Determination of synthetic food colorants in fish products by an HPLC-DAD method

Food Chemistry 177 (2015) 197–203

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Analytical Methods

Determination of synthetic food colorants in fish products by an HPLC-DAD method q G. Karanikolopoulos ⇑, A. Gerakis, K. Papadopoulou, I. Mastrantoni General Chemical State Laboratory, Chemical Division of Piraeus and Aegean, 32 Etolikou Str., 18510 Piraeus, Greece

a r t i c l e

i n f o

Article history: Received 28 November 2013 Received in revised form 12 March 2014 Accepted 3 January 2015 Available online 8 January 2015 Keywords: Liquid chromatographic analysis Diode-array detector Synthetic dyes Food analysis Fish products

a b s t r a c t Reliable methods for quantification of synthetic water-soluble colors in complex food matrices are currently not available. The present work describes the development and validation of an improved protocol for the analysis of synthetic food colorants in complex food matrices presenting high protein and/or fat content. The method developed employs an extraction stage, followed by a subsequent sonification, centrifugation and concentration step. A final clean up via SPE on polyamide cartridges was also employed. The isolated colorants were separated and analyzed by an RP-HPLC/DAD system. High and consistent recoveries (min. 81%) and low RSDs (max. 6%) were achieved for all studied colorants. The issue of high fat content matrices was also addressed showing the need for an additional defatting step in the procedure. Overall, the protocol presented shows high precision and accuracy of detection and can provide the basis for future development of similar methods in other complex food matrices. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Color has been added to food to enhance the visual appeal for several hundreds of years. In the developing food industry of the late 19th century a wide range of synthetic colors were used, but issues over the toxicity of these additives has led to the development of legislation regulating the types and quantities of synthetic colors in different food products. As a result of these efforts, there is currently in European Union (EU) a solid framework of legislation controlling the use of color additives in food (Directive EC, Regulation EC). Still the recommended levels of these colorants are not well defined, and new evidence from in vitro and in vivo studies has been presented concerning both carcinogenicity and genotoxicity for some of these substances (EFSA Journal). Moreover, the techniques and protocols used for the determination of these colorants in complex food matrices are not robust enough, and further improvements are required. The accurate and reliable determination of synthetic colorants in different food matrices is of paramount importance to ensure safety and concordance with regulations. Many analytical techniques have been developed for the identification and quantification of various synthetic food colorants, such

q Part of this work has been presented at 8th International Conference on Instrumental methods of Analysis: Modern trends and Applications – IMA 2013. ⇑ Corresponding author. Tel.: +30 2104613991; fax: +30 2104613998. E-mail addresses: [email protected] (G. Karanikolopoulos), [email protected] (I. Mastrantoni).

http://dx.doi.org/10.1016/j.foodchem.2015.01.026 0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.

as spectrometry (Altinoz and Toptan, 2002), thin-layer chromatography (TLC) (Oaka et al., 1987; Oaka et al., 1994), adsorptive voltammetry (Ni et al., 1997), and differential pulse chromatography (Combeau et al., 2002). However, all of them present major drawbacks as they require time-consuming extensive pretreatment of the samples or/and cannot be applied to complex colorant mixtures. To overcome these limitations, the use of capillary electrophoresis (CE) (Liu et al., 1995; Razee et al., 1995; Perez-Urquiza and Beltran, 2000; Huang et al., 2003; Huang et al., 2002) and ion chromatography (IC) (Chen et al., 1998) techniques has been proposed as an alternative. However, the small injection volumes utilized in those techniques and/or background noise, results to sensitivity problems limiting the robustness of these methods. High-performance reversed-phase liquid chromatography (Nordic Committee on Food Analysis (NMKL), 1989; Prado and Godoy, 2002; Kirchbaum et al., 2003; Kirschbaum et al., 2006; Minioti et al., 2007; Vachirapatama et al., 2008; Garcia-Falcon and SimalGandara, 2005; Zhang et al., 2005) and ion-pair liquid chromatography (Gennaro et al., 1997; Fuh and Chia, 2002; Kiseleva et al., 2003) coupled with UV or diode-array detectors (DAD) are currently the most preferred analytical techniques as they provide excellent robustness combined with unrivalled resolution, sensitivity and selectivity. These approaches, although perfectly suitable for the determination of different colorants in liquid and water-soluble matrices (e.g. soft drinks or jams), when applied to complex food matrices of high protein and/or high fat content, such as meat or fish

