Keywords: Red Allura, Red Ponceau, synthetic food dyes, HPLC, soft drinks

Development of a Reversed-phase High Performance Liquid Chromatographic Method for Simultaneous Determination of Allura Red AC and Ponceau 4R in Soft Drinks MARIA MADALINA JURCOVAN, ELENA DIACU* University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, 1-7 Polizu Str., 011061, Bucharest, Romania Red Allura and Ponceau 4R are two of the most used red pigments in food and beverages. As they are synthetic organic substances that bears azo functional group in their molecule, which, generally, is incriminated as potential cause of malformations and tumours, these colors are perceived by consumers as harmful substances. For this reason, European regulators have limited the content of Red Alura and Ponceau 4R in beverages, and increased attention have been given to the development of methods of analysis for the determination of this kind of additives permitted in soft drinks. This work presents the possibility of simultaneous determination of Red Allura and Ponceau 4R in soft drinks by liquid chromatographic HPLC technique. The sample preparation is quite simple, without solvent extraction, requiring before chromatographic analysis only filtration and bringing the soft drink sample to a ph of 6.50. The values of the analytical performances criteria (detection limit, quantification limit, and recovery, repeatability, reproducibility and uncertainty measurement) have been determined, which shows that the described method is accurate and appropriate to be used. for the determination at legal requirements regarding the level of concentration (μg/l) of Red Alura and Ponceau 4R in soft drinks. Keywords: Red Allura, Red Ponceau, synthetic food dyes, HPLC, soft drinks In order to impart or to improve the visual aspect and flavour of food and beverages, manufacturers have used food coloring for centuries, being known that people associate certain colours with certain flavours [1, 2]. Among food pigments, the synthetic ones have certain properties that recommend them to be used in food and soft drinks, instead of the natural ones: less cost, a higher coloring power, a better stability on time, the offset colour loss due to exposure to light, temperature, air conditions, moisture and long storage, a vast range of new colours, a better distribution of color to the dyed food or drinks. Therefore, today colour additives are recognized as an important part of almost all processed foods and drinks; even if they are recognized as harmful for human health in case they are consumed excessively. Nowadays, effects of synthetic food pigments on health consumers are still insufficiently known, especially regarding the effect of azo-dyes food colors over time, and only long-term studies and experiments performed on a number of subjects could provide accurate data. Being able to improve overall sensory quality of foodstuff, synthetic colors are accepted to be used in the food industry as additives in a reduced number, only in a maximum allowable content and they are needed to be listed on the food labels. Therefore, all these colours were subject to certification and they are strictly controlled by European Union legislation and national laws [3-5]. Among synthetic colors, Red Allura and Ponceau 4 R are two of the most used red pigments in food and soft drinks. Red Allura AC (RA), known as E129, is according to the IUPAC name, disodium 6-hydroxy-5-((2-methoxy-5- methyl-4-sulfophenyl)azo)-2-naphthalenesulfonate and is a water soluble monoazo dye with good compatibility to other food constituents. This dye can be used in soft drinks either separately, or in binary combination with other red * email:elena_diacu@yahoo.co.uk synthetic dye, such as Ponceau 4R (P 4R), known as E124, whose IUPAC name is trisodium (8Z)-7-oxo-8-[(4- sulfonatonaphthalen-1-yl)hydrazinylidene]naphthalene- 1,3-disulfonate. This last dye is a suspected harmful substance, but permitted in Europe [3]. A Fig. 1. Chemical structure of Red Allura (A) and Ponceau 4R (B) In the context of increase of consumer concern regarding the global consumption of synthetic dyes, considerable interest is given to development of such analytical methods, able to separate and quantitatively determine these dyes, whose substances show, in most cases, quite similar physicochemical properties. In the literature, there is a large number of references regarding the analytical methods for synthetic food dyes determination in different food matrices, either separately, or in combination with other additives. Here one can mention few of them: spectral methods [8-11], capillary electrophoresis [12-14], electrochemical methods [15-17] and the most numerous, chromatographic methods [18-24]. Among all mentioned methods used for the analysis of food dyes, HPLC methods are by far the most promising technique regarding the accuracy, precision, repeatability and detection limit. This paper describes the development and in-housevalidation of a reversed-phase high-performance liquid B REV. CHIM. (Bucharest) 65 No. 2 2014 http://www.revistadechimie.ro 137

