteristics such as
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61 high stability to light, oxygen, pH, colour uniformity, less microbiological contamination and
62 low production cost (Wu et al., 2013; Llamas, Garrido, Di Nezio, & Band, 2009). Synthetic
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colours are mainly included as azo, triphenylmethane, xanthene, indigotine, and quinolone
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64 colours. Azo colours contained azo groups (-N=N-) as the chromophore in the molecular
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65 structure is the largest group of colours accounting more than half of global dyes production.
66 An approximately 65 % of azo colours are used as food additives in foodstuffs (Yamjala,

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67 Nainar, & Ramisetti, 2016; Li, Zhang, Ma, Li, & Guo, 2015; Rebane, Leito, Yurchenko, &
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68 Herodes, 2010).
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70 Amaranth is soluble in water (approximately 70 g L-1 at 25oC) and slightly soluble in
71 ethanol (up to 4 g L-1), but insoluble in vegetable oils. The powders or granules are a red-
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brown shade; meanwhile it has a bluish-red colour in liquid form and also Amaranth
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73 normally supplied as sodium, potassium or calcium salts (Yamjala et al., 2016; EFSA, 2010).
74 Amaranth is widely used in many industries such as food, cosmetic, textile, medical as well
75 as paper production (Mpountoukas et al., 2010). Therefore, several research have been
76 conducted for determination of Amaranth in food and beverage products by using numerous
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77 analytical methods such as high performance liquid chromatography (HPLC) (Li et al., 2015;
78 Shen, Zhang, Prinyawiwatkul, & Xu, 2014; Wu et al., 2013), thin layer chromatography
79 (TLC) (Andrade et al., 2014), liquid chromatography/tandem mass spectrometry (LC-
80 MS/MS) (Martin, Oberson, Meschiari, & Munari, 2016; Tsai, Kuo, & Shih, 2015),
81 spectrophotometric (Sha & Zhu, 2015; Llamas et al., 2009), electrochemical sensor (Wang,
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82 Sun, Yang, Zhao, 2015a; Wang, Gao, Sun, & Zhao, 2015b; Chandran, Lonappan, Thomas,
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83 Jos, & Kumar, 2014), capillary electrophoresis (Patsovskii, Rudometova, & Kamentsev,
84 2004), enzyme-linked immunosorbent assay (ELISA) (Zhang et al., 2013) as well as
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85 molecularly imprinted polymers (MIPs) (Han et al., 2014). Hence, this review paper is
86 critically discussed the existing methods applied for extraction and determination of
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Amaranth in foods and beverages. Additionally also discussed in detailed the toxicological
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88 effects and safety level of Amaranth.
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90 2.0 Amaranth (E 123)

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92 Colours always play important roles towards consumer acceptance and represent the
93 freshness or the acceptable parameter in foods. Across the century, natural colours are
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94 extracted from natural sources which widely used in cooking purposes. Natural colours also
95 showed some flaw about its properties such as unstable towards heat, light and temperature,
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easy decolourisation during cooking. Thus, synthetic colours have possibility to replace
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97 natural colours due to their several advantages such as low cost, high storage stability and
98 freshness as well as low microbiological contamination (Wu et al., 2013; Li et al., 2015).
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100 Amaranth belongs to synthetic azo dyes and comprised by coupling 4-amino-1-
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101 naphthalenesulphonic acid with 3-hydroxy-2, 7-naphthalenedisulphonic acid (Fig. 1).
102 Amaranth has several synonyms names such as E 123, Food Drug & Cosmetic (FD&C) Red
103 No. 2, Color Index (C.I) 16185, Food Red 9 and trisodium (4E)-3-oxo-4-[(4-sulfonato-1-
104 naphthyl)hydrazono] naphthalene-2,7-disulfonate (C20H11N2Na3O10S3) (Yamjala et al., 2016;
105 Basu, & Kumar, 2015; EFSA, 2010). Amaranth has reddish or brownish colour abundantly
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106 used in soft drinks, ice creams, cake mixes, wines, tinned fruit pie fillings, soups, prawns,
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107 cereals, salad dressings, chewing gums, jams, chocolate and coffee (Mpountoukas et al.,
108 2010). Even though some European countries are banned Amaranth colours as additives, up
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109 to now, still applied of Amaranth in textiles dyes for wool and silk as well as for photography
110 (Tariq, Faisal, & Muneer, 2005).
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Fig. 1
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113 3.0 Toxicological Study of Amaranth (E 123)
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115 Several researches have been investigated on the effect of Amaranth in living organism.
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116 Holmberg (1977) has found on the effects of Amaranth, Ponceau 4R and Vitamin A on
117 enzyme activities of the rat liver. Male rats are treated daily for 9 days with 0.25 M sucrose as
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118 control, Amaranth (85 mg/kg bw/day) in sucrose, Vitamin A (115 mg/kg bw/day) in arachis
119 oil. The result found that the body weight increase is lower in the Amaranth-Vitamin A and
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Vitamin A groups than in the control and the Amaranth-treated groups. Furthermore,
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121 Sarikaya, Selvi, & Erkoç (2012) conducted a study about the potential genotoxicity of five
122 food colours including Amaranth using Somatic Mutation and Recombination Test (SMART)
123 of Drosophila melanogaster which showed positive results with different concentrations of
124 Amaranth. The lowest concentration (1 mg mL-1) found inconclusive results for small single
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125 spots with total multiple wing hair (mwh) and spots. However, high concentration (50 mg
126 mL-1) of Amaranth clearly increased the frequency of all mutation types which indicate that
127 Amaranth may cause genotoxic effects. Mpountoukas et al. (2010) conducted on the
128 cytogenetic and DNA interaction between Amaranth, Erythrosine and Tartrazine in human
129 peripheral blood cells (HPBC). About 0.02 to 8 mmol L-1 of colouring agents are tested in
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130 HPBC to investigate their genotoxic, cytotoxic and cytostatic potential. It found that
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131 Amaranth had a genotoxic effect at all concentrations tested and at the highest concentration,
132 which over to the control level that may lead to break on chromosome and high genotoxicity.
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133 Cytostaticity of Amaranth has observed at the highest concentrations of 4 to 8 mmol L-1, and
134 found strong positive correlation between Proliferation Rate Index (PRI) and Mitotic Index
135 (MI).
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137 Hashem, Atta, Arbid, Nada, & Asaad (2010) conducted the immunological studies on
138 azo colours in albino rats to investigate the effect of oral administration where the rats are
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139 given doses of 47 mg/kg synthetic colours for 4 weeks. After 2 weeks, all animals are
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140 immunostimulated by intra peritoneal injection of sheep RBCs 10 %. The percentage of
141 neutrophils and monocytes are significantly decreased. For the enumeration of mononuclear
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142 cells, number of circulating mononuclear cells in peripheral blood is significantly increased.
They concluded that Amaranth compound did not alter humoral immunity even it does affect
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143
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144 certain parameters. Moreover, Zhang, & Yadi (2013) investigated the mechanistic and
145 conformational studies on the interaction of Amaranth with human serum albumin (HSA).
146 The results showed the protein surface hydrophobicity (PSH) increase prior to interaction of
147 Amaranth analyte. The binding of Amaranth to HSA induced the conformational change of
148 HSA which represent the disturbance in hydrogen bonding networks. The results showed
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149 Amaranth quenched the fluorescence of human hemoglobin (Hb) efficiently and the
150 quenching mechanism found to be static in nature. Thus, Amaranth has expected to be able to
151 induce unfolding and loss large part of the helical stability of Hb which significantly affects
152 the secondary structural changes in Hb (Basu, & Kumar, 2015).
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154 4.0 Safety Level of Amaranth (E 123)
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156 Amaranth has permitted under governmental regulations in every country, and it differs
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157 from countries. In 1908, United State (US) allowed the usage of Amaranth in foodstuffs by
158 the permitted level. In 1976, Food and Drugs Administration (FDA) conducted and
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confirmed the carcinogenic and embryotoxic effects of Amaranth. The panels were agreed to
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160 reduce the acceptable daily intake (ADI) of Amaranth to 0.15 mg/kg bw/day from 0.8 mg/kg
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161 bw/day (EFSA, 2010; Perez-Urquiza, & Beltran, 2000). However, World Health
162 Organization (WHO) with Food and Agriculture Organization (FAO) has recommended the
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163 ADI of Amaranth between 0 to 0.5 mg kg-1 (Zhang et al., 2013). The joint of FAO/WHO
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164 Expert Committee on Food Additives (JEFCA) has recommended the ADI of Amaranth
165 between ranged of 0 to 1.5 mg kg-1 (Mpountoukas et al., 2010). Besides, Brazilian National
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166 Agency for Public Health Surveillance (ANVISA) has allowed the presence of Amaranth in
167 non-alcoholic beverages is 0.005 g in 100 mL (Andrade et al., 2014). In china, the maximum
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permissible amount of Amaranth in food products is between 0.025 to 0.3 g kg-1 (Zhang et
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169 al., 2013). In Canada, Australia, New Zealand and Brazil have permitted of Amaranth to be
170 used in soft drinks, edible ices, preserves canned foods and confectionary. Interestingly,
171 Canadian government has permitted to use of Amaranth because the chemical structure of
172 Amaranth is same like other dyes considered as non-carcinogenic (EFSA, 2010; Barros,
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173 Steter, Lanza, & Motheo, 2014). However, Taiwan, Japan and Italy and some European
174 countries have banned the usage of Amaranth in food and beverage products due to the
175 scientifically proven on the toxicologically effect (Tsai et al., 2015; Barros et al., 2014;
176 Gennaro, Gioannini, Angelino, Aigotti, & Giacosa, 1997).
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178 5.0 Extraction Methods of Amaranth
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179 5.1 Solid Phase Extraction (SPE)
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181 Solid-phase extraction (SPE) commonly used to extract synthetic colour. This method
182 has been applied in several studies for the analysis of specific synthetic colour in food
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products due to its rapidity and simplicity. SPE is used to isolate analytes and purify the
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184 extraction where any interfering components will be eliminated for cleaner extraction which
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185 containing target analyte (Rovina, Acung, Siddiquee, Akanda, & Shaarani, 2016; Żwir-
186 Ferenc, & Biziuk, 2006). This method has involved four simple steps which are conditioning
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187 step for removing trapped air or activating the ligands on the surface of SPE particle, loading
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188 of samples, washing to eliminate contaminants which are not target analyte, and lastly elution
189 step for releasing target analyte from sorbent. Recently, Chai, Wang, Zhang, & Ding (2016)
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190 developed a method of dispersive SPE for extraction and enrichment of four colours
191 including Amaranth in powder products. A 0.4 mg mL-1 of polydopamine-coated Fe3O4