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products, result to various problems including in low and inconsistent recoveries. This behavior can be attributed to interactions of the analytes with food components (i.e. binding of the studied colorant to protein matrices) as well as due to food components interference with the chemical analyses itself (Nordic Committee on Food Analysis (NMKL), 1989; Kirschbaum et al., 2006). Importantly, reliable methods for quantification of synthetic water-soluble colors in complex food matrices are currently not available. The aim of this study was to develop and optimize an efficient experimental protocol based on a reversed-phase high-performance liquid chromatography technique for the simultaneous determination of seven water-soluble synthetic colorants (E 110, E 122, E 123, E 124, E 127, E 128 and E 129), in food matrices of high protein and/or high fat content. Different experimental procedures were tested and evaluated towards efficiency and consistency of the isolation and separation step in precooked crustaceans, crab imitation products (e.g. surimi) and fish roe matrices. All the studied colorants, permitted in the EU market, were efficiently separated using an optimized gradient elution in a single run within less than 19 min. The analytical method based on the developed protocol was fully validated in crustacean matrices and was successfully applied to real samples obtained from the market. 2. Materials and methods 2.1. Apparatus The HPLC instrumentation consisted of a Shimadzu LC-20AD prominence gradient pump capable of mixing up to four solvents, a DGU-20A5 prominence mobile phase degasser, a SIL-20A prominence autosampler, a CTO-20AC prominence column oven and a UV–Vis Spectra system SPD-M20A prominence diode-array detector. The chromatographic data were collected and processed using a personal computer running LC solution 1.21 (Shimadzu Corporation, Kyoto, Japan). A microprocessor pH-meter (HACH, HQ 40d multi) equipped with a combined glass-calomel electrode was employed for pH measurements. The determination of colorants maximum absorbance wavelength (kmax) was carried out with a double beam UV–Visible spectrophotometer, UV-1700 Pharma Spec, with 1 cm quartz cells (Shimadzu, Japan). An Omnilab D-78224 thermostatic ultrasonic bath (Elma, Germany), an ALC PK 131R multispeed refrigerated centrifuge (ALC International, Italy) and a laboratory disperser IKA Ultra-Turrax 25 basic (IKA, Germany) were used for the pretreatment of food samples. 2.2. Reagents All solutions were prepared with deionized water and all chemicals were of analytical grade, unless stated otherwise. Ultrapure water from P.Nix Power System I (Human Corporation, Korea – water purity P 18.0 MXcm) was used for the preparation of the aqueous mobile phase as well as for the preparation of the standard solutions. Aqueous ammonia (25% w/v) (Reag. USP, Ph. Eur.) p.a. was purchased from Panreac Quimica, ammonium acetate P97% p.a. ACS from Roth, petroleum ether (40–60 °C) (PAR) from Panreac Quimica and n-hexane P99% from Merck. The HPLC grade organic solvents methanol (MeOH) and acetonitrile (ACN) were purchased from Fisher Chemical and Sigma Aldrich, respectively. Solution I is a mixture of aqueous NH3 and MeOH (5/95 v/v). The food colorants E 110 (Sunset Yellow FCF), E 122 (Azorubin), E 123 (Amaranth), E 124 (Ponceau 4R), E 127 (Erythrosine), E 129 (Allura Red AC) produced by IPS (Institute of Leather Industry,

