Determination of food dyes in soft drinks containing natural pigments by liquid chromatography with minimal clean-up

Food Control 16 (2005) 293–297 www.elsevier.com/locate/foodcont

Determination of food dyes in soft drinks containing natural pigments by liquid chromatography with minimal clean-up M.S. Garc
ıa-Falc
on, J. Simal-G
andara

*

Nutrition and Bromatology Group, Analytical and Food Chemistry Department, Faculty of Food Science and Technology, Ourense Campus, University of Vigo, E-32004 Ourense, Spain Received 19 October 2003; received in revised form 20 March 2004; accepted 23 March 2004

Abstract The essential aim of this work was the optimisation and characterisation of a method for the determination of five synthetic food colours added to soft drinks with natural colours by liquid chromatography with minimal clean-up. The method is simple, rapid, inexpensive and broadly applicable to soft drinks. The results show that tartrazine (E-102), quinoline yellow (E-104), yellow orange (E-110), azo rubine (E-122) and ponceau (E-124) can be determined at low levels simultaneously. The method was evaluated by constructing calibration lines, measurement of recovery and precision, and the limits of quantification and detection. It involves direct injection of a beverage solution (homogenized and degassed) for reversed-phase HPLC analysis with visible detection at a single wavelength. Synthetic colours determination in soft drinks was not affected by the presence of other natural colours contained in drinks with fruit juice and flavour extracts. The results obtained with the samples confirm that the proposed method works well and is useful for serious control or screening of the addition of synthetic colours in soft drinks; the method is therefore recommended for use by the quality control departments of soft drink producers using synthetic food colours.
2004 Elsevier Ltd. All rights reserved. Keywords: Synthetic food colours; Minimal clean-up; Direct injection RP-HPLC with visible detection; Soft drinks

1. Introduction It is possible that synthetic colours added to food and drinks exceed the authorised levels. Monitoring of the levels of dyes in high consumption products such as beverages becomes therefore of paramount importance. Within one of the comprehensive schemes regulating the use of food colours, the European Union (Directive 94/ 36/EC, 1994) issued legal provisions for foods. The above regulations set the scene for the analytical chemist who has to test for the levels of dyes added to food. Levels of interest are in the mg/kg range for many of the additives used in food manufacture. In the case of nonalcoholic beverages with added juices and/or flavours, five synthetic food colours are mainly used (E-102, E104, E-110, E-122 and E-124; Table 1) and the total concentration of these synthetic colours studied should not exceed 100 mg/l. For some of these colours (E-110, E-122 and E-124; Table 1), the individual limits are at 50

*

Corresponding author. E-mail address: [email protected] (J. Simal-G
andara).

0956-7135/$ - see front matter
2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2004.03.009

mg/l. In other geographical areas there are also food colour certification programs and regulations limiting the level of colour that can be used in foods. Regarding the five colours studied in this paper, in the USA, the Food and Drug Administration has only approved colours E-102 and E-110 for use in foods generally (CFR, 2002). Implementation of the regulations carries an obligation for the different states to carry out the necessary tests to ensure that foods comply with their colour additives requirements. This task often falls on the official food control laboratories, but the food industry also has a responsibility to ensure that colouradded foods are manufactured in accordance with good manufacturing practice and comply with the relevant legislation. A scientific literature survey of methods indicate that a large number of techniques have been employed for the analysis of synthetic colours in beverages. The principal problems associated with the determination of colour additives in beverages are the mixtures of colours and the diversity of potential interferences present. The analytical techniques frequently used for the determination of the colours include Visible Spectrophotometry

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ıa-Falc
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andara / Food Control 16 (2005) 293–297

Table 1 Selected food colours Code

Colour name

Colour index

CAS No.