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nanoparticles (Fe3O4@PDA NPs) has used as an extraction agent. The results showed the
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193 excellent extraction performances found the specific area of nanomaterial and multiple
194 interactions between PDA and colorants. The operation is more convenient and rapid for D-
195 SPE procedure due to the magnetic property. It concluded that the proposed method exhibited
196 high sensitivity and good repeatability for the analysis in real samples with satisfactory
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197 recoveries between ranges of 91.9 % to 112.5 %.
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199 Wu et al. (2016) have used ionic liquid cross-linked polymers coated with Fe3O4
200 nanoparticles (Fe3O4@IL-CLP) to form new adsorbent for magnetic SPE (MSPE) (Fig. 2) to
201 extract Amaranth. The Fe3O4@IL-CLP has found high selectively absorbed Amaranth from
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202 aqueous samples, which easily diluted with recoveries, ranged from 94.3 % to 104.5 %. Tang
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203 et al. (2015) addressed green extraction method based on-plate SPE using solvents with low
204 toxic, easy to biodegradable. The proposed method is capable to sustain from natural sources
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205 as ethanol-water-acetic acid (50:50:1) and ethanol-water-ammonia aqueous solution
206 (75:14:1). The method showed high sensitivity and limit of detection (LOD) of 9.59 ng.
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209 Andrade et al. (2014) have used SPE cartridge Sep-Pack C18 with isopropyl alcohol 18
210 % (v/v) to extract Amaranth from soft drinks. The acceptable recovery range obtained
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211 between from 81 to101 %. Besides, Tang et al. (2014) have developed an extraction method
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212 of 16 synthetic dyes in hotpot condiment using 2 mol L-1 carbamide solution containing 5 %
213 of ammonia and methanol-acetone. The average recoveries found in the range of 63.2 to 97.1
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214 %, with relative standard deviations (RSD) of 1.5 to 10.6 %. Bonan, Fedrizzi, Menotta, &
215 Elisabetta (2013) used methanol/ammonia solution (1:1 v/v) as solvent to extract the
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colorants from food samples as meat and fishery products, pastries, cakes, jam, bakery
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217 products and fruit and vegetables sauces. The result showed high repeatability and
218 reproducibility.
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221 5.2 Liquid-Liquid Extraction (LLE)
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223 Liquid-liquid extraction (LLE) is known as solvent extraction. The method has
224 involved the separation of compounds based on their solubility with two different immiscible
225 liquids (Yamjala et al., 2016). Martin et al. (2016) conducted the study on the extraction of
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226 Amaranth from ice cream and chocolate using LLE technique with SPE clean-up. Methanol-
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227 acetic acid solution was used as extracting agent before the samples undergo clean-up using
228 SPE technique. The recoveries rates of 18 colours including Amaranth were found from 84.3
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229 % to 166.0 %, with highly sensitivity. Furthermore, Li et al. (2015) have used polyamide
230 cartridge eluted with 0.9 % of ammonia ethanol solution for desorption purposes in LLE
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method. Amaranth compound was extracted from chewing gum, carbonated beverage,
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232 alcoholic beverage and syrup. The recovery value found from 90.9 % to 92.2 %.
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234 Sha, Zhu, Feng, & Ma (2015) constructed a simple, fast, highly efficient and non-toxic
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235 method for separation and enrichment of azo colours including Amaranth in food samples.
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236 The method has used aqueous two-phase system based on ionic liquid (1-alkyl-3-
237 methylimidazolium bromide ([C4MIM][Br])) microextraction which interact with

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238 dipotassium phosphate that act as salt in order to obtain quantitative extraction and higher
239 separation efficiency. Similarly, Wu et al. (2013) used 1-octyl-3-methylimidazolium

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tetrafluoroborate ([C8MIM][BF4]) dispersive liquid phase micro extraction (IL-DLPM)

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241 technique to extract Amaranth in soft drinks, sugar and gelatin based confectionary. The
242 [C8MIM][BF4] has found easily disperse in the aqueous phase simply by manual shaking,
243 which decrease greatly the time of extraction. The recoveries rates obtained from 95.8 % to
244 104.5 %. Liao, Li, & Luo (2012) used accelerated solvent extraction method to extract
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245 Amaranth. They reported that ethanol-water-ammonia (75:24:1) are effective and suitable for
246 the extraction process at temperature of 85 °C. The precision results of intra-day and inter-
247 day repeatability for Amaranth was found of 1.7 %.
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249 5.3 Membrane Filtration
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250
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251 Membrane is a thin layer of semi-permeable substance that is driving forces applied
252 across the membrane. Membrane filtration method is one of the efficient methods for
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253 removing interference materials in food matrices. The membrane has ability to control
254 permeation rate of chemical species where widely used in separation, purification, and
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concentration processes (Hong et al., 2016; Yamjala et al., 2016; Wang, & Chung, 2005).
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256 Tsai et al. (2015) successfully extracted among 20 types of synthetic dyes including
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257 Amaranth from Chili powder and raisin samples. The samples were flow through single-step
258 extraction protocol using 0.45 µm nylon membrane filter to the supernatant before it
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259 transferred into amber auto sampler vial. Acetonitrile was used as an extraction solvent due to
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260 the good extraction yield, less fat solubility, precipitation of carbohydrates and precipitations
261 of proteins. The results showed high recoveries value obtained for both Chili powder and
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262 raisin for Amaranth between 91.9 to 95.2 %. Llamas et al. (2009) used membrane filtration
263 extraction method to extract Amaranth, Sunset Yellow and Tartrazine from beverage
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samples. Two isotonic drinks and seven soft drinks are homogenized first before filtered
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265 through glass fiber filter with 0.45 µm in pore size. The result of individual prediction errors
266 was found below 5.6 % and overall prediction error was 3.0 %. Prado, Boas, Bronze, &
267 Godoy (2006) used cellulose ester membrane (0.45 µm pore) as a filter for beverage samples
268 and the recoveries value for Amaranth was found of 101.0 %.
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269 5.4 Other Extraction Methods
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271 Other extraction method such as green extraction is available for extraction of
272 Amaranth. Bazregar, Rajabi, Yamini, & Asghari (2015) addressed the green extraction of
273 four artificial colour including Amaranth in fizzy drinks, fruit juices, tea powder and fruit
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274 jelly powder by in-tube electro-membrane extraction (IEME) using organic solvent.
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275 Electrical driving force has been applied as green auxiliary energy and the natural
276 purification of three phase methods brought a proper clean-up. The proposed method
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277 exhibited a simple and convenient method for pre-treatment of samples with recoveries
278 values from 91 % to 108 %. Sun, Sun, Li, Zhang, & Yang (2013) used microwave-assisted
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extraction (MAE) to extract colours from meat samples and clean-up using SPE. Methanol-
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280 water (95:5) is used as extraction reagent that found better recovery for 21 synthetic colorants
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281 at 60 °C. MAE method only used 15 mL of extraction solvent with 5 min of extraction time,
282 which is faster compare to conventional soxhlet extraction.

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284 Moreover, Shen et al. (2014) developed extraction method using methanol and acetone
285 as solvent for oily and viscous food samples including salad dressing, tomato sauce and
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286 ketchup in ultrasound-assisted extraction (UAE) method. The proposed method showed that
287 increasing of extraction recovery for both hydrophilic and hydrophobic pigments in different
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food products. The recovery of Amaranth was obtained in fifteen different types of products
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289 range from 80.5 % to 97.2 %. Pourreza, & Elhami (2009) used UAE method that showed
290 high sensitivity, selectivity, simplicity and faster for the extraction of synthetic colorants. The
291 UAE method has modified by addition of an ion pair with tetrabutylammonium from aqueous
292 solution using Triton X-100 as nonionic surfactant. The recoveries value was obtained from
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293 93.2 % to 100.6 % for beverage and jelly samples. All the recent extraction methods are
294 summarized in Table 1.
295 Table 1
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297 6.0 Analytical Methods for Detection of Amaranth
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298 6.1 High Performance Liquid Chromatography (HPLC)
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300 High performance liquid chromatography (HPLC) is one of the chromatography based
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301 technique that used to analyse colorants. Most of HPLC are coupled with UV-vis (Sha et al.,
302 2015; Bazregar et al., 2015; Wu et al., 2013), photodiode array (PDA) (Shen et al., 2014;
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Liao et al., 2012) and diode-array detector (DAD) (Karanikolopoulos, Gerakis,
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304 Papadopoulou, & Mastrantoni, 2015; Li et al., 2015; Tang et al., 2014; Bonan et al., 2013)
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305 detectors. There are several types of solvents used as mobile phase and stationary phase,
306 where octadecyl (C18) and monomeric octyl (C8) stationary phases are widely used efficient
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307 packing for reversed phase separations. Recently, Wu et al. (2016) developed HPLC
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308 combined with spectrophotometric method for determination of five different colours in
309 carbonated drink, cocktail, solid beverage, fruit-flavoured candy, fruit-flavoured jelly and ice
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310 cream products. The LOD and LOQ for Amaranth were found of 6.4 ng mL-1 and 19 ng mL-1,
311 respectively. Chai et al. (2016) addressed the HPLC method with satisfactory results of
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reliability and feasibility in powdered beverage, lollipop and juice samples. The LOD and
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313 LOQ were obtained of 0.00025 mg L-1 and 0.001 mg L-1, respectively.
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315 Sha et al. (2015) conducted simultaneous determination of five food colorants in soft
316 drink, sugar-based, instant powdered drink and gelatin-based confectionary by using HPLC-
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317 UV with variable wavelength. The proposed method showed LOD for Amaranth was 0.056
318 ng mL-1. Wu et al. (2013) constructed an analysis for determination of six food colourings
319 using HPLC-UV in carbonated drink, fruit flavoured drink, fruit flavoured candy, lollipop,
320 lactic acid jelly and fruit flavoured jelly. The LOD was found for Amaranth was 0.017 ng
321 mL-1, meanwhile LOQ was 0.05 ng mL-1. Besides, Bazregar et al. (2015) determined
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322 Amaranth compounds in fizzy drinks, fruit juices, tea powder and fruit jelly powder using
HPLC-UV with LOD and LOQ were found 0.3 ng mL-1 and 1.0 ng mL-1, respectively.
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324
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325 Li et al. (2015) used DAD coupled with HPLC for analysis of 34 water-soluble
326 synthetic colorants in carbonated drinks, alcoholic beverages, syrup and chewing gum. The
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LOD and LOQ for Amaranth were found of 0.009 µg mL-1 and 0.045 µg mL-1. Apart from
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328 that, Karanikolopoulos et al. (2015) used HPLC-DAD in simultaneous determination of
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329 seven water-soluble synthetic colours in fish products such as precooked crustaceans, surimi
330 and fish roe with LOD was found of 1.3 µg mL-1. Furthermore, Bonan et al. (2013) used
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331 HPLC-DAD to determine 17 synthetic dyes in solid and liquid matrices such as fishery
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332 products, pastries, cakes, jam, bakery products, fruit, vegetable sauces, soft drink, alcoholic
333 aperitifs and clear fruit juices. The recovery value of Amaranth was found in solid food
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334 matrices was 60.4 %, however, in beverages samples was found of 98.7 %. Qi et al. (2015)
used HPLC-MS/MS to analyse the level of Amaranth in corn steamed bun, barbecued pork
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336 and roasted duck. The LOD and LOQ were detected for Amaranth in the range of 0.007 to
337 0.096 mg kg-1 and 0.023 to 0.32 mg kg-1, respectively. The mass spectrometric detector was
338 used for further confirmation to validate the accuracy of the proposed method.
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340 The ultrahigh performance liquid chromatography (UHPLC) method can identify and
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341 quantify the natural and artificial colours with high sensitivity and repeatability (Shen et al.,
342 2014). Liao et al. (2012) used UHPLC method in determination seven artificial food dyes in
343 the meat products. Amaranth was detected at wavelength of 522 nm with LOD of 0.02 mg kg-
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344 and LOQ of 0.05 mg kg-1. Besides, Sun et al. (2013) developed UHPLC with satisfactory
345 recovery, good precision and short analytical time for the analysis of Amaranth in meat
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346 samples including fish, beef, pork and chicken sausage. About 21 colours are identified and
quantified with LOD and LOQ were found of 2.11 and 7.04 µg kg-1. Kong et al. (2015) used
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348 freeze deproteinization and chitosan purification to determined eight food colour in surimi
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349 products. The LOD was found in the range of 0.1 to 0.4 mg kg-1 for all colours with the linear
350 range of 0.6 to 10 mg kg-1. The results indicated that the efficiency in the extraction and
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purification method is important to achieve precise and accurate results.
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353 6.2 Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS)
354
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355 Liquid chromatography/tandem mass spectrometry (LC-MS/MS) is one of the