Poland) and E 128 (Red 2G) produced by IMPM (Institute of Polymer materials and Dyes, Poland), were all of known analytical purity (ranging from 80% to 94%) and purchased from LGC standards. The chemical formulas of the studied colorants along with common names, European Community numbers (E numbers), Color Index dominations (CI numbers), Maximum absorbance wavelength (kmax) and dye percentage of crude colorants are reported in Table 1. 2.3. Chromatographic conditions For chromatography a Symmetry C18 (Waters, Milford, USA) column (150 mm  4.6 mm i.d.) 5 lm particle size, was used together with C18 (25 mm  4.6 mm i.d., 5 lm) guard column (Waters). The injection volume was 20 lL, the flow rate was kept at 1.5 mL min 1 and the column oven temperature was set at 30 °C. The mobile phase consisted of an aqueous ammonium acetate buffer (1% w/v) (0.13 M) titrated to pH: 7.5 by addition of 0.1 M aqueous NH3 (solvent A), methanol (MeOH, solvent B) and acetonitrile (ACN, solvent C). Solvent A is prepared daily and filtered by vacuum through a 0.22 lm membrane filter (Millipore, Type GVWP) prior to HPLC analysis. In order to achieve a successful resolution of all colorants a number of gradient elution programs were tested, the final optimized gradient consists of the following steps: 0–2 min: isocratic elution of 100% solvent A; 2–20 min: linear gradient from 100% solvent A to 20% solvent A, 65% solvent B, 15% solvent C; 20–22 min: isocratic elution of 20% solvent A, 65% solvent B and 15% solvent C; 22–24 min: linear gradient from 20% solvent A, 65% solvent B, 15% solvent C back to the initial condition of 100% solvent A and 24–26 min: equilibrium to the initial conditions of the following injection. The diode-array detector was programmed to monitor the seven colorants at a range of 300–750 nm. The detection and quantification of each analyte was performed at its maximum absorbance wavelength (kmax) (see Table 1). A characteristic chromatogram of a mixed standard solution of all studied colorants using the optimized gradient program described above is displayed in Fig. 1. 2.4. Colorants standards solutions and sample preparation Standard stock solutions of 1000 mg L 1 for each individual food colorant were prepared by dissolving approximately 25 mg of unpurified colorant in 25 mL deionized water, taking into consideration the purity of the colorant. The solutions were kept in the fridge (4–8 °C) for a maximum time period of 3 months. Standard working solutions of 100 mg L 1 were prepared by appropriate dilution of the stock solutions in deionized water and kept stable for 1 month at 4–8 °C. The mixed standard solutions containing all colorants at concentrations between 0.8 and 100 mg L 1 were also prepared by mixing and diluting appropriate aliquots from standard working or standard stock solutions of each colorant. All the above solutions were kept at 4–8 °C for 1 month. All samples were obtained from internal market control as well as from the border control of imported goods from third countries (outside EU) and included crustaceans, crab imitation products and other fish products (i.e. fish roe). Samples were homogenized prior to analysis and time between homogenization and analysis did not exceed 24 h. In particular, for the preparation of the spiked samples about 50 g of a color free crustaceans sample was weighed and spiked with the appropriate amount of a mixed standard solution depending on the desired concentration level (for example: for a final colorants concentration of 3 ppm a 4 mL aliquot of a 100 mg L 1

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Table 1 Chemical formulas, common names, European community numbers (E numbers), color index dominations (CI numbers), molecular weights (MW), maximum absorbance wavelength (kmax) and dye percentage of all studied colorants.

*

Color (E-No)

Common names

CI number

Formula

MW

kmax (nm)

Dye content* (% w/w)

E E E E E E E

Sunset Yellow FCF, FD&C Yellow No. 6, Food Yellow 3, Orange Yellow S Azorubin, Carmoisine, Food Red 3, Acid Red 14 Amaranth, FD&C Red No. 2, Naphtol Red S, Food Red 9, Acid Red 27 Ponceau 4R, New Coccine, Cochineal Red A, Food Red 7, Acid Red 18 Erythrosine, FD&C Red No. 3, Food Red 14, Acid Red 51 Red 2G, Acid Red 1, Food Red 10 Allura Red AC, FD&C Red No. 40, Food Red 17