chromatography method for the simultaneous determination of two red synthetic dyes, Red Allura and Ponceau 4R (fig 1). Both these food colors are approved by European regulations in the maximum allowable concentration of 100 mg/l, used either individually, or in binary combination [3-6]. It is worth mentioning that Ponceau 4R, due to the carcinogenic suspicion is not allowed in USA [7]. Experimental part Chemicals and reagents Allura Red AC (purity 99%) and Ponceau 4R (purity 98%), both analytical standards, were purchased from Germany. NaOH and acetonitrile were Chromasolv purity from Germany. All aqueous solutions were prepared using ultrapure water obtained through a Millipore Milli-Q system. Mixed stock solution of the standard containing 1000 mg/l was prepared by weighing 0.1010 g of each colour to 100 ml and preservation at 4 8 o C temperature (valid for 3 months). The mixed working solutions of calibration standards were prepared on the day of determination, by taking the appropriate volumes of stock solution after its equilibration to room temperature and then diluted in ultrapure water. Apparatus The determination of RA and P 4R content was performed with an Agilent Liquid chromatograph-series 1100, equipped with DAD detection, on a column with stationary phase as Nucleosyl 5 μ MOS C 8 on an analytical column length of 250 x 4.60 mm. The flow rate was set up at 1.0 ml/min and the injection volume was 10 μl. The detection was achieved at 520 ± 2 nm, as the optimum wavelength for monitoring both red solutes. Peak identification was done by the retention times of the samples with food color standards and the quantification by peak areas. Samples Preparation Soft drink samples were filtered in vacuum on a microfiltration membrane (pore size of 0.45 μm). If the sample was carbonated, it was degassed by ultrasonication for 10 min. The ph sample was adjusted at 6.50 values, with a NaOH solution 10 %, using a ph-meter equipped with a combined glass electrode. The chromatographic separation of RA and P 4R was achieved through a gradient-elution system consists of two components: component A was ultrapure water, and B, a mixture of acetonitrile and ultrapure water, in 1:4 volumes. Results and discussions The gradient-elution system used for mobile phase was programmed in four steps: i) 0-1 min isocratic elution at 5% A:95% B; ii) 1-15 min linear gradient elution from 5% A:95% B to 45% A:55% B; iii) 15-16 min linear gradient to recover initial conditions of 5% A:95% B and iv) is the conditioning step at 5% A:95% B for 15 min. Experiments were performed on either standard solutions or on samples fortified soft drinks with RA and P 4R, and a significant difference between retention times of the two dyes was recorded on the chromatogram (5.7 ± 0.2 min for P 4R and 11.4 ± 0.2 min for RA), meaning a proper identification, as can be seen in figure 2. The calibration curves for RA and RP were obtained by fortification of a juice sample with both colorants, and were drawn based on the peak areas of different working calibration standard solutions. From figure 3, A and B, it Fig. 2. Typical chromatogram for a fortified juice sample with RA and P 4R 138 http://www.revistadechimie.ro REV. CHIM. (Bucharest) 65 No.2 2014

Fig. 3. Calibration curves for Red Allura (A) and Ponceau 4R(B) B can be seen that there is a very good linearity of the dependency of the analytical signal with both colorants concentration in the range of 10-100 mg/l. In the development of analytical methods care should be taken for interferences. Study of possible interferences (other food colorants, or other additives usually present in beverages, such as preservatives and sweeteners) was realized, and no significant interference with the method here proposed was not observed. In addition, the selectivity of the developed HPLC method was proven by investigation of the spectral purity of chromatogram using a fortified beverage sample, and good values were obtained for the purity factors of both colorants, respectively 999.929 for P 4R, and 999.920 for RA. The HPLC method was then used to simultaneously determine the pairs of colorants RA and P 4R in ten real soft drink samples, with results that showed that the content did not exceed the legal limits, respectively 100 mg/l. Validation of the method Next step of this study was the validation of HPLC method for the determination of RA and P 4R in beverage samples. The following analytical performance criteria: linearity of calibration curve, detection limit (LOD), quantification limit (LOQ), recovery, repeatability, reproducibility and measurement uncertainty, (U) were determined in accordance with the references [25-27] for in-house-validation procedure. Detection limit, LOD, and quantification limit, LOQ For the developed HPLC method, LOD was considered as the colorant concentration giving an analytical signal equal to the blank plus 3 times the standard deviation s r of the blank. The equipment used here offers excellent