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356 chromatographic techniques used to determine colours in food matrices. The principle is the
357 same with ordinary HPLC method which is required certain types of mobile phase and other
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358 condition to perform. The mass spectrometers are used to overcome spectral interventions of
359 PDA/UV-Vis detectors. Martin et al. (2016) developed chromatography method based on
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LC-MS/MS to determine 18 water-soluble artificial dyes in ice-cream, chocolate sweets and

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361 sugar and gummy confectionary. The LOD and LOQ values obtained of 5 µg kg-1 and 10 µg
362 kg-1. Tsai et al. (2015) addressed LC-MS/MS method to determine synthetic dyes in Chili
363 powders and syrup-preserved fruits. About 20 dyes are identified and quantified including
364 Amaranth. The LOQ for Amaranth was found of 1 mg kg-1 which was the highest as compare
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365 to other synthetic colours.
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367 6.3 Thin Layer Chromatography (TLC)
368
369 Thin layer chromatography (TLC) is another fundamental type of chromatography that
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370 applied as simple procedure, low costs and rapid detection of food colouring present in real
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371 samples by compare the Rf (ratio of fronts) values with the colours of the extract samples in
372 relation to the standards. TLC is proven convenient separations technique for quality control
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373 and screening unknown compound especially in complex food matrix. The separation process
374 occurs in the stationary phase in present of suitable mobile phase. Lately, TLC method has
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studied and analysed the present of water soluble food colorants in food samples. The TLC
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376 plates are consisted of a thin layer of solid silica gel coated onto a flexible plastic material
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377 (Andrade et al., 2014). Tang et al. (2015) determined five synthetic colours from various
378 beverages samples by using polyamide TLC method with on-plate SPE. The LOD of
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379 Amaranth was found of 9.59 ng which showed the method is a rapid, low cost, sensitive and
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380 lower detection limit. Andrade et al. (2014) conducted TLC method to determine the
381 occurrence of four colours in food products with LOD and LOQ found to be 0.04 µg mL-1
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382 and 0.09 µg mL-1. They compared the results with ion-pair HPLC-PDA method to validate
383 the identification and quantitative measurement of Amaranth in real samples.

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385 6.4 Spectrophotometric
386
387 Food colours are highly light absorbing species in visible region and each compound
388 has specific absorption spectra which indicated their presence in certain places. UV-visible
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389 method is widely used for the analysis of specific synthetic colours in food and drink
390 samples. This method is preferable an analytical method in colour determination because of
391 low instrumentation cost and does not require any skilled labour to handle the procedure
392 (Yamjala et al., 2016). Rossinia, Milani, Pezza, & Pezza, (2016) developed environmentally
393 friendly analytical technique and successfully applied in syrup, ice cream and gelatin which
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394 offering low consumption of reagents, simple and fast identification of Amaranth. The
environmentally spectrophotometric method showed a linear range between 1.0 x 10-5 to 5.0
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395
396 x 10-4 mol L-1 with LOD and LOQ were found of 1.13 x 10-6 and 1.25 x 10-5 mol L-1,
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397 respectively. The sensitivity of the environmental friendly method showed sufficient to
398 identify and quantify Amaranth in food products with less usage of reagents and waste
399
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generation. Besides, Wu et al. (2016) determined specific synthetic colours by using both
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400 HPLC and spectrophotometric methods. The LOD and LOQ achieved were 220 ng mL-1 and
600 ng mL-1, respectively. Sha et al. (2015) also used spectrophotometric method for
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401
402 determination of Amaranth and Brilliant Blue in beverage and fruit candy samples with LOD
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403 for Amaranth was 10 µg L-1. The method showed high selectivity, wider linearity and able to
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404 detect low concentration. Besides, Zhu, Huang, & Wang, (2014) identified Amaranth analyte
405 by developing fluorescence methods with the presence of sodium dodecyl benzene sulfonate
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406 (SDBS). The SDBS is able to enhance the fluorescence intensity when present of Amaranth.
The linear range was found from 1.0 x10-7 to 1.0 x 10-3 mol L-1 (R =0.9920) with LOD of
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3.27 x 10-7 mol L-1.

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410 Pourreza, & Elhami (2009) used spectrophotometric method that able to detect lower
411 concentration of the colours in beverage and jelly samples with good reliability. The
412 calibration graph was linear in the range of 20 to1600 ng mL-1 with detection limit of 13.0 ng
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413 mL-1. Besides, Llamas et al. (2009) used a combination of direct UV-visible
414 spectrophotometric measurement and multivariate curve resolution-alternating least squares
415 (MCR-ALS) for determination of three food dyes in soft drinks and isotonic drinks. The
416 developed method represented an interesting, rapid, environmental friendly and cheap
417 alternative for the separation methods. Ni, Wang, & Kokot (2009) applied kinetic
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418 spectrophotometric aid with chemometrics method to analyse five synthetic food colorants in
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419 fruit flavoured drink, alcoholic drinks and fruit jelly. The detection limit was found of 0.095
420 mg L-l with linearity range of 0.2 to 8.0 mg L-1. The developed method showed satisfactory
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421 result with dependent on the reduction of iron (III) with synthetic dyes followed by the
422 production of Prussian blue species with the reaction of the iron (II) and the hexacyanoferrate
423 (III) ion.

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424
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425 6.5 Electrochemical Sensors
426
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427 Electrochemical behaviours of food additives, drugs, biomolecules and advances in

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428 electrochemical measuring systems have resulted in detection of specific compound in food,
429 agriculture and environment for the analysis. Electrochemical sensor emerged in many fields
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430 like proteomics, biochemistry, pharmaceutical and food analysis due to high sensitivity, low-
431 cost, simplicity, selectivity and easy to miniaturization. In recent years, efforts have exerted
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in researches of electrochemical methods for determination of synthetic colorants. In

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433 electrochemical system, the main important process is to design and modify the working
434 electrodes that highly sensitive and selective towards specific compound. The modified
435 working electrode are extensively increased the roughness surface area for improving the
436 performance electron transfer, lower detection limit, high stability and reproducibility (Lalo
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437 et al., 2012; Wang, Zhou, Zhang, Boey, & Zhang, 2009; Baravik, Tel-Vered, Ovits, &
438 Willner, 2009).
439
440 Recently, Huang, Zhang, & Hu (2017) developed electrochemical sensor platform
441 based on graphene nanosheets through ultrasonic exfoliation of graphite powder in N-methyl-
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442 2-pyrrolidone (NMP) for determination of Amaranth in soft drinks. The wide linear range
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443 found from 2.5 to 125 nM, with LOD as low as 0.75 nM. The proposed electrochemical
444 method manifest that this new sensing system possesses good accuracy and promising
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445 practical applications in monitoring synthetic food colourings. Additionally, He et al. (2015)
446 addresses electrochemical sensor based on single-walled carbon nanotube-tin (SWCNT-Tin)
447
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nanocomposite for determination of Amaranth in beverage samples. The method exhibited a
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448 novel, sensitive and faster due to the excellent electrocatalytic activity of SWCNT-Tin
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449 nanocomposites. Under optimal condition, SWCNT-Tin showed wider linear range and LOD
450 were 0.1 to 100 µmol L-1 and 40 nmol L-1, respectively. Wang et al. (2015a) developed a
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451 sensitive, stable and robust electrode modified by carbon nanotube (CNT) that dispersed in
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452 graphene oxide (GO) with the aid of prototype ionic liquid of 1-butyl-3-methylimidazolium
453 hexafluorophosphate ([BMIM][PF6]). The LOD was calculated to be 0.1 nmol L-1 which is
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454 much lower than the permitted level of Amaranth in soft drinks. Similarly, Wang et al.
(2015b) developed ultrasensitive and simultaneous determination of isomers of Amaranth and