15985 14720 16185 16255 45430 18050 16035

C16H10N2Na2O7S2 C20H12N2O7S2Na2 C20H11N2O10S3Na3 C20H11N2Na3S3O10 C20H6I4Na2O5⁄H2O C18H13N3O8S2Na2 C18H14N2Na2O8S2

452.4 502.4 604.5 604.5 897.9 504.9 496.4

494 520 520 505 532 532 505

93.7 86.8 85.5 81.7 88.1 80.1 86.5

110 122 123 124 127 128 129

+/ +/ +/ +/ +/ +/ +/

0.5 0.5 0.5 0.5 0.5 0.5 0.5

Based on certificates of analysis provided by the supplier of the colorants.

mAU Erythrosin E127

400

350

Red 2G E128

Allura Red E129

E122 Azorubin

200

E124 Ponceau 4R

Amaranth E123

250

E110 Sunset Yellow

300

150

100

50

0

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0

22.5

25.0 min

Fig. 1. Chromatogram (extracted chromatogram of four different channels) of a mixed standard solution using the optimized gradient program (concentration of all colorants were 30 mg L 1).

mixed standard solution is utilized). Finally, ammonium acetate buffer (solution A) was added to the resulting mixture until a final weigh of 150 g and the whole was homogenized. The resulting spiked pulp was divided to appropriate subsamples and analyzed following the protocol described below. Extraction of the colorants for a 5 g sample portion was carried out by adding 10 mL of a solution containing aqueous NH3 (25% w/ v) and MeOH (5/95 v/v) (Solution I) and 10 mL of deionized H2O or ammonium acetate buffer (0.13 M), depending on the protocol. The sample was then subsequently vortexed for 1 min and stirred for 1 h or sonificated for 20 min at 30 °C, depending on the experimental protocol. The resulting dispersion was centrifuged at 5000 rpm/ 15 °C for 10 min utilizing vials fitted with screw cap in order to eliminate loss of the colored extract. The colored supernatant solution was then decanted and the extraction was repeated at least twice or until the resulting extract is colorless. A total of three extractions was usually sufficient for the color to be extracted quantitatively from the food matrices studied. The resulting clear

colored solutions were collected together and evaporated (usually in a water bath or a vacuum evaporator) to about 15 mL until all traces of methanol and ammonia were removed and the pH of the solution turns neutral. The concentrate was then filtered through a glass fiber filter (Macherey–Nagel, GF) and a subsequent clean step was carried out on solid phase extraction cartridges (SPE) of Polyamide 6 (Macherey–Nagel, Chromabond PA, 6 mL/ 500 mg). Cartridges were initially conditioned with hot water (60–70 °C), and then the colored concentrates were forced through the cartridges under vacuum. By this procedure synthetic colors are adsorbed on polyamide while natural colors are eluted. A washing up step with MeOH further elutes natural colors (if present). Finally, the synthetic colors were desorbed with 10 mL of Solution I. At the resulting clear colored liquid 2 mL of deionized water were added and a final concentration step is taking place until a volume of 1–2 mL was reached (removal of all traces of MeOH). The final concentrates were diluted to 5, 10 or 20 mL volume with deionized water, depending on the expected

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concentration of the colorants determined, filtered through 0.45 lm PVDF filters (Millipore, Millex-HV) and analyzed using an RP-HPLC/DAD technique. In the cases of high fat content food matrices a defatting step was employed in the experimental procedure. In this process petroleum ether or n-hexane (20 mL) is added in 5 g of homogenized sample and the whole is vortexed for 1 min, the supernatant liquid is discarded and the step is repeated if necessary. A series of three extraction steps (with the organic solvent) were usually adequate for the quantitative removal of the fatty phase. Finally, the remains of the organic solvent were evaporated via a gentle nitrogen stream. After the defatting step the sample were subjected to the procedure described above for the extraction, concentration and purification of the isolated food colorants.