FD&C

Maximum wavelength (nm)

E-102

Tartrazine 1H-Pyrazole-3-carboxylic acid, 4,5-dihydro-5-oxo1-(4-sulfophenyl)-4-[(4-sulfophenyl)azo]-, trisodium salt Quinoline Yellow 1,3-Isobenzofurandione, reaction products with methylquinoline and quinoline, sulfonated Yellow Orange S 2-Naphthalenesulfonic acid, 6-hydroxy-5-[(4-sulfophenyl)azo]-, disodium salt Azo Rubine 1-Naphthalenesulfonic acid, 4-hydroxy-3-[(4-sulfo-1-naphthalenyl)azo]-, disodium salt Ponceau 4R 1,3-Naphthalenedisulfonic acid, 7-hydroxy-8-[(4sulfo-1-naphthalenyl)azo]-, trisodium salt

19140 Yellow 4

1934-21-0

Yellow No. 5

430

47005 Yellow 13

8004-92-0

Yellow No. 10

414

15985 Yellow 3

2783-94-0

Yellow No. 6

476

14720 Red 3

53026-69-9

Red No. 10

516

16255 Red 7

2611-82-7

Red No. 8

508

E-104

E-110

E-122

E-124

(Berzas-Nevado, Rodr
ıguez-Flores, Villase~ nor-Llerena, & Rodr
ıguez-Fari~ nas, 1999), Thin Layer Chromatography (Oka et al., 1987), Capillary Electrophoresis (Chou, Lin, Cheng, & Hwang, 2002; Kuo, Huang, & Hsieh, 1998; Liu et al., 1995; Mas
ar & Kaniansky, 1996; Mas
ar, Kaniansky, & Madajov
a, 1996; Suzuki et al., 1994), and mainly HPLC (Chaytor & Heal, 1986; Chen, Mou, Hou, Riviello, & Ni, 1998; De la Cruz-Garc
ıa et al., 1998; Greenway, Kometa, & Macrae, 1992; Griffin, Kee, & Adams, 1988; Weaver & Neale, 1986). Common reversed-phase high performance liquid chromatography, better than the more expensive high-performance ion chromatography (Qing-Chuan, Shi-Fen, Xiao-Ping, Riviello, & Zhe-Ming, 1998), is the technique of choice for the rapid analysis of colour additives in beverages, especially when an extensive sample treatment is not required, and with the possibility of selective detection by visible spectrophotometry. In this paper, we propose a simple HPLC method for the determination of synthetic colours (Table 1) in beverages with excellent performance characteristics. No time-consuming sample pre-analytical treatment is necessary. This reduces the chances for low recoveries in solid phase extraction followed by reversed-phase liquid chromatography (Sung-Kwan et al., 2000) and, moreover, increases the sample throughput.

2. Methods 2.1. Chemicals and standard solutions Tartrazine (E-102), Quinoline Yellow (E-104), Yellow Orange S (E-110), Azo Rubine (E-122) and Ponceau 4R (E-124) for use as standards (Table 1) were obtained from the food additive suppliers Analema, ChromaGesellchaft or Fluka. Water, ammonium acetate and

methanol were of analytical grade and supplied by Panreac or Merck. Individual standard stock solutions containing each colour at 1000 mg/l in water, were prepared. Subsequently mixed intermediate standard solutions containing all colours were prepared at 100 mg/l in water. The calibration standard solutions were then prepared by appropriate dilution of intermediate mixed standard solutions in water to give concentrations between 1 and 100 mg/l. All solutions were stored at 4 C in amber glass bottles and were stable for at least 2 months; there was no trend indicative of compound instability over this period. 2.2. Apparatus and instrumental parameters All HPLC measurements for colours determination were taken using a Thermo Separations Product liquid chromatograph, equipped with a SCM1000 vacuum membrane degasser, a P200 binary pump, an AS-1000 autosampler and a Microuvis 20 Carlo Erba variable wavelength ultraviolet–visible detector. The chromatographic data were collected and processed using a personal computer running Chrom-Card for Windows multitasking software. The instrumental parameters are recorded in Table 2. A double beam UV/visible spectrophotometer (Uvikon 923 from Kontron Instruments) was used to obtain visible spectra of the selected colours in order to look for the best compromise wavelength within detection sensitivity for all colours. A Sartorius analytical balance and a Selecta ultrasonic bath were also used. 2.3. Determination of colours in soft drinks The concentrations of the calibration standards were in the ranges given in Table 3. The quantification by

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ıa-Falc
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andara / Food Control 16 (2005) 293–297

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Table 2 Instrumental parameters of the proposed method for the determination of the food colours in beverages HPLC method Injection volume Guard column