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455
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456 Ponceau 4R using CNT and polypyrrole (ppy) composite modified electrode in fruit drink
457 samples. The detection limit obtained for Amaranth was 5.0 x 10-10 mol L-1. The proposed
458 method represented high electrocatalytic activities towards the oxidation of isomers which
459 then lead to high rate of dispersibility and effective surface area of CNT.
460
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461 Ji, Zhang, Yu, Hu, & Wu (2016) have used three kinds of resorcinol-formaldehyde
462 carbonized polymers (RF) which prepared with different types of acidic solutions, and
463 applied in electrochemical system. The RF carbonized is three-dimensional network
464 polymers that able to enhance the peak current due to their attractive properties such as large
465 surface areas, high porosities, controllable pore structures, remarkable electrical conductivity
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466 and outstanding thermal and mechanical properties (Al-Muhtaseb, & Ritter, 2003; Elkhatat,
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467 & Al-Muhtaseb, 2011). Different preparation of RF polymers displayed different
468 electrochemical reactivity towards the peak oxidation when interact with Amaranth in
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469 electrochemical system. Based on their results, the oxidation signals of Amaranth are more
470 active and significantly increased when interact with surface of RF-1 carbonized polymer in
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0.2 M HCl. Under optimum condition of RF-1 carbonized polymer, the oxidation peak
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472 currents are increased linearly over the range from 0.5 to 100 nM with sensitivity value of
14.98 µA µM-1. The developed method successfully applied in the drink sample as compared
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473
474 to HPLC method with satisfactory recoveries values (Ji et al., 2016).
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475
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476 Furthermore, Steter, Barros, Lanza, & Motheo (2014) developed method based on
477 sonoelectrochemical process by using boron-doped diamond anode BDD. The Amaranth
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478 concentration decayed as a function of electrolysis time and the reactions followed pseudo
first-order kinetics, with an apparent constant rate between range 10-1 to 10-3 min-1. After 90
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479
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480 min, the yielded of TOC removal values ranged from 92.1 % to 95.1 %. The current
481 efficiency values obtained were 18.2 % and 23.6 %, respectively. Chandran et al. (2014) used
482 multi-walled carbon nanotube (MWCNT) to determine Amaranth in soft drinks samples. The
483 LOD was found of 6.8 x 10-8 mol L-1 with a good correlation value of 0.998. Zhang, Wang,
484 Shentu, Wang, & Chen (2014a) used expanded graphite paste electrode (EGPE) instead of
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485 CNT to detect Ponceau 4R and Amaranth in grape juice samples. The LOD and LOQ for
486 Amaranth were found of 36 nmol L-1 and 109.1 nmol L-1, respectively. The proposed
487 procedures showed wide linear range, low concentration, good reproducibility and stability,
488 reusability, and successfully applied for simultaneously determination of Amaranth in soft
489 drinks. Zhang, & Yadi (2013) used alumina microfiber-modified carbon paste electrode
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490 (CPE) to develop rapid and sensitive electrochemical method for determination of Amaranth
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491 in drink samples. The alumina microfibers exhibited stronger enhancement effect to the
492 oxidation and significantly increased the oxidation signal. The linear range was from 1 to 150
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493 nmol L-1 with LOD was found of 0.75 nmol L-1.
494
495
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Wang, Zhang, Ding, Zhu, & Chen (2013) developed a simple electrochemical method
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496 by square-wave adsorptive stripping voltammetry based on EGPE. The EGPE showed strong
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497 and sensitive voltammetric responses to Amaranth, owing to the multiporous structure and
498 large surface area. Under optimum conditions, the oxidation peak current has detected with
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499 different concentrations of Amaranth from 0.01 to 4.0 µmol L-1, with LOD of 0.005 µmol L-1.
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500 Ju, & Guo (2013) determined the level of Amaranth based on the GCE modified with ordered
501 mesoporous carbon (OMC) by utilizing sucrose as carbon source. Compared with the bare
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502 GCE and carbon nanotubes/GCE, OMC/GCE have displayed better electroactive surface
area, and an enhanced electrochemical response toward the oxidation of Amaranth in the
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503
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504 neutral solution. The peak current found to be linear with Amaranth concentration in the
505 ranged from 1.0 × 10-7 to 3.0 × 10-6 mol L-1, with LOD of 3.2 × 10−8 mol L-1. Wang et al.
506 (2010) developed an electrochemical sensor based on multiwall carbon nanotube (MWNT) as
507 the sensing film for monitoring and rapid detection of Amaranth. The sensor showed a strong
508 enhancement effect on the oxidation of amaranth, and largely increased the current peaks due
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509 to the roughness surface area and high accumulation efficiency of MWNT. The linear range
510 found from 40 nM to 0.8 µM, with LOD of 35 nM. The proposed method has successfully
511 employed to identify and quantify of Amaranth in soft drinks.
512
513 6.6 Molecularly Imprinted Polymers (MIPs)
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514
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515 Molecularly imprinted polymers (MIPs) are biomimetic synthetic receptors possessing
516 specific cavities designed for the target molecule. MIPs method are capable to bind target
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517 molecules with specificities and affinities comparable to those of natural receptors (Haupt,
518 Linares, Bompart, & Tse, 2012; Bui, & Haupt, 2010; Alexander et al., 2006). MIPs normally
519
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used in optical sensors which known as optodes based on optical fibers where the MIP
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520 immobilized on the surface (Ton et al., 2013; Marazuela, & Moreno-Bondi, 2002). The
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521 applications of optical fibers sensors permeated several fields of countless importance in
522 analytical chemistry including environment, food, chemical and clinical analyses (Wang, &
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523 Wolfbeis, 2013; Orellana, & Haigh, 2008). Optodes represent some benefits such as simple
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524 manufacturing, low cost, high selectivity and sensitivity, real-time analysis and environment
525 friendly.
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526
527 Han et al. (2014) fabricated a novel molecularly imprinted electrochemical sensor
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(MIES) by magnetic field induced self-assembly of reduced oxide graphene-based

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528
529 composites with Fe3O4 (Fe3O4@rGO) as shown in Fig. 3. The Fe3O4@rGO composites have
530 synthesized by a facile method using hydrazine as reducer could be fast oriented and
531 assembled on the surface of magnetic glassy carbon electrode (MGCE) by magnetic field
532 induction. In molecular imprinting, the complex including the function monomer of aniline,
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533 the template of Amaranth and Fe3O4@rGO are pre-assembled through π-π stacking and
534 hydrogen bonding interactions, then self-assembled on the surface of MGCE with help the
535 magnetic field induction before electropolymerization. The prepared MIES exhibited good
536 sensitivity, selectivity, reproducibility and efficiency for detecting Amaranth in fruit drinks.
537 The average recoveries were found between 93.15 % to 100.81 %, with RSD less than 3.0 %.
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538 Fig. 3
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539
540 6.7 Capillary Electrophoresis (CE)
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541
542 There are several separation techniques have been developed to detect the presence of
543
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excess synthetic colorants in foodstuffs. Electrophoresis is a generic name given to a series of
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544 separation techniques that involved the application of electric field in a capillary which filled
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545 with buffer solution. Capillary electrophoresis (CE) has become a valuable method for
546 separating analytes from large molecules to small ions in complex matrix. This method
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547 showed high efficiency, low waste production, and fast separation for the analysis of natural
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548 food pigments and synthetic food colorants (Frazier, Inns, Dossi, Ames, & Nursten, 2000;
549 Hsieh, & Huang, 1997). Generally, CE technique become a low sample capacity and optical
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550 path due to the narrow internal diameter of the capillary tube, resulting low detection limit of
551 the analyte. To date, most research using CE focused on the detection of colorants in soft
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drinks and popsicles, with combination of SPE extraction methods (S’adecka, & Polonsky,
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553 2000). Capillary electrophoresis (CE) has different separation modes which included
554 capillary zone electrophoresis, micellar electrokinetic capillary chromatography (MEKC),
555 and capillary isotachophoresis. These methods showed to be low costs, better column
556 resistance, shorter analysis time and less samples volume required (Yamjala et al., 2016;
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557 Prado et al., 2006). Perez-Urquiza, & Beltran (2000) used CZE method coupled with PDA
558 detector to determine the synthetic dyes in beverages, syrups and jellies. At wavelength of
559 280 nm, the LOD and LOQ for Amaranth were found of 1.1 and 3.6 µg mL-1, respectively.
560 Additionally, Patsovskii et al. (2004) stated that CE method effectively used for
561 determination of 11 synthetic dyes in various alcoholic beverages. The RSD was found below
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562 10 % in the dyes concentration range between 5 to 200 mg L-1. Chou, Lin, Cheng, & Hwang
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563 (2002) developed MEKC method to quantify Amaranth in cola, carbonated water and candy.
564 The results gave satisfactory outcome in term of detection limit, and recovery value. About
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565 14 artificial colours analyzed including Amaranth with detection limit was found of 2.50 µg
566 g-1. Prado et al. (2006) used MEKC connected with UV-Vis to determine 11 colours in
567
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alcoholic beverages. The technique represents very simple and practical efficient, without any
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568 sample pretreatment. The detection limit was found of 0.8 µg mL-1 with quantification limit
of 2.7 µg mL-1. Ryvolová, Táborský, Vrábel, Krásenský, & Preisler (2007) developed a
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569
570 capillary electrophoresis method with laser-induced fluorescence detection (CE-LIF) to

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571 determine red food colorants including Amaranth from sour cherry syrup. The proposed
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572 method was efficiently used for separation and detection of certain red colorants. The LOD
573 and LOQ obtained for Amaranth were found of 0.2 µg mL-1 and 0.6 µg mL-1, respectively.
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574
6.8 Enzyme-Linked Immunosorbent Assay (ELISA)

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577 Enzyme-linked immunosorbent assay (ELISA) technique is widely used for detection of
578 various additives in food industries for monitoring adventitious contamination of food
579 products by allergenic ingredients. ELISA is one of the rapid technique with high sensitivity
580 and specificity, low detection limit, mobility, simple, and convenience to field test (Sreenath,
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581 & Venkatesh, 2008; Monaci & Visconti, 2010; Li et al., 2011). The immunogen and the
582 coating antigen are designed by introducing a carboxyl group into Amaranth for the
583 conjugation with carrier proteins. Zhang et al. (2013) conducted indirect ELISA method
584 which showed high sensitivity, specificity and high accuracy for rapid determination for
585 Amaranth. The linear standard curve found on the range of 3 to 243 ng mL-1 with LOD of
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586 3.35 ng mL-1. In addition, the method is more convenient since it is based on the oxidation
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587 signal, and the influence of oxygen is negligible. Among the studied colorants, the cross-
588 reactivity value of anti-amaranth MAbs against Ponceau 4R is extraordinarily low (<1 %),
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589 might be because of the Amaranth sodium sulfonate has modified at position C4′ during
590 reaction process with carrier protein. Hence, the different substituent of disulfonate between
591
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Amaranth (C2, C7) and Ponceau 4R (C5, C7) effect the cross-reactivity value of both
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592 compounds. Table 2 shows the summary of analytical method for detection of Amaranth in
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593 food and beverage products.
594
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595 Table 2
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596 7.0 Conclusion
597
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598 Amaranth is widely used as food additives in food products to make them more
599 attractive and restore their original appearance when it has lost colorant during manufacture
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processes. Amaranth is an organic large compound containing azo group (-N=N-) and
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601 aromatic ring that have potential risk toxicity effects on human health. Amaranth reported
602 may cause several adverse health effects such as genotoxicity, cytotoxicity, and cytostaticity.
603 Hence, World Health Organization (WHO) with Food and Agriculture Organization (FAO)
604 has recommended the permitted level of Amaranth added on the range of 0 to 0.5 mg kg-1.
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605 The reported effect of overused and over consumption of Amaranth has caused a rise in the
606 detection method of this food colorant in food matrices. In this present review, the recent
607 study regarding extraction and detection methods for Amaranth analyte in complex food
608 matrix successfully discussed. Before analysing any samples, the extraction procedure is
609 important to ensure there is no interferences substance available during analysing the food
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610 matrices. Previously several extraction methods applied such as solid-phase extraction,
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611 liquid-liquid extraction, cloud point extraction, ultrasound assisted solvent extraction and
612 some other extraction methods. Up to now, several analytical methods have been developed
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613 including capillary electrophoresis, spectrophotometry, chromatography, MIPs, and ELISA
614 for the determination of Amaranth. Recently, electrochemical sensor based on the modified
615
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working electrode has gained high attention in the field of food safety analysis because of the
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616 unique characteristics such as simple procedure, fast, high sensitivity and selectivity, low cost
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617 and good reproducibility. Through all these previous studies, new methods are required to
618 develop for the detection of food colorants considering the potential and appropriate method
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619 affordability.
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620
621 Acknowledgement
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623 This work was supported by grants from the Ministry of Education Malaysia,
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Fundamental Research Grant Scheme (FRGS), (No. FRGS/1/2014/SG05/UMS/02/4).

