Food Chemistry 158 (2014) 521–526
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Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
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Quantitative determination of carmine in foods by high-performance liquid chromatography Ho-Soo Lim, Jae-Chon Choi, Sung-Bong Song, Meehye Kim ⇑ Food Additives and Packages Division, Ministry of Food and Drug Safety, Osong Health Technology Administration Complex, 187 Osong, Chungcheongbuk-do 363-951, Republic of Korea
a r t i c l e
i n f o
Article history: Received 11 November 2013 Received in revised form 29 January 2014 Accepted 19 February 2014 Available online 3 March 2014 Keywords: Carmine Carminic acid Cochineal extract HPLC
a b s t r a c t A simple and rapid method has been developed and validated for the determination of carmine in foods. Samples were homogenised and extracted with 0.05 M NaOH, followed by centrifugation. The resulting solution was filtered and injected to HPLC. Carmine was separated by HPLC using an NovaPak C18 column coupled to a photodiode array detector. The contents of carmine were finally quantified using corresponding calibration curves over ranges of 1.0–100 lg ml 1, with good correlation coefficients (r2 = 0.9999). The recoveries of carmine from foods spiked at levels of 10, 50, and 100 lg g 1 which ranged from 90.4% to 96.2% with relative standard deviations between 2.8% and 6.8%. Limit of detection and limit of quantification of carmine were 0.4 and 1.0 lg ml 1, respectively. This method was found to be useful to distinguish carmine from carminic acid, a major component of cochineal extract. The method has been successfully applied to various foods. Ó 2014 Elsevier Ltd. All rights reserved.
Contents 1. 2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Chemicals and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Preparation of standard solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Sample collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Preparation of samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. High performance liquid chromatography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Method validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Method development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Liquid chromatographic conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. Solvents for the extraction of carmine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Method performance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Linearity, limit of detection and limit of quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Accuracy and precision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Analytical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
⇑ Corresponding author. Tel.: +82 043 719 4351; fax: +82 043 719 4350. E-mail address: [email protected] (M. Kim). http://dx.doi.org/10.1016/j.foodchem.2014.02.122 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
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1. Introduction Synthetic food dyes are food additives that can impart colour when added to foods. These substances must be preapproved by the Korea Ministry of Food and Drug Safety and listed in Korea Food Additives Code (MFDS, 2013a) in order to be legally used in food products retailed in Korea. The MFDS lists 18 permitted synthetic colour additives in synthetic substances part of the Code. The use of food colours is strictly controlled by laws and regulations (MFDS, 2013b). Carmine, one of the synthetic food dyes, is authorised for use in Korea, USA (FDA, 2012), and European Union (European Commission, 1994) except for Japan. The acceptable daily intake (ADI) value of carmine, which is formulated by Joint FAO/WHO Expert Committee on Food Additives, is 5 mg kg 1 based on weight (JECFA, 2001). As the Committee’s conclusion, carmine in foods may initiate or provoke allergic reactions in some individuals. Carmine is a lake pigment used in many different products such as juice, candies, and confectioneries. It is usually produced by precipitating carminic acid in aluminium hydroxide in the form of aluminium or aluminium-calcium salt. Carminic acid is obtained from dried bodies of the female insect Cocus cacti L., which lives on various cactus plants (Feller, 1986). Carminic acid also is a major component of cochineal extract, which is used as a natural food colour in Korea. Carmine and cochineal extract permitted in Korea should not be used for the food items as followings: natural food (meat, seafood (whale meat included), fruits, vegetables, marine algae, bean and their simply processed food (peeled or cut)); tea and coffee; red pepper powder or shredded red pepper; Kimchi; Gochujang (fermented red pepper pastes) or seasoned Gochujang (seasoned soybean paste with red peppers); vinegars; spice products (limited to products containing red pepper or red pepper powder). In addition, the permissible level is not established in Korea Food Additive Code. Carmine might be present as a non-permitted colour additive in food products imported to Korea and illegally added to food products by manufacturers to improve appearance and colour. Cochineal extracts also may be marked on food products where carmine was actually used. Accordingly, monitoring of carmine in foods is necessary to ensure food safety and consumer confidence. Most research has been related to the analysis of carminic