20 ll Pelliguard LC-18 40 lm (5 cm · 4.6 mm i.d.) Tracer Anal
ıtica ODS 5 lm (15 cm · 4 mm i.d.) Solvent A: Methanol Solvent B: 40 mM Ammonium acetate aqueous solution (pH ¼ 5) 0–3 min: isocratic elution at 10%A:90%B 3–5 min: linear gradient from 10%A:90%B to 25%A:75%A 5–8 min: isocratic elution at 25%A:75%B 8–18 min: linear gradient from 25%A:75%B to 75%A:25%B 18–20 min: isocratic elution at 75%A:25%B 20–21 min: linear gradient to recover initial conditions of 10%A:90%B 21–30 min: conditioning step at 10%A:90%B 1 ml min
1 Visible detection at 414 nm peak 1: 7.6 (E-102) peak 2: 11.5 (E-124) peak 3: 12.9 (E-104, 1st isomer) peak 4: 13.1 (E-110) peak 5: 14.9 (E-104, 2nd isomer) peak 6: 17.1 (E-122) peak 7: 18.1 (E-104, 3rd isomer) peak 8: 18.4 (E-104, 4th isomer)

Analytical column Mobile phase Mobile phase gradient programme

Flow Detection wavelength Retention times (min); ref. Fig. 1

against concentrations. Since samples were analysed directly without any pre-analytical step, such as extraction, clean-up or concentration, recoveries were kept at about hundred percent. Five identical aliquots of beverages containing the additives were analysed to estimate method precisions. Quantification and detection limits were calculated following ACS guidelines (ACS, 1980) and correspond to the analyte concentrations equivalent to a signal-to-noise ratio of ten and three, respectively. These limits were then experimentally confirmed by analysis of blank beverages spiked at the calculated level (Table 3). Fig. 1. HPLC chromatogram of the colour additives. See references for peaks in Table 2: 1 at 7.6 min (E-102), 2 at 11.5 min (E-124), 3 at 12.9 min (E-104, 1st isomer), 4 at 13.1 min (E-110), 5 at 14.9 min (E-104, 2nd isomer), 6 at 17.1 min (E-122), 7 at 18.1 min (E-104, 3rd isomer), and 8 at 18.4 min (E-104, 4th isomer).

HPLC used the chromatographic peak area (Fig. 1), and calibration lines were constructed by plotting peak areas

2.4. Investigation of commercial beverages with added synthetic food colours A total of 9 commercial samples of soft drinks with added juices and/or flavours were analysed (Table 4). Once sample packages were open, samples were poured

Table 3 Chromatographic performance of the proposed method for the determination of the food colours in beverages Calibration range (mg/l) r2 Precision (RSD%, n ¼ 5) Quantification limit (mg/l, n ¼ 7) Detection limit (mg/l, n ¼ 7)

E-102

E-104 (isomers sum)

E-110

E-122

E-124

1–100 (n ¼ 9 levels, injected twice)

4–100 (n ¼ 6 levels, injected twice)

2–100 (n ¼ 8 levels, injected twice)

3–100 (n ¼ 7 levels, injected twice)

<1.8

<2.1

1–100 (n ¼ 9 levels, injected twice) >0.9992 <2.1

<1.1

<0.8

1.0

4.0

1.0

2.0

3.0

0.3

1.0

0.3

0.6

1.0

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Table 4 Food colours concentrations in analysed beverages (min.-max.; n ¼ 3) Samples

Soft drinks (trade name)

Colour

Concentration (mg/l)

1

With fruit juice (Radical orange)

2 3

With fruit juice (Radical apple temptation) With fruit juice (Radical wild fruits seduction)

4 5 6

With fruit juice (Radical pineapple emotion) With fruit juice (Kas lemon) With fruit juice (Kas orange)

7 8 9

With fruit juice (Fanta Lemon) With flavour extracts (Casera lemon) With flavour extracts (Casera orange)

E-102 E-110 E-102 E-122 E-124 E-102 E-102 E-102 E-110 E-104 E-104 E-102 E-110

21.5–22.5 3.9–4.1 7.6–8.3 32.7–33.4 17.7–18.2 5.8–6.1 2.0–2.1 11.9–12.3 2.0–2.1 3.8–4.0 4.0–4.3 17.8–18.1 6.0–6.2

into amber glass bottles, sealed and stored under refrigerated conditions below 4 C. Following sample homogenization and degassing in an ultrasonic bath, sample aliquots were transferred directly to HPLC vials and subsequently injected into the HPLC system. The quantification of colours used the chromatographic peak area and was performed by interpolation of the colour response on the standard solutions calibration line by means of the corresponding equation. For the specific case of E-104, the total area of any of the four isomers found was calculated by assuming equal response for all isomers because they have a common visible-absorbing structure. After quantification, dyestuff purity corrections for pure pigment were performed as required by the Colours in Food Regulations and Purity Directive 95/54/EC (1995).