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629 References
630 Al-Muhtaseb, S. A., & Ritter, J. A. (2003). Preparation and Properties of Resorcinol–
631 Formaldehyde Organic and Carbon Gels, Advanced Materials, 15, 101-114.
632 Alexander, C., Andersson, H. S., Andersson, L. I., Ansell, R. J., Kirsch, N., Nicholls, I. A.,
633 O'Mahony, J., & Whitcombe, M. J. (2006). Molecular imprinting science and
634 technology: a survey of the literature for the years up to and including 2003. Journal of
635 Molecular Recognition, 19, 106-180.
PT
636 Andrade, D. F. I., Guedes, M. I. F., Vieira, Í. G. P., Mendes, F. N. P., Rodrigues, P. A. S.,
637 Maia, C. S. C., Ávila, M. M. M., & de Matos Ribeiro, L. (2014). Determination of
RI
638 synthetic food dyes in commercial soft drinks by TLC and ion-pair HPLC. Food
639 Chemistry, 157, 193-198.
SC
640 Baravik, I., Tel-Vered, R., Ovits, O., & Willner, I. (2009). Electrical contacting of redox
641 enzymes by means of oligoaniline-cross-linked enzyme/carbon nanotube composites.
642 Langmuir, 15, 13978-13983.
U
643 Barros, W. R., Steter, J. R., Lanza, M. R., & Motheo, A. J. (2014). Degradation of amaranth
644 dye in alkaline medium by ultrasoniccavitation coupled with electrochemical oxidation
AN
645 using a boron-doped diamond anode. Electrochimica Acta, 143, 180-187.
646 Basu, A., & Kumar, G. S. (2015). Interaction of toxic azo dyes with heme protein:
M
647 biophysical insights into the binding aspect of the food additive amaranth with human
648 hemoglobin. Journal of Hazardous Materials, 289, 204-209.
D
649 Bazregar, M., Rajabi, M., Yamini, Y., & Asghari, A. (2015). In-tube electro-membrane
650 extraction with a sub-microliter organic solvent consumption as an efficient technique
TE
651 for synthetic food dyes determination in foodstuff samples. Journal of Chromatography
652 A, 1410, 35-43.
653 Bonan, S., Fedrizzi, G., Menotta, S., & Elisabetta, C. (2013). Simultaneous determination of
EP
654 synthetic dyes in foodstuffs and beverages by high-performance liquid chromatography

655 coupled with diode-array detector. Dyes and Pigments, 99, 36-40.
C
656 Bui, B. T. S., & Haupt, K. (2010). Molecularly imprinted polymers: synthetic receptors in
657 bioanalysis. Analytical and Bioanalytical Chemistry, 398, 2481-2492.
AC
658 Chai, W., Wang, H., Zhang, Y., & Ding, G. (2016). Preparation of polydopamine-coated
659 magnetic nanoparticles for dispersive solid-phase extraction of water-soluble synthetic
660 colorants in beverage samples with HPLC analysis. Talanta, 149, 13-20.
661 Chandran, S., Lonappan, L. A., Thomas, D., Jos, T., & Kumar, K. G. (2014). Development of
662 an electrochemical sensor for the determination of amaranth: a synthetic dye in soft
663 drinks. Food Anal Method, 7, 741-746.
664
27
ACCEPTED MANUSCRIPT
665 Chou, S. S., Lin, Y. H., Cheng, C. C., & Hwang, D. F. (2002). Determination of synthetic
666 colors in soft drinks and confectioneries by micellar electrokinetic capillary
667 chromatography. Journal of Food Science, 67, 1314-1318.
668 Elkhatat, A. M., & Al-Muhtaseb, S. A. (2011). Advances in tailoring

669 resorcinol‐formaldehyde organic and carbon gels. Advanced Materials, 23, 2887-2903.
670 European Food Safety Authority (EFSA). Scientific opinion on the re-evaluation of
671 Amaranth (E 123) as a food additive, EFSA Journal 8 (2010) 1649.
PT
672 Frazier, R. A., Inns, E. L., Dossi, N., Ames J. M., & Nursten, H. E. (2000). Development of a
673 capillary electrophoresis method for the simultaneous analysis of artificial sweeteners,
RI
674 preservatives and colours in soft drinks. Journal Chromatography A, 876, 213-220.
675 Gennaro, M. C., Gioannini, E., Angelino, S., Aigotti, R., & Giacosa, D. (1997). Identification
SC
676 and determination of red dyes in confectionery by ion-interaction high-performance
677 liquid chromatography. Journal of Chromatography A, 767, 87-92.
678 Han, Q., Wang, X., Yang, Z., Zhu, W., Zhou, X., & Jiang, H. (2014). Fe3O4@rGO doped
U
679 molecularly imprinted polymer membrane based on magnetic field directed self-
680 assembly for the determination of Amaranth. Talanta, 123, 101-108.
AN
681 Hashem, M. M., Atta, A. H., Arbid, M. S., Nada, S. A., & Asaad, G. F. (2010).
682 Immunological studies on amaranth, sunset yellow and curcumin as food colouring
M
683 agents in albino rats. Food and Chemical Toxicology, 48, 1581-1586.
684 Haupt, K., Linares, A.V. and Bompart, M., Tse S. B. B. 2012. Molecularly imprinted
D
685 polymers. In: Haupt, K. (ed.), Topics in Current Chemistry. Springer:

686 Berlin/Heidelberg, pp. 1-28.
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687 He, J. L., Kou, W., Li, C., Cai, J. J., Kong, F. Y., & Wang, W. (2015). Electrochemical
688 sensor based on single-walled carbon nanotube-tin nannocomposites for detecting
689 amaranth. International Journal of Electrochemical Science, 10, 10074-10082.
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690 Holmberg, D. (1977). Effect of amaranth, ponceau 4r and/or vitamin A on enzyme activities
691 of the rat liver. Food and Cosmetics Toxicology, 16, 1-5.
C
692 Hong, J., Kawashima, A., Okamoto, M., Kanetsuki, K., Makino, T., & Hamada, N. (2016).
AC
693 Fundamental study of a novel membrane filtration clean up method for pesticide
694 analysis in agricultural products. Food Control, 64, 1-9.
695 Hsieh, Y. Z., & Huang, H. Y. (1997). Determination of saikosaponins by micellar

696 electrokinetic capillary chromatography. Journal Chromatography A, 759, 193-201.
697 Huang, W., Zhang, M., & Hu, W. (2017). N-methyl-2-pyrrolidone-exfoliated graphene
698 nanosheets as sensitive determination platform for amaranth at the nanomolar level.
699 Ionics, 23, 241-246.
28
ACCEPTED MANUSCRIPT
700 Ji, L., Zhang, Y., Yu, S., Hu, S., & Wu, K. (2016). Morphology-tuned preparation of
701 nanostructured resorcinol-formaldehyde carbonized polymers as highly sensitive
702 electrochemical sensor for amaranth. Journal of Electroanalytical Chemistry, 779, 169-
703 175.
704 Ju, J., & Guo, L. (2013). Sensitive voltammetric sensor for Amaranth based on ordered
705 mesoporous carbon. Chinese Journal of Analytical Chemistry, 41, 681-686.
706 Karanikolopoulos, G., Gerakis, A., Papadopoulou, K., & Mastrantoni, I. (2015).
PT
707 Determination of synthetic food colorants in fish products by an HPLC-DAD method.
708 Food Chemistry, 177, 197-203.
RI
709 Kong, C., Fodjo, E. K., Li, D., Cai, Y., Huang, D., Wang, Y., & Shen, X. (2015). Chitosan-
710 based adsorption and freeze deproteinization: improved extraction and purification of
711 synthetic colorants from protein-rich food samples. Food Chemistry, 188, 240-247.
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712 Lalo, H., Bon-Saint-Côme, Y., Plano, B., Etienne, M., Walcarius, A., & Kuhn, A. (2012).
713 Site selective generation of sol-gel deposits in layered bimetallic macroporous electrode
714 architectures. Langmuir, 5, 2323-2326.
715
U
Li, W. H., Meng, M. He, F. Y., Wan, Y. P., Xue, H. Y., Liu, W., Yin, W. W., Xu, J., Feng, C.
AN
716 W., Wang, S. L., Lu, X., Liu, J. T. & Xi, R. M. (2011). Preparation of an anti-
717 diethylstilbestrol monoclonal antibody and development of an indirect competitive
718 ELISA to detect diethylstilbestrol in biological samples. Chinese Science Bulletin, 56,
M
719 749-754.
720 Li, X. Q., Zhang, Q. H., Ma, K., Li, H. M., & Guo, Z. (2015). Identification and
D
721 determination of 34 water-soluble synthetic dyes in foodstuffs by high performance

722 liquid chromatography-diode array detection-ion trap time-of-flight tandem mass
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723 spectrometry. Food Chemistry, 182, 316-326.
724 Liao, Q. G., Li, W. H., & Luo, L. G. (2012). Applicability of accelerated solvent extraction
725 for synthetic colorants analysis in meat products with ultrahigh performance liquid
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726 chromatography–photodiode array detection. Analytica Chimica Acta, 716, 128-132.
727 Llamas, N. E., Garrido, M., Di Nezio, M. S., & Band, B. S. F. (2009). Second order
C
728 advantage in the determination of amaranth, sunset yellow fcf and tartrazine by uv-vis
729 and multivariate curve resolution-alternating least squares. Analytica Chimica Acta,
AC
730 655, 38-42.
731 Marazuela, M., & Moreno-Bondi, M. (2002). Fiber-optic biosensors- An overview.