possibility to establish the LOD, the value being obtained directly from the chromatographic software by analyzing four different concentrations of P 4R and RA standard solutions (100-1000 μg/l). For each colorant, LOQ found was 0.1 mg/l. Consequently, the LOQ is 0.3 mg/l, considering LOQ = 3 LOD. Calibration curve linearity Linearity of calibration curve for HPLC method was checked on the basis of the chromatographic peak areas using red juice samples fortified with various concentrations of mixed standard solutions RA and P 4R in a dynamic concentration range 0-100 mg/l (as can be in red beverages). The calibration curve was linear for all the ranges of interest, with a good regression coefficient, of 0.99996 for RA and 0.99991 for P 4R, respectively [fig. 3]. Recovery % Achievement of the percent recovery for RA and P 4R determination from beverages was carried out considering the maximum accepted level by law for these dyes, using a set of 12 spiked soft drinks samples for each colorant, at two concentration levels (50 and 100 mg/l), 6 samples for each level. An unfortified sample of soft drink red, was analyzed also here, its content being considered 0.0 mg/l, because no analytical signal was generated. The recovery percentage obtained is between 93-102% for P 4R and 90-103% for RA, which shows that the sample matrix does not affect the determination of both colorants from soft drinks. Repeatability, r Repeatability r is the measure of agreement between results obtained under repeatability conditions. This parameter was obtained under the requirements (i.e., same method, same sample, work done by one operator, in the same laboratory, with the same apparatus, and with short time interval) using the formula according to which r = 2.8 x s r. (2.8 is the number derived from Gaussian distribution, according with ISO 5725). Standard deviation s r was calculated for a number of data n 10, with 95% confidence. The obtained results for s r and r are displayed in table 1, where it can be seen that the values for s r are REV. CHIM. (Bucharest) 65 No. 2 2014 http://www.revistadechimie.ro 139

Table 1 DETERMINATION OF REPEATABILITY FOR RED ALLURA AND PONCEAU 4R DETERMINATION Table 2 RDS VALUES FOR RED ALLURA AND PONCEAU 4R RECOVERY Table 3 RED ALLURA AND PONCEAU 4R CONTENT IN DIFFERENT KINDS OF SOFT DRINKS less than 0.14, and for r are between 0.18 and 0.38, which shows that repeatability of analytical data for both colorants at all fortification levels is very good. Reproducibility, R The reproducibility R is a measure of the dispersion of results obtained with the same method but under different conditions, such as: work done by different persons, with different equipment, laboratories, and times. R was calculated after performing two sets of experiments, in two different days, by two persons, with different chromatographs, for both colorants, at two fortification levels (50 and 100 mg/l) with formula R = 2.8 x s R, where s R is the standard deviation of these results calculated for a number of data n 8 (with 95% confidence). For both fortification levels of P 4R and RA determination, the results for R are less than 1 (s R ranges between 0.05 and 0.21 and from 0.14 to 0.6 for R), values that gives good reproducibility to the proposed method. Measurement uncertainty, U In order to evaluate the measurement uncertainty in the determination of RA and P 4R by HPLC in soft drinks, according with in-house-validation procedure [25-27], the following uncertainty components have been identified and evaluated: uncertainty on standard solutions on the recovery, on the reproducibility, and on HPLC instrument. Uncertainty on standard solutions In order to obtain the associated uncertainty on the preparation of standard solutions, two components should be considered: the uncertainty on the preparation of the 140 http://www.revistadechimie.ro stock solution and the uncertainty on the preparation of the working solutions itself. Taking into account all combined uncertainties involved here, respectively: due to the purity of the two dyes powders (RA 99% and P 4R 98%), due to the weighing of analytical balance (calibration certificate of Mettler Toledo AT 261), to the use of a 100 ml, 10 ml volumetric flasks Class A and of micropipette (100-1000 μl), due to variation of the temperature, the value for the associated uncertainty on standard solutions u standards was found as 0.36 mg/l. The uncertainty due to the recovery percentage To obtain the uncertainty due to the recovery percentage, the relative standard deviations, RSD P 4R and RSD RA were calculated separately for the two dyes and were integrated for 8 series of analyses within 1 day. Table 2 presents the analytical data for both standard deviation (SD) and relative standard deviation (RSD) of recovery percentage. Therefore, the individual values obtained for RSD P 4R = 0.098% and RSD RA = 0.157%, are useful for the evaluation of associated uncertainty related to the recovery in simultaneous determination of RA and P 4R, considered as their sum, respectively 0.258. The uncertainty due to the to the reproducibility The uncertainty related to reproducibility is achieved from the sum of the averages RSD values of eight time analyses of a fortified soft drink sample at 100 mg/l level of P 4R and 50 mg/l RA, performed in two different days (RSD P 4R = 0.128%, RSD RA = 0.175%. The Associated uncertainty related to the reproducibility in this case is 0.303%. REV. CHIM. (Bucharest) 65 No.2 2014