2.5. Experimental protocols For the purposes of this study different extraction procedures and solvents’ mixture were tested for the optimization of the extraction stage: Typical Protocol (Stages): 3 extraction steps were utilized each employing 10 mL of Solution I and 10 mL of deionized H2O, mechanical stirring of the dispersion for each extraction step for at least 1 h (manually or magnetic stirrer), centrifugation at 3000 rpm at RT (employing vials without cap) for 5 min, concentration in water bath (at about 90 °C) to a final volume of 15 mL, acidification of the concentrate with dropwise addition of a diluted solution of HCl or CH3COOH (0.1 M) to pH: 4–5, final SPE clean up (Nordic Committee on Food Analysis (NMKL), 1989). Alternative Protocol (Stages): 4 extraction steps were utilized each employing 10 mL of Solution I and 10 mL of deionized H2O, Sonification at RT for 20 min, centrifugation at 3000 rpm/RT/ 10 min, concentration using incubation in a water bath to about 15 mL, neutral pH (after concentration step, pH: 7), final SPE clean up. New Protocol (Stages): 4 extraction steps were utilized each employing 10 mL of Solution I and 10 mL of ammonium acetate solution (0.13 M), sonification at about 30 °C for 20 min, centrifugation at 5000 rpm/15 °C for 10 min (screw cap vials), concentration using incubation in a water bath to about 15 mL, neutral pH (after concentration step, pH: 7), final SPE clean up.

2.6. Determination of repeatability and reproducibility Intra-day and inter-day precision was assessed by analyzing six replicates of a sample during one working day (n = 6, df = 12, intraday precision-repeatability) or three replicates by two analysts in two different days (n = 3, df = 6, inter-day precision-reproducibility), respectively.

3. Results and discussion The present work describes the development and optimization of a new and efficient protocol for the simultaneous analysis of seven red water-soluble synthetic dyes (E 110, E 122, E 123, E 124, E 127, E 128 and E 129) which are currently allowed for use in EU in precooked crustaceans, crab imitation products (e.g. surimi) and fish roe. Crustaceans were chosen as representative of complex food matrices due to their widespread consumption worldwide. In addition, they have high protein content along with negligible fat, thereby presenting a unique substrate for the study of food colorants analysis in the presence of high protein content.