732 Analytical and Bioanalytical Chemistry, 372, 664-682.
733 Martin, F., Oberson, J. M., Meschiari, M., & Munari, C. (2016). Determination of 18 water-
734 soluble artificial dyes by LC–MS in selected matrices. Food Chemistry, 197, 1249-
735 1255.
736
29
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737 Monaci, L., & Visconti, A. (2010). Immunochemical and DNA-based methods in food
738 allergen analysis and quality assurance perspectives. Trends in Food Science and
739 Technology, 21, 272-283.
740 Mpountoukas, P., Pantazaki, A., Kostareli, E., Christodoulou, P., Kareli, D., Poliliou, S., &
741 Lialiaris, T. (2010). Cytogenetic evaluation and DNA interaction studies of the food
742 colorants amaranth, erythrosine and tartrazine. Food and Chemical Toxicology, 48,
743 2934-2944.
PT
744 Ni, Y., Wang, Y., & Kokot, S. (2009). Simultaneous kinetic spectrophotometric analysis of
745 five synthetic food colorants with the aid of chemometrics. Talanta, 78, 432-441.
RI
746 Orellana, G., & Haigh, D. (2008). New trends in fiber-optic chemical and biological sensors.
747 Current Analytical Chemistry, 4, 273-295.
SC
748 Patsovskii, A. P., Rudometova, N. V., & Kamentsev, Y. S. (2004). Electrophoretic
749 determination of synthetic dyes in alcoholic beverages. Journal of Analytical
750 Chemistry, 59(2), 150-154.
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751 Perez-Urquiza, M., & Beltran, J. L. (2000). Determination of dyes in foodstuffs by capillary
752 zone electrophoresis. Journal of Chromatography A, 898, 271-275.
AN
753 Pourreza, N., & Elhami, S. (2009). Cloud point extraction and spectrophotometric
754 determination of amaranth in food samples using nonionic surfactant Triton X-100 and
M
755 tetrabutylammonium hydrogen sulfate. Journal of the Iranian Chemical Society, 6(4),
756 784-788.
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757 Prado, M. A., Boas, L. F. V., Bronze, M. R., & Godoy, H. T. (2006). Validation of
758 methodology for simultaneous determination of synthetic dyes in alcoholic beverages
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759 by capillary electrophoresis. Journal of Chromatography A, 1136, 231-236.
760 Qi, P., Lin, Z. H., Chen, G. Y., Xiao, J., Liang, Z. A., Luo, L. N., Zhou, J., & Zhang, X. W.
761 (2015). Fast and simultaneous determination of eleven synthetic color additives in flour
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762 and meat products by liquid chromatography coupled with diode-array detector and
763 tandem mass spectrometry. Food Chemistry, 181, 101-110.
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764 Rebane, R., Leito, I., Yurchenko, S., & Herodes, K. (2010). A review of analytical techniques
765 for determination of Sudan I–IV dyes in food matrixes. Journal of Chromatography A,
AC
766 1217, 2747-2757.
767 Rossinia, E. L., Milani, M. I., Pezza L., & Pezza H. R. (2016). New eco-friendly
768 methodology for determination of Amaranth dye in foodstuffs using diffuse reflectance
769 spectroscopy. Analytical Methods, 8, 4086-4092.
770 Rovina, K., Acung, L. A., Siddiquee, S., Akanda, J.H., & Shaarani, S. M. (2016). Extraction
771 and Analytical Methods for Determination of Sunset Yellow (E110)-A Review. Food
772 Analytical Methods, 1-15.
30
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773 Ryvolová, M., Táborský, P., Vrábel, P., Krásenský, P., & Preisler, J. (2007). Sensitive
774 determination of erythrosine and other red food colorants using capillary
775 electrophoresis with laser-induced fluorescence detection. Journal of Chromatography
776 A, 1141, 206-211.
777 S’adecka, J., & Polonsky, J. (2000). Electrophoretic methods in the analysis of beverages.
778 Journal Chromatography A, 880, 243-279.
779 Sarıkaya, R., Selvi, M., & Erkoç, F. (2012). Evaluation of potential genotoxicity of five food
PT
780 dyes using the somatic mutation and recombination test. Chemosphere, 88, 974-979.
781 Sha, O., & Zhu, X. (2015). Simultaneous ionic liquid aqueous two-phase extraction and
RI
782 spectrophotometric determination of amaranth and brilliant blue in food samples.
783 Analytical Chemistry, 70, 558-565.
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784 Sha, O., Zhu, X., Feng, Y., & Ma, W. (2015). Aqueous two-phase based on ionic liquid
785 liquid–liquid microextraction for simultaneous determination of five synthetic food
786 colorants in different food samples by high-performance liquid chromatography. Food
787 Chemistry, 174, 380-386.
788
U
Shen, Y., Zhang, X., Prinyawiwatkul, W., & Xu, Z. (2014). Simultaneous determination of
AN
789 red and yellow artificial food colorants and carotenoid pigments in food products. Food
790 Chemistry, 157, 553-558.
M
791 Sreenath, K., & Venkatesh, Y. P. (2008). Analysis of erythritol in foods by polyclonal
792 antibody-based indirect competitive ELISA. Analytical and Bioanalytical Chemistry,
793 391, 609-615.
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794 Steter, J. R., Barros, W. R. P., Lanza, M. R. V., Motheo A. J. (2014). Electrochemical and
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795 sonoelectrochemical processes applied to amaranth dye degradation. Chemosphere,

796 117, 200-207.
797 Sun, H., Sun, N., Li, H., Zhang, J., & Yang, Y. (2013). Development of multiresidue analysis
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798 for 21 synthetic colorants in meat by microwave-assisted extraction–solid-phase

799 extraction–reversed-phase ultrahigh performance liquid chromatography. Food
800 Analytical Methods, 6, 1291-1299.
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801 Tang, B., Xi, C., Zou, Y., Wang, G., Li, X., Zhang, L., Chen, D., & Zhang, J. (2014).
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802 Simultaneous determination of 16 synthetic colorants in hotpot condiment by high

803 performance liquid chromatography. Journal of Chromatography B, 960, 87-91.
804 Tang, T. X., Xu, X. J., Wang, D. M., Zhao, Z. M., Zhu, L. P., & Yang, D. P. (2015). A rapid
805 and green limit test method for five synthetic colorants in foods using polyamide thin-
806 layer chromatography. Food Analytical Methods, 8, 459-466.
807 Tariq, M. A., Faisal, M., & Muneer, M. (2005). Semiconductor-mediated photocatalysed
808 degradation of two selected azo dye derivatives, amaranth and bismarck brown in
809 aqueous suspension. Journal of Hazardous Materials, 127, 172-179.
31
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810 Ton, X. A., Tse Sum Bui, B., Resmini, M., Bonomi, P., Dika, I., Soppera, O., & Haupt, K.
811 (2013). A versatile fluorescence fiber optic sensor based on in-situ polymerized
812 molecularly imprinted microstructures. Angewandte Chemie International Edition, 52,
813 8317-8321.
814 Tsai, C. F., Kuo, C. H., & Shih, D. Y. C. (2015). Determination of 20 synthetic dyes in chili
815 powders and syrup-preserved fruits by liquid chromatography/tandem mass
816 spectrometry. Journal of Food and Drug Analysis, 23, 453-462.
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817 Wang, K. Y., & Chung, T. S. (2005). The characterization of flat composite nanofiltration
818 membranes and their applications in the separation of Cephalexin. Journal of
819 Membrane Science, 247(1), 37-50.
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820 Wang, M. L., Zhang, J., Ding, N. N., Zhu, X. L., & Chen, Z. D. (2013). Electrochemical
821 detection of Amaranth in food based on the expanded graphite paste electrode. Journal
SC
822 of AOAC International, 96, 625-629.
823 Wang, M., Gao, Y., Sun, Q., & Zhao, J. (2015b). Ultrasensitive and simultaneous
824 determination of the isomers of amaranth and ponceau 4R in foods based on new
U
825 carbon nanotube/polypyrrole composites. Food Chemistry, 172, 873-879.
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826 Wang, M., Sun, Y., Yang, X., & Zhao, J. (2015a). Sensitive determination of amaranth in
827 drinks by highly dispersed CNT in graphene oxide ‘‘water’’ with the aid of small
828 amounts of ionic liquid. Food Chemistry, 179, 318-324.
M
829 Wang, P., Hu, X., Cheng, Q., Zhao, X., Fu, X., & Wu, K. (2010). Electrochemical Detection
830 of Amaranth in Food Based on the Enhancement Effect of Carbon Nanotube Film.
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831 Journal of Agricutural and Food Chemistry, 58, 12112-12116.

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832 Wang, X. D., & Wolfbeis, O. S. (2013). Fiber-optic chemical sensors and biosensors (2008–
833 2012). Analytical Chemistry, 85, 487-508.
834 Wang, Z., Zhou, X., Zhang, J., Boey, F., & Zhang, H. (2009). Direct electrochemical
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835 reduction of single-layer graphene oxide and subsequent functionalization with glucose
836 oxidase. Journal of Physical Chemistry C, 113, 14071-14075.
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837 Wu, H., Gao, N., Zhang, L., Li, Y., Shi, Y., & Du, L. (2016). Automated magnetic solid-
838 phase extraction for synthetic food colorant determination. Food Analytical Methods, 9,
AC
839 614-623.
840 Wu, H., Guo, J. B., Du, L. M., Tian, H., Hao, C. X., Wang, Z. F., & Wang, J. Y. (2013). A
841 rapid shaking-based ionic liquid dispersive liquid phase microextraction for the
842 simultaneous determination of six synthetic food colourants in soft drinks, sugar-and
843 gelatin-based confectionery by high-performance liquid chromatography. Food
844 Chemistry, 141(1), 182-186.
845 Yamjala, K., Nainar, M. S., & Ramisetti, N. R. (2016). Methods for the analysis of azo dyes
846 employed in food industry – a review. Food Chemistry, 192, 813-824.
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847 Zhang, B., Du, D., Meng, M., Eremin, S. A., Rybakov, V. B., Zhao, J., Yin, Y., & Xi, R.
848 (2013). Determination of amaranth in beverage by indirect competitive enzyme-linked
849 immunosorbent assay (ELISA) based on anti-amaranth monoclonal antibody. Food
850 Analytical Methods, 7, 1498-1505.
851 Zhang, G., & Yadi, M. (2013). Mechanistic and conformational studies on the interaction of
852 food dye amaranth with human serum albumin by multispectroscopic methods. Food
853 Chemistry, 136, 442-449.
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854 Zhang, J., Wang, M., Shentu, C., Wang, W., & Chen, Z. (2014a). Simultaneous determination
855 of the isomers of ponceau 4R and amaranth using an expanded graphite paste electrode.
856 Food Chemistry, 160, 11-15.
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857 Zhu, H., Huang, W., & Wang, F. (2014). Determination of amaranth by fluorescence
858 spectrometry. Advanced Materials Research, 1010-1012, 835-838.
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859 Żwir-Ferenc, A., & Biziuk, M. (2006). Solid phase extraction technique – Trends,
860 opportunities and applications. Polish Journal of Environmental Studies, 15, 677-690
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876 List of Figures
877
878 Fig. 1. Molecular Structure of Amaranth
879 Fig. 2. Schematic of automated MSPE (Wu et al., 2016)
880 Fig. 3. Schematic representation for the preparation of molecularly imprinted electrochemical
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881 sensor (Han et al., 2014).
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901 Figure 1
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904 Fig. 1. Molecular Structure of Amaranth
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923 Fig. 2. Schematic of automated MSPE (Wu et al., 2016)