acid, a major component of cochineal extract, using high-performance liquid chromatography (Ishikawa, Shigeoka, Nagashima, Takahashi, & Kamimura, 2003; Lancaster & Lawrence, 1996; Merino, Edberg, & Tidriks, 1997), capillary electrophoresis (Liu et al. 1995), and spectrophotometric methods (Tripathi, Khanna, & Das, 2004). Recently, a method used to determine carmine itself in foodstuffs by stripping voltammetry was reported (Alghamdi, Alshammery, & Ahdalla, 2009). This method had an advantage that is suitable for the analysis of very diluted samples and decrease the real time of determination. However, the proposed method was applied to determine carmine only in spiked commercial ice cream and soft drinks. This electrochemical procedure is known to be influenced by factors such as temperature, pH, and organic substances (Kalvoda & Kopanica, 1989). The stripping voltammetry is also not widely used in the analysis of food additives. To the best of our knowledge, no chromatographic method which can separate carmine from carminic acid in foods, has been reported in the literature for the determination of this dye by highperformance liquid chromatography. This is due to insolubility in water and organic solvents. For this reason, carmine cannot be effectively extracted from food samples with usual liquid extraction. Thus, the development of a specific method for the quantification of carmine in foods is required. In the study described in this paper, we have developed a simple and quantitative method for the analysis of carmine in food products by high-performance liquid chromatography coupled to
a diode array detector. The new method separates carmine from carminic acid in food samples. The procedure is quantitatively characterised and linearity, accuracy, limits of detection and quantification are satisfactory. 2. Materials and methods 2.1. Chemicals and reagents Carmine and carminic acid used as standards were purchased from Sigma–Aldrich (St. Louis, MO, USA). All other chemicals used in extraction and preparation of mobile phase, such as sodium hydroxide, sodium phosphate dibasic, and sodium phosphate monobasic were of analytical reagent grade and also supplied by Sigma–Aldrich. Water for all applications in our study was obtained from an Milli-Q ultra-pure water system (Millipore, Bedford, MA, USA) with resistivity equal to or higher than 18.2 MO cm. 2.2. Preparation of standard solutions The stock solution (1000 lg ml 1) of carmine was prepared by transferring 100 mg of carmine into a 100 ml beaker and adding 0.05 M NaOH solution to give a final volume of 100 ml. Calibration standard solutions at serial concentrations of carmine were obtained by mixing subsequent dilution (1–100 lg ml 1) with water. 2.3. Sample collection A total of 124 samples were purchased from retail stores in big cities including Seoul, Incheon, Daejeon, Gwangju, Daegu and Busan in South Korea. The samples were categorised into 16 food types. Processed milk (4), confectionery (50), functional food (20), processed fruit vegetable product (4), tea (1), sugaring product (5), meat product (4), fish meat product (7), processed cheese (4), beverage (10), fermented soybean paste (2), salted seafood (5), seasoned food (4), Kimchi (2), chocolate (1) and coffee (1) in commercial products were purchased after checking the labels and colours written on the products. All samples were stored at 4 °C. 2.4. Preparation of samples A 5.0 g of ground food samples was transferred into a 100 ml beaker and 0.05 M NaOH solution was added to give a final volume of 50 ml. After it was mixed by a homogenizer for 2 min, the sample was shaken mechanically for 10 min with a shaking rate of 300 min 1. The extract was centrifuged for 5 min at 5000 rpm. The 10 ml supernatant was then filtered with 0.45 lm membrane filter for injection into HPLC. 2.5. High performance liquid chromatography HPLC analysis was performed on an Agilent HPLC 1200 series (Santa Clara, CA, USA) coupled to a photodiode array detector. The LC system consisted of degasser, binary pump, autosampler, and column oven. An NovaPak C18 column (150 3.9 mm, 5 lm) purchased from Waters Corporation (Milford, MA, USA) was used for chromatographic separation. All separations were carried out isocratically at room temperature with a mobile phase consisting of methanol-phosphate buffer (pH 6.0) at ratios of 15:85 (v/v). The flow-rate was maintained at 0.8 ml min 1 and a 20 ll sample volume was injected into HPLC. Carmine, eluted from the column, was monitored by photodiode array detector set at 281 nm. The absorption spectra of carmine
H.-S. Lim et al. / Food Chemistry 158 (2014) 521–526
were recorded between 200 and 500 nm. Peak identification was performed by comparing the retention time and absorption spectra of samples with the standard solution. Separation of carmine from carminic acid, a major component of cochineal extracts, was carried out in the same conditions as above. 2.6. Method validation The method was validated for linearity, accuracy (recovery), precision, limit of detection (LOD) and limit of quantification (LOQ) using carmine standard solutions and control samples. The linearity was determined with working standard solutions of carmine over ranges of 1.0–100 lg ml 1 (1, 10, 20, 40, 60, 80, 100 lg ml 1). Integrated peak areas of the absorbing wavelength of carmine at 281 nm were used to construct seven-point calibration curves, which were applied for quantification of