3.1. Optimization of the experimental protocol For all three different protocols tested spiking experiments were conducted to determine precision and accuracy of detection. Spiking experiments were performed in crustacean matrices, utilizing a mixture of four different colorants (E 110, E 122, E 124 and E 129) in three fortification levels (3, 7 and 15 ppm). The spiking levels were chosen in the range of 10 ppm based on laboratory observations from real samples analysis. Specifically, more than 300 fish products have been analyzed in our laboratory for synthetic food colorants the past two years derived both from internal market control and Customs authorities. The great majority of these samples (>98%) presented artificial colorants in concentrations up to 15 ppm, while for the remaining cases (almost 2% of the total) synthetic colorants concentration ranged from 20 to 30 ppm. These data suggested that spiking levels in the range of 10 ppm would be sufficient to simulate real samples found in EU market. Every spiking experiment was performed in duplicate and the variations of recovery, as well as RSD values, were recorded. The results are summarized in Table 2. The variations of recovery values among the different protocols are also depicted. The first protocol tested was based on standard conditions (Typical Protocol, see Table 2) suitable for the analysis of synthetic colors in liquid and water-soluble matrices (e.g. soft drinks, jams or confectionery) (Nordic Committee on Food Analysis (NMKL), 1989; Kirschbaum et al., 2006; Minioti et al., 2007; Vachirapatama et al., 2008; Zhang et al., 2005). However, when tested in complex food matrices (i.e. crustaceans) this protocol resulted in poor recoveries for all the studied colorants, usually in the range of 20–40%, accompanied by poor precision characteristics (RSD values ranging from 14% to 25%). In more detail, the extraction step of this protocol proved inadequate for the substrates tested leaving the samples significantly colored even after three extractions. The use of an additional protein splitting step before the extraction procedure, as proposed in the literature (Nordic Committee on Food Analysis (NMKL), 1989), did not improve the resulting recoveries (values remained in the range of 30–40%). In addition, significant color loss was observed during acidification of the concentrate, after NH3 and MeOH removal. Acidification is usually employed to stabilize the colorants (which are unstable in alkaline pH) prior to sample clean up, but in our case resulted in colored precipitates. The color loss at this stage can be attributed to protein coagulation in the acidic conditions resulting in the precipitation of colored sediment. Accordingly, a differentiated protocol (Alternative Protocol, see Table 2) was tested next. In this protocol acidification was omitted and improvements in extraction stage were added. In particular, there was an increase of the number of extraction steps from 3 to 4 along with sonification of the resulting dispersion. Using this protocol, the solution remained neutral (pH: 7) after the complete removal of MeOH and NH3 under heating, thus preventing protein precipitation and consequently color loss. The synthetic colors studied were stable at neutral pH and were retained efficiently on the polyamide material of the cartridge during sample clean up. Overall use of the Alternative Protocol resulted in increased recovery values ranging in the area of 40–80%, for all four colorants. Further improvement of recoveries was obtained by additional experiments leading to optimization of the extraction step (New Protocol, see Table 2). These improvements included: (a) replacement of H2O with ammonium acetate solution (0.13 M, pH: 7.5) in the extraction solvent mixture (increase of the ionic strength); (b) modifications in sonification step (carried out at 30–40 °C); and (c) centrifugation (5000 rpm/15 °C and use of screw cap vials). These modifications resulted in significant improvement in recovery and RSD values (Table 2). Using this optimized New Protocol led to an increase of mean recoveries up to 90%.

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Table 2 The three experimental protocols – a comparative presentation for recoveries (%), standard deviation (Sr) and relative standard deviation (RSDr%) values derived from spiked food matrices, in all cases six independent replicates were analyzed for each concentration level (n = 6, df = 12). Recovery% Protocol

Concentration level (mg kg

Typical protocol

3 7 15 Mean values Sr RSDr% 3 7 15 Mean values Sr RSDr% 3 7 15 Mean values Sr RSDr%

Alternative protocol

New protocol

1

)

E 110

E 122

E 124

E 129

29 31 39 33 5 16 39 38 42 40 2 5 91 89 85 88 3 3

26 30 40 32 7 23 58 71 63 64 7 10 82 83 86 84 2 2

30 36 44 37 7 19 92 86 78 85 7 8 94 89 90 91 3 3

31 36 41 36 5 14 63 72 61 65 6 9 84 84 88 85 2 3

Table 3 Validation data derived from spiking experiments conducted in duplicates using the finalized experimental protocol (new protocol) (mean values, n = 6, df = 12), and calibration curves (n = 10) – recoveries (%), RSDr (%), rel. repeatability (%), rel. reproducibility (%), relative combined expanded uncertainty (U%) and xc, xd values for all studied colorants. Colorant (E-No)