Associated uncertainty for HPLC instrument The associated uncertainty for liquid chromatograph, u HPLC can be expressed as average between the standard deviation for P 4R and RA, with a value of 0.2925 mg/l. Measurement uncertainty-u calculation In the end, the measurement uncertainty U was calculated as expanded uncertainty by the formula U = u k, where k, the coverage factor, has the value 2, taking into account a normal distribution of the experimental data. For the concentration of 100mg/L for both colorants, U = 2.62 mg/l. This U-value which defines the interval between RA and RP content and may be acceptable to be included in soft drinks and encompasses the conditions of food safety and of food authenticity detection. Developed HPLC method was applied in the determination of RA and RP either separately and simultaneously in 10 real soft drinks samples commercially available, and the obtained values exhibited in table 3 show that the content of the two studied dyes did not exceed the legal limit. Conclusions An HPLC method was developed for determination of red synthetic colors Allura Red AC and Ponceau 4R in soft drinks. The described method was in-house validated, and the analytical performance criteria and the critical parameters on measurement uncertainty were determined. The method has proven its ability to obtain reliable results, precise and accurate, with a satisfactory limit of quantification of mg/l, in a relatively short time (14 min) and with a slight sample preparation process for analysis. All these recommend the method for use in monitoring the legal limits of the content of synthetic dyes, Ponceau 4R and Allura Red in soft drinks and in determining the authenticity of the natural ones. References 1. GHORPADE, V.M.,. DESHPANDE, S.S.,. SALUNKHE,D.K., in: J.A. Maga, A.T. Tu (Editors), Food Additive Toxicology, Marcel Dekker, New York, 1995. 2. ALI, M.A.,.BASHIER,S.A., Food Additives & Contaminants 2006, 1, p.1 3. *** Council Directive 94/36, European Parliament, Official Journal of the European Communities, 1994, No. L 273 4. *** Council Directive 95/45/EC, European Parliament, Official Journal of the European Communities, 1995, No. L226 5. *** Commission Regulation (EU) No. 1129, Official Journal of the European Union,,2011, L 295. 6. *** ORDIN Nr. 438/295/2002, Monitorul Oficial R.A., nr. 722/2002 7. *** Food and Drug Administration Compliance Program Guidance Manual, 2011, p.10 8. CAPITAN-VALLVEY, L.F., FERNANDEZ,M.D., DE ORBE, I., AVIDAD,R.,Talanta, 1998, 47, 4, p.861 9. DINÇ, E., BAYDAN, E., KANBUR, M., ONUR, F., Talanta, 58, 3, 2002, p.579. 10. KISELEVA, M. G., PIMENOVA, V. V., ELLER, K. I., J.Anal. Chem., 58, 7, 2003, p. 685. 11. POURREZA, N., RASTEGARZADEH, S., LARKI, A., Food Chemistry, 126, 3, 2011, p.1465. 12. WATANABE, T., TERABE, S., Journal of Chromatography A, 880, 1 2, 2, 2000, p 311. 13. FRAZIER, R.A., Electrophoresis, 2001, 22, p.4197. 14. FRAZIER, R.A., PAPADOPOULOU, A., Electrophoresis, 24, 22-23, 2003, p. 4095. 15. DIACU, E., UNGUREANU, E.M., ENE, C. P., IVANOV, A.A., Rev. Chim. (Bucharest), 62, no.11, 2011, p. 1085. 16. DIACU, E., UNGUREANU, E.M., JURCOVAN, M.M., ENE, C. P., IVANOV, A.A., Rev. Chim. (Bucharest), 63, no. 6, 2012, p. 580. 17. MEDEIROS R. A., LOURENCAO B. C., ROCHA-FILHO, R. C., ORLANDO, F-F., Talanta 99, 2012, p, 883. 18. CORNET, V., GOVAERT, Y., MOENS, G., JORIS V. L., DEGROODT, J.- M., J. Agric. Food Chem., 54, 3, 2006, p 639. 19. ERTAS, E., OZER, H., ALASALVAR, C., Food Chemistry, 2007, 105, p. 756. 20. KUCHARSKA, M., Talanta, 80, 3, 2010, p 1045 1051 21. MINIOTIA, K.S., SAKELLARIOUB, C.F., THOMAIDIS, N.S., Anal. Chim. Acta, 583, 1, 2007, p.103. 22. DIACU, E., ENE, C. P., Rev Chim. (Bucharest), 60, no. 8, 2009, p. 745. 23.MOLDOVEANU, S., DAVID, V., Essentials in Modern HPLC Separations, R.J. Reynolds Tobacco Co., Winston-Salem, NC, 2013. 24. YOSHIOKA, N., ICHIHASHI, K., Talanta,74, 5, 15, 2008, p.1408. 25. *** ISO/IEC 17025:1999. General Requirements for the Competence of Calibration and Testing Laboratories. ISO, Geneva (1999) EURACHEM, Quantifying Uncertainty in Analytical Measurement. Laboratory of the Government Chemist, London ISBN 0-948926-08-2 (1995). 26. DIACU, E., ENE, C. P., Rev Chim. (Bucharest), 61, no 12, 2010, p. 1177 27. *** ISO GUM, Guide to the Expression of Uncertainty in Measurement, 2nd edn. (1995), with Supplement 1, Evaluation of measurement data, JCGM 101:2008, Organization for Standardization, Geneva, Switzerland (2008). ttp://www.bipm.org/en/publications/ guides Manuscript received: 1.07.2013 REV. CHIM. (Bucharest) 65 No. 2 2014 http://www.revistadechimie.ro 141