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937 Fig. 3. Schematic representation for the preparation of molecularly imprinted electrochemical
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938 sensor (Han et al., 2014).
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952 List of Tables
953
954 Table 1. Extraction methods available for Amaranth
955 Table 2. Analytical methods based detection of Amaranth
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977 Table 1. Extraction methods available for Amaranth
Extraction procedure Solvents Samples References
Dispersive solid-phase 0.4 mg/mL of polydopamine- Powdered beverage, (Chai et al.,
extraction (D-SPE) coated Fe3O4 nanoparticles lollipop, and juice 2016)
(Fe3O4@PDA NPs).
Automated Magnetic Ionic liquid cross-linked Carbonated drinks, (Wu et al.,
Solid-Phase Extraction polymers coated with Fe3O4 cocktail, solid beverage, 2016)
(MSPE) nanoparticles (Fe3O4@IL- fruit-flavoured candy,
CLP). fruit-flavoured jelly and
ice cream.
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Liquid-liquid extraction Methanol-acetic acid solution. Ice cream and chocolate (Martin et
(LLE) al., 2016)
On-plate Solid phase Ethanol water-acetic acid Beverages (Tang et al.,
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extraction (SPE) (50:50:1) and ethanol-water- 2015)
ammonia aqueous solution
(75:14:1).
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Ionic liquid 1-alkyl-3-methylimidazolium Foodstuffs and beverages (Sha et al.,
microextraction (ILME) bromide ([C4MIM][Br]) + 2015)
dipotassium phosphate
(K2HPO4).
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Liquid-liquid extraction 0.9 % of ammonia ethanol Chewing gum, carbonated (Li et al.,
(LLE) solution. beverage, alcoholic 2015)
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beverage and syrup.
Single-step extraction Acetonitrile Chili powder and raisin (Tsai et al.,

protocol using 0.45-µm 2015)
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nylon membrane filter

In-tube electro- Organic solvent Fizzy drinks, fruit juices, (Bazregar et
membrane extraction tea powder and fruit jelly al., 2015)
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(IEME) powder
Solid phase extraction Isopropyl alcohol 18 % (v/v) Soft drinks (Andrade et
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(SPE) al., 2014)
Solid phase extraction 2 mol/L carbamide solution Hotpot condiment (Tang et al.,
(SPE) containing 5 % ammonia and 2014)
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methanol-acetone.
Purified using SPE column

eluted with 7 mL of 5 %
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ammonia-methanol
Ultrasound-assisted Methanol and acetone Salad dressing, tomato (Shen et al.,
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extraction (UAE) sauce and ketchup 2014)

technique
Solid phase extraction Methanol/ammonia solution Meat and fishery products, (Bonan et
(SPE) (1:1 v/v) pastries, cakes, jam, al., 2013)
bakery products and fruit
and vegetables sauces
Ionic liquid dispersive 1-octyl-3-methylimidazolium Soft drinks, sugar and (Wu et al.,
liquid phase tetrafluoroborate gelatin based 2013)
microextraction (IL- ([C8MIM][BF4]). confectionary
DLPM)
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Microwave-assisted Methanol-water (95:5) Meat (Sun et al.,
extraction (MAE) 2013)
Accelerated solvent Ethanol-water ammonia Meat products (Liao et al.,

extraction (ASE) (75:24:1). 2012)
Membrane filtration Glass fibre filter (0.45 µm in Beverages (Llamas et
pore size). al., 2009)
Cloud Point Extraction Triton X-100 Beverage and jelly (Pourreza &
(CPE) Elhami,
2009)
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Membrane filtration Cellulose ester membrane Beverage (Prado et
(0.45 µm pore). al., 2006)
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997 Table 2. Analytical method for detection of Amaranth
Method of Detection Limit of References
Detection
(LOD)
HPLC C18 column (5 µm; 4.6 mm×250 mm). 6.4 ng mL-1 (Wu et al.,
Mobile phase: (A): 0.02 mol L-1 ammonium acetate aqueous 2016)
solution (pH 7.5, adjusted with 10 mol L-1 sodium hydroxide).
Mobile phase: (B): methanol-acetonitrile (30:70, v/v).
Flow rate: 1.0mL/min.
Temperature of the column: 30°C.
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Sample injection volume: 10 µL.
UV-visible spectrophotometer at 510 nm. 220 ng mL-1 (Wu et al.,
2016)
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HPLC-DAD C18 column (150 x 4.6 mm i.d., 5 µm). 0.00025 mg L-1 (Chai et al.,
Mobile phase: (A): 20 mmol L-1 ammonium acetate. 2016)
Mobile phase: (B): methanol.
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Flow rate: 1.0 mL/min.
LC-MS/MS C18 column (2.1 x 100 mm, 1.7 µm). 5.0 µg kg-1 (Martin et al.,
Mobile phase: (A): water containing 40 mM ammonium acetate 2016)
with 2.5% acetonitrile at pH 7.8 (adjusted with ammonia solution
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2.5%).
Mobile phase: (B): acetonitrile
Flow rate: 0.4 mLmin−1
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Injection volume: 40 µl.
HPLC-UV ODS III column (250 mm × 4.6 mm i.d., 5 µm particle 0.3 ng mL-1 (Bazregar et
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diameter). al., 2015)

Mobile phase: (A): ammonium acetate 1% m/v.
Mobile phase: (B): acetonitrile.
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Mobile phase: (C): methanol.

Flow rate: 1 mL min−1.
UV-visible spectrophotometer at 400-700 nm 10 µg L-1 (Sha & Zhu,
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2015)
HPLC-UV Eclipse plus-C18 column (4.6 mm x 150 mm x 3.5µm). 0.056 ng mL-1 (Sha et al.,
Mobile phase: (A): 0.02 mol/L ammonium acetate aqueous 2015)
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solution (pH 4.5, adjusted by glacial acetic acid).

Mobile phase: (B): methanol.
Flow rate: 0.8 mL min−1
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HPLC-DAD-IT-TOF/MS C18 (4.6 mm x 250 mm x 5 µm). 0.009 µg mL-1 (Li et al.,

Mobile phase: (A): 20 mM ammonium formate buffer. 2015)
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Mobile phase: (B) methanol/acetonitrile, 1/1 (v/v); gradient

elution.
Flow rate: 1.0 mL min−1
Sample injection volume: 20 µL.
HPLC-DAD Symmetry C18 (150 mm x 4.6 mm i.d.) 5 µm used 1.3 µg mL-1 (Karanikolop
together with C18 (25 mm x 4.6 mm i.d., 5 µm) guard column. oulos et al.,
Mobile phase: (A): aqueous ammonium acetate buffer (1% w/v) 2015)
(0.13 M) titrated to pH: 7.5 by addition of 0.1 M aqueous NH3.
Mobile phase: (B): methanol (MeOH)
Mobile phase: (C): acetonitrile (ACN)
HPLC-UV C18 column 0.1 – 0.4 mg kg- (Kong et al.,
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1
Mobile phase: (A): methanol (all colors 2015)
Mobile phase: (B): 20 mM of ammonium acetate including
Amaranth)
TLC plate of 10×20 cm clean with 20 ml of the solvent solution 9.59 ng (Tang et al.,
(ethanol (95 %)–water–acetic acid (50:50:1)). 2015)
The plate then dried with hot air.
LC-MS/MS C16 column (3 mm, 120 Å, 4.6 x 150 mm). - (Tsai et al.,
Mobile phase: (A): acetonitrile 2015)
Mobile phase: (B): 20Mm ammonia acetate buffer with 1% acetic
acid.
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Flow rate: 1 mL min−1
HPLC-DAD C18 column 0.007 – 0.096 (Qi et al.,
(4.6 x 150 mm, 5 µm) with a C18 guard column (4.6 x 12.5 mm, 5 mg kg-1 2015)
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µm). (all colors
Mobile phase: (A): 20 mM ammonium acetate buffer at pH 7. including
Mobile phase: (B): methanol Amaranth)
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HPLC-MS/MS C18 column (3.0 x 75 mm, 2.2 µm).
Mobile phase: (A): methanol
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Mobile phase: (B): 5 mM ammonium acetate solution.
Electrochemical sensor (SWCNT-Tin) nanocomposite modified 40 nmol L-1 (Wang et al.,
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glassy carbon electrode (GCE, 3 mm in diameter). 2009)
Modified electrode was immersed in 0.1 M acetate buffer solution.
DPV parameters: increment potential, 0.004 V; pulse amplitude,
0.05 V; pulse width, 0.05 s; sample width, 0.0167 s; pulse period,
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0.2 s; quiet time, 2 s.

Electrochemical sensor: CNT/GO-[BMIM][PF6]/GCE 0.1 nmol L-1 (Wang et al.,
Working electrode: bare or modified glassy carbon electrode 2015a)
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(GCE; diameter = 3 mm).

Reference electrode: saturated calomel electrode (SCE)
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Counter electrode: platinum wire.

0.1 M BR buffer with pH of 7.0 was used as the supporting
electrolyte.
Square wave stripping voltammetry (SWSV): 0.4 to 1.0 V.
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Electrochemical sensor 5.0 x 10-10 mol (Wang et al.,

Working electrode: CNT–ppy/CE L-1 2015b)
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0.1 M pH 7.0 BR buffer solutions.