Recovery%

E 110

84

E 122

RSDr%

rel. r%

rel. R%

U%

Concentration level (mg kg

5

13

21

85

5

15

24

E 123

81

6

16

25

E 124

93

6

17

28

E 127

52

10

27

44

E 128

85

6

15

24

E 129

88

4

13

20

43 20 39 19 40 20 43 22 46 33 38 19 38 16

<10 P10 <10 P10 <10 P10 <10 P10 <10 P10 <10 P10 <10 P10

Next we tested the finalized New Protocol for the analysis of spiked shrimp samples with a mixture of all seven studied colorants (E 110, E 122, E 123, E 124, E 127, E 128 and E 129) at various fortification levels (3, 7 and 15 ppm). The results of these multiple spiking experiments are displayed in Table 3. The results show that the finally adopted experimental protocol for the analysis of colorants (New Protocol) is characterized by significantly high and consistent recovery values (min. 81%) as well as low RSD values (max. 6%) for all studied colors. Exception to this general picture was the case of Erythrosine (E 127). In all experiments, this colorant showed poorer recovery characteristics (generally values in the range of 50–60%) accompanied with higher RSD values (in the area of 10%). This behavior could be partially attributed to the relatively higher sensitivity of the specific colorant to elevated temperature conditions. To account for this, milder temperature conditions were applied in the concentration step of the New Protocol by the use of a vacuum evaporator at 60 °C. Under these conditions, the recovery for E 127 reached values as high as 91% (Fig. 2). It should be noticed that all the studied colorants presented the same trend and a general increase in recoveries was observed (generally 5–15% with regard to usual experimental procedure – New Protocol, Fig. 2). However, the use of vacuum evaporator was proved time consuming for application in everyday laboratory practices. The vacuum

1

)

Critical value, xc (lg mL

1

)

MDV, xd (lg mL

0.7

1.4

0.7

1.3

0.7

1.3

0.7

1.4

0.7

1.4

0.5

1.0

0.7

1.3

1

)

110 R e c o v e r y

100 90 80 70

concentraon in water bath

% 60 concentraon ulizing mild condions (vacuum evaporator at 60°C)

50 40 E 127

E 128

E 124

E 122

E 129

E 110

E 123 Colorants

Fig. 2. Temperature effect on recovery values.

evaporator concentration step could last for as long as 45 min for each sample, when the water bath alternative last no more than 20–30 min for the analysis of 4–6 samples. Thus, in real samples analysis the vacuum evaporator step was not adopted. Instead, conditions of temperature and time were adjusted during

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Table 4 The fish roe issue – the effect of the deffating step on Recovery values for fish roe samples (n = 2, df = 6). Colorant (E-No)

Experimental protocol

Concentration level (mg kg

E 122

New New New New New New

17

E 124 E 110

protocol protocol with defatting step protocol protocol with defatting step protocol protocol with defatting step

17 17

concentration stage in order to achieve the highest possible recoveries at reasonable analysis time.

3.2. Method validation The New Protocol procedure was fully validated for the analysis of all seven colorants in crustacean matrices. The final combined expanded uncertainty (U) was calculated as the square root of the sum of squares of the component standard uncertainties, U(xi), multiplied by a coverage factor (k = 2, confidence level: 95%). The uncertainty sources in the analytical procedure were identified and quantified. The contributions of the following component uncertainties were estimated: relative standard deviation in terms of reproducibility (RSDL), recovery uncertainty u(rec), uncertainty of the reference materials concentration u(CS), uncertainty of the calibration curve u(Co), uncertainty due to volume measurements u(Vd) and uncertainty due to mass measurements u(m). The component with the highest contribution to the combined expanded uncertainty (U) was the calibration curve uncertainty, u(Co) at low concentration levels (<10 ppm), whereas the relative standard reproducibility (RSDL) had the highest impact on uncertainty at concentration levels higher than 10 ppm. The results are presented in Table 3. The quantification of uncertainty is of high importance for analytical measurements, offering, to different laboratories across the world, the ability to compare analytical results through traceability. This is the first study in the field quantifying and reporting an estimation of uncertainty for the proposed protocol. Additionally, critical values (xc) and minimum detectable values (MDV, xd) (according to ISO 11843) were calculated for all studied colorants (see Table 3). For this, data sets from calibration equations (n = 10) of mixed standards solutions of all colorants in six calibration levels (concentration range: 0.8–30 lg mL 1) were utilized. The calibration curves of each colorant were used for the validation experiments (estimation of u(Co)) as well as quantification. The corresponding data were acquired in a time period of over one year. Finally, the correlation coefficient (R2) for all calibration curves was determined at 0.999 or higher.