3. Results and discussion The development of chemical and instrumental methods for the separation, identification and quantitative analysis of synthetic food colours has become extremely important for the food and beverages industry, academic and governmental institutions to assess the quality and safety of food products. In this study, an HPLC method minimizing pre-analytical sample treatment was developed (Table 2) and characterised (Table 3) for the determination of synthetic colours added to soft drinks containing juices and/or flavour extracts. Sample filtration with syringe filters (0.45 lm; cellulose acetate or nylon membranes) or centrifugation (3000 rpm for 10 min) was abandoned since there were significant losses of the colours (losses ranged from about 30–40 to 60–70%). A guard column was efficient in protecting the analytical column from contamination build-up for at least one hundred analyses after sample homogenization and degassing. It was found that use of an internal standard was not necessary since the method performance characteristics were already satisfactory

calibrating the analytical system (injection, separation and detection) with external standards of colours. The HPLC method uses inexpensive mobile phase components (methanol and aqueous ammonium acetate), which are compatible with atmospheric pressure ionisation techniques of mass spectrometric detection for confirmation identity purposes. Single wavelength visible detection was selected in the search for a compromise amongst the colour absorption maxima responses (430 nm for E-102, 414 nm for E-104, 476 nm for E-110, 516 nm for E-122, and 508 nm for E-124), while avoiding to programme wavelength with elution time. High throughput chromatography was possible with the use of an automatic injector (total procedure time per sample was 30 min). Nine soft drinks containing different levels of synthetic colours were selected to investigate the method behaviour with a variety of real samples in the search for potential co-eluting interferences. No interferences were found, and all samples contained levels below the maximum legal limit for synthetic colours in beverages. The presence of the different synthetic colours in each sample was confirmed by recording its visible absorption spectra and comparing it with that obtained previously in the standard solutions. Given the biological variability in the pigment content of the fruit and vegetable extracts used in these beverages, and variable losses during processing, manufacturers will usually use colour additives to ensure that the product will always provide a certain degree of colour uniformity. Therefore, it seems necessary the quality control of soft drinks by measuring the level of the synthetic colours added, taking into account the differences between regulations in the EU and the USA. This is important for specific groups of people, especially children, since hyperkinesis (a condition characterized by hyperactivity) has been shown to be related in several clinical studies (Berdonces, 2001; Ward, 1997) with the consumption of high levels of synthetic food colours.

M.S. Garc
ıa-Falc
on, J. Simal-G
andara / Food Control 16 (2005) 293–297

4. Conclusions The versatility of HPLC as an analytical tool makes it an ideal technique for analytical quality control and research and development laboratories in the food and beverage industry, especially when minimal clean-up needs to be performed. We propose a rapid and interference-free HPLC method developed for the quality control department of soft drink producers using synthetic food colours. The description of the method is accompanied by a detailed characterization of its performance. The determination of the synthetic colour additives in beverages at the mg/L level was run automatically and performed in a short time. The analytical procedures proved not to decompose the colours as indicated by the method precisions obtained with standards and soft drink samples. Detection limits for the additives in the beverages were found to be satisfactory and in all cases lower than fifty or one hundred times the restrictions given in EU Directives. The method is recommended for the determination of those additives in soft drinks. The results obtained with the samples confirm that the proposed method works well and is useful for a serious control or screening of the addition of synthetic colours in soft drinks. References ACS, American Chemical Society––Subcommittee on Environmental Analytical Chemistry (1980). Guidelines for data acquisition and data quality evaluation in environmental chemistry. Analytical Chemistry, 52(14), 1980, 2242–2249. Berdonces, J. L. (2001). Attention deficit and infantile hyperactivity. Revista de Enfermer
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