SWSV: 0.708 V
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Electrochemical sensor: CNT–ppy/SCE 5.0 x 10-10 mol (Baravik et

L-1 al., 2009)
-1
HPLC-DAD C18 column (4.6 × 250 mm, 5 µm). 0.002 mg kg (Tang et al.,
Mobile phase: (A): methanol 2014)
Mobile phase: (B): 0.01 mol L-1 phosphate buffer solution, pH 7.5
HPLC-PDA C18 column (id 250 x 4.60 mm 5 micron). 20 ng mL-1 (Shen et al.,
Mobile phase: (A): ammonium acetate 2014)
Mobile phase: (B): methanol
TLC and HPLC-IP-PDA C18 column 0.04 µg mL-1 (Andrade et
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(150 mm x 4.6 mm; 5 µm) and guard al., 2014)
column (20 mm x 4 mm; 5 µm).
Purified by TLC-PET 20 x 20 silica gel chromatography plates
with mobile phase (8 mL of isopropyl alcohol and 3 mL of
ammonium hydroxide).
Mobile Phase I: (A): Water; (B) methanol
(70:30; 61:39; 63:37, v/v).
Mobile Phase II: (A): Ammonium acetate
solution 0.08 m; (B) methanol (70:30;
61:39; 63:37, v/v).
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Electrochemical sensor (MWCNT), 0.1 M acetate buffer solution, 6.8 x 10-8 mol (Chandran et
pH 5.0. L-1 al., 2014)
Electrochemical sensor expanded graphite paste electrode (EGPE). 36 nmol L-1 (Zhang et al.,
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Working electrode: EGPE. 2014a)
SWSV: 0.4 V for 400 s in 0.05 mol L-1 BR buffer solutions (pH 7).
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Then, EGPE was regenerated by cycling voltammetry (10 times)
from 0.3 to 1.0 V in 0.05 mol L-1 BR buffer solutions (pH 6).
Indirect Competitive Enzyme-linked Immunosorbent Assay 3.35 ng mL-1 (Zhang et al.,
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(ELISA). 2013)
Amaranth (10.0 mmol) was treated with thionyl chloride (2.5 mL)
in anhydrous DMF (100 µL) to produce sulfonyl chloride. Then,
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the resulted amaranth-sulfonyl chloride (2.0 mmol) was allowed to
react with glycine (2.0 mmol) for the introduction of carboxyl
group, and the resultant was used as the hapten of amaranth.
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After that, 7.0 mL of PBS containing 0.1-mmol amaranth hapten

was mixed with 5.0 mL of PBS containing 1.0-mmol EDC and
0.5-mmol NHS, which were allowed to react for 2h at room
temperature.
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Then the mixture was added dropwise to 10.0 mL of PBS

containing 0.003-mmol cBSA.
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HPLC-UV C18 column (5 µm; 4.6 mm x 250 mm). 0.017 ng mL-1 (Wu et al.,
Mobile phase: (A): 0.1 mol L-1 ammonium acetate aqueous 2013)
solution (pH 7.5, adjusted by 10 mol L-1 sodium hydroxide).
Mobile phase: (B): methanol–acetonitrile (30:70, v/v).
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HPLC-DAD C8 column (150 mm x 4.6 mm id, 3 µm). - (Bonan et al.,

Mobile phase: (A): acetonitrile. 2013)
Mobile phase: (B): 100 Mm sodium acetate buffer at pH 7.
Flow rate: 1 ml min-1
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Injection volume: 20 µl.

UHPLC-DAD C18 column (2.1 × 50 mm, 1.7 µm). 2.11 µg kg-1 (Sun et al.,
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Mobile phase: (A): 20 mmol L-1 acetic ammonium, 0.02 % acetic 2013)
acid (pH=5).
Injection volume: 2 µL.
Electrochemical sensor alumina microfibers-modified carbon paste 0.75 nmol L-1 (Zhang &
electrode. Yadi, 2013)
Working electrode: alumina microfibers-modified carbon paste
electrode.
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Counter electrode: platinum wire
0.1 M phosphate buffer with pH of 6.0 was used as the supporting
electrolyte.
Oxidation peak current: 0.72 V.
Pulse amplitude: 50 mV.
Pulse width: 40 ms.
Scan rate: 40 mV s−1.
UHPLC-PDA C18 column (1.7 µm, 2.1 mm × 100 mm). 0.02 mg kg-1 (Liao et al.,
−1
Mobile phase: (A): 0.02 mol L of ammonium acetate aqueous 2012)
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solution
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Injection volume: 5 µL.
UV-visible spectrophotometer at 518 nm 13.0 ng mL-1 (Pourreza &
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Elhami,
2009)
UV-vis/ MCR-ALS at 359-600 nm - (Llamas et
al., 2009)
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Kinetic spectrophotometric at 300–700 nm 0.095 mg L-1 (Ni et al.,
2009)
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Capillary electrophoresis with laser-induced fluorescence detection 0.2 µg ml-1 (He et al.,
(CE–LIF). 2015)
Separation was carried out in fused
silica capillary with I.D. 50 µm, O.D. 360 µm, effective length 30
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cm and total length 38.5 cm.

Fluorescence was taken orthogonally by 60 × microscope
objective and focused on a photomultiplier tube.
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The separation was performed at V = +17 kV (intensity of

electrical field was 460Vcm−1).
TE
MEKC with fused silica capillary of 73 cm effective length, at 35 0.8 µg mL-1 (Prado et al.,
◦C, buffer phosphate solution of 10 mmol L-1 with sodium dodecyl 2006)
sulphate of 10 mmol/L, pH 11, and +25 kV of voltage.
Electrophoretic separation-PDA (effective length 56 cm, inner - (Patsovskii et
EP
diameter 75mm) at a constant voltage of 20 kV, using detection at al., 2004)

254 nm.
Micellar electrokinetic capillary chromatography (MEKC) of 57 2.50 µg g-1 (Lalo et al.,
cm x 75 micrometer uncoated fused-silica capillary column, 2012)
C
operating at 25 kV and detected by UV at 214 nm.

Solution comprised of 18% acetonitrile and 82% 0.05 M sodium
AC
deoxycholate in borate-phosphate buffer (pH 7.8).

Capillary zone electrophoresis (CZE) using bare CElect-FS75 CE 1.1 µg mL-1 (Perez-
column, 10 mmol L-1 phosphate buffer at pH 11.0. Urquiza &
Hydrodynamic injections at 0.5 p.s.i. for 4 s (21 nL of sample) and Beltran,
20 kV separation voltages were used. 2000)
998
999
44
ACCEPTED MANUSCRIPT
Review of Highlight
1) Amaranth belongs to the family of azo dyes and widely used in food industry.
2) To elaborate extraction and analytical methods for determination of Amaranth.
PT
3) To discuss on the acceptable daily intake and toxicology.
4) Azo dyes are aromatic compounds with one or more –N = N– groups.
RI
U SC
AN
M
D
TE
C EP
AC

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Accepted Manuscript

Toxicology, extraction and analytical methods for determination of Amaranth in food

Kobun Rovina, Shafiquzzaman Siddiquee, Sharifudin Md Shaarani

To appear in: Trends in Food Science & Technology

Received Date: 20 September 2016

2 and Beverage Products

5 Kobun Rovina1,2, Shafiquzzaman Siddiquee*1, and Sharifudin Md Shaarani2

22 Fax: 006088 320993

38 (HPLC), thin layer chromatography

40 electrophoresis (CZE). Additionally, a brief description on the uses of different extraction

43 guideline or idea in choosing an appropriate method for detection of Amaranth in foodstuffs.

45 Keywords: Amaranth; synthetic dyes; analytical methods; chromatography; extraction;

66 An approximately 65 % of azo colours are used as food additives in foodstuffs (Yamjala,

70 Amaranth is soluble in water (approximately 70 g L-1 at 25oC) and slightly soluble in

79 (TLC) (Andrade et al., 2014), liquid chromatography/tandem mass spectrometry (LC-

84 2004), enzyme-linked immunosorbent assay (ELISA) (Zhang et al., 2013) as well as

90 2.0 Amaranth (E 123)

104 naphthyl)hydrazono] naphthalene-2,7-disulfonate (C20H11N2Na3O10S3) (Yamjala et al., 2016;

110 (Tariq, Faisal, & Muneer, 2005).

113 3.0 Toxicological Study of Amaranth (E 123)

140 immunostimulated by intra peritoneal injection of sheep RBCs 10 %. The percentage of

152 the secondary structural changes in Hb (Basu, & Kumar, 2015).

176 Gennaro, Gioannini, Angelino, Aigotti, & Giacosa, 1997).

191 including Amaranth in powder products. A 0.4 mg mL-1 of polydopamine-coated Fe3O4

237 methylimidazolium bromide ([C4MIM][Br])) microextraction which interact with

239 separation efficiency. Similarly, Wu et al. (2013) used 1-octyl-3-methylimidazolium

tetrafluoroborate ([C8MIM][BF4]) dispersive liquid phase micro extraction (IL-DLPM)

247 day repeatability for Amaranth was found of 1.7 %.

249 5.3 Membrane Filtration

282 which is faster compare to conventional soxhlet extraction.

294 summarized in Table 1.

297 6.0 Analytical Methods for Detection of Amaranth

353 6.2 Liquid Chromatography/Tandem Mass Spectrometry (LC-MS/MS)

355 Liquid chromatography/tandem mass spectrometry (LC-MS/MS) is one of the

LC-MS/MS to determine 18 water-soluble artificial dyes in ice-cream, chocolate sweets and

367 6.3 Thin Layer Chromatography (TLC)

383 the identification and quantitative measurement of Amaranth in real samples.

385 6.4 Spectrophotometric

3.27 x 10-7 mol L-1.

414 spectrophotometric measurement and multivariate curve resolution-alternating least squares

423 (III) ion.

425 6.5 Electrochemical Sensors

427 Electrochemical behaviours of food additives, drugs, biomolecules and advances in

in researches of electrochemical methods for determination of synthetic colorants. In

438 Willner, 2009).

446 addresses electrochemical sensor based on single-walled carbon nanotube-tin (SWCNT-Tin)

(2015b) developed ultrasensitive and simultaneous determination of isomers of Amaranth and

463 applied in electrochemical system. The RF carbonized is three-dimensional network

511 employed to identify and quantify of Amaranth in soft drinks.

513 6.6 Molecularly Imprinted Polymers (MIPs)

(MIES) by magnetic field induced self-assembly of reduced oxide graphene-based

540 6.7 Capillary Electrophoresis (CE)

554 capillary zone electrophoresis, micellar electrokinetic capillary chromatography (MEKC),

570 capillary electrophoresis method with laser-induced fluorescence detection (CE-LIF) to

6.8 Enzyme-Linked Immunosorbent Assay (ELISA)

593 food and beverage products.

596 7.0 Conclusion

Fundamental Research Grant Scheme (FRGS), (No. FRGS/1/2014/SG05/UMS/02/4).

654 synthetic dyes in foodstuffs and beverages by high-performance liquid chromatography

668 Elkhatat, A. M., & Al-Muhtaseb, S. A. (2011). Advances in tailoring

685 polymers. In: Haupt, K. (ed.), Topics in Current Chemistry. Springer:

695 Hsieh, Y. Z., & Huang, H. Y. (1997). Determination of saikosaponins by micellar

721 determination of 34 water-soluble synthetic dyes in foodstuffs by high performance

723 spectrometry. Food Chemistry, 182, 316-326.