3.3. The high fat matrices issue The issue of high fat content matrices was also addressed in the present study. The high fat content of a food matrix can pose an additional problem during the extraction stage, especially in regard to the separation of the two phases, aquatic and lipophilic, after the sonification and centrifugation step. To overcome those difficulties an additional defatting step was employed in the New Protocol, utilizing an organic solvent (either petroleum ether or n-hexane). In this analysis, samples of colorized fish roes (commercially available) or spiked fish roes (prepared in the lab) fortified with a mixture of three colors (E 110, E 122 and E 124) were used and analyzed following the optimized experimental protocol (New Protocol) with and without a defatting step. All samples were analyzed in duplicates.

1

)

Recovery% (mean values)

RSDr%

70 81 71 80 75 84

7 4 11 3 15 4

In the absence of a defatting step, poor separation took place and in some samples emulsions were formed, which made the quantitative decant of the aquatic phase extremely difficult. However, a significant increase in recovery values was observed when a defatting step was employed (Table 4). These are the first published data recording and estimating the performance characteristics of the method applied (accuracy – as Recovery%, and precision – as RSD%) in food products of high fat and/or high protein content based on real samples analysis utilizing an RP-HPLC/DAD technique. 4. Conclusions The existing methods for the analysis of synthetic food colorants are mainly suitable for liquid and water-soluble matrices. These methods do not allow accurate and reproducible analysis of complex food matrices of high protein content such as fish and meat products. In the present work, we developed and optimized a new protocol dedicated to food matrices of high protein and/or high fat content. This method was validated using crustacean matrices allowing robust and efficient measurements of Recovery and RSD values. Moreover, it allowed the simultaneous determination of seven water-soluble synthetic food colorants (E 110, E 122, E 123, E 124, E 127, E 128 and E 129) at very low concentration (<1 ppm). Overall, the protocol presented here provides a simple and relatively fast method for the determination of food colorants in the presence of analytically challenging matrices. This protocol shows high precision and accuracy of detection and can provide the basis for future development of similar methods in other complex food matrices such as meat products. References Altinoz, S., & Toptan, S. (2002). Determination of tartrazine and ponceau-4R in various food samples by vierordt’s method and ratio spectra first-order derivative UV spectrophotometry. Journal of Food Composition and Analysis, 15, 667–683. Chen, Q. C., Mou, S. F., Hou, X. P., Riviello, J. M., & Ni, Z. M. (1998). Determination of eight synthetic food colorants in drinks by high-performance ion chromatography. Journal of Chromatography A, 827(1), 73–81. Combeau, S., Chatelut, M., & Vittori, O. (2002). Identification and simultaneous determination of Azorubin, Allura red and Ponceau 4R by differential pulse polarography: Application to soft drinks. Talanta, 56(1), 115–122. (a) Directive EC 94/35/EC.(b) Directive EC 94/36/EC.(c) Directive EC 95/2/EC. (a) . EFSA Journal, 515, 1–28(b). EFSA Journal, 7(11), 1327(c). EFSA Journal, 7(11), 1331. Fuh, M. R., & Chia, K. J. (2002). Determination of sulphonated azo dyes in food by ion-pair liquid chromatography with photodiode array and electrospray mass spectrometry detection. Talanta, 56(4), 663–671. Garcia-Falcon, M. S., & Simal-Gandara, J. (2005). Determination of food dyes in soft drinks containing natural pigments by liquid chromatography with minimal clean-up. Food Control, 16(3), 293–297. Gennaro, M. C., Gioannini, E., Angelino, S., Aigotti, R., & Giacosa, D. (1997). Identification and determination of red dyes in confectionery by ion-interaction high-performance liquid chromatography. Journal of Chromatography A, 767 (1–2), 87–92. Huang, H. Y., Chiu, C. W., Sue, S. L., & Cheng, C. F. (2003). Analysis of food colorants by capillary electrophoresis with large-volume sample stacking. Journal of Chromatography A, 995(1–2), 29–36. Huang, H. Y., Shih, Y. C., & Chen, Y. C. (2002). Determining eight colorants in milk beverages by capillary electrophoresis. Journal of Chromatography A, 959(1–2), 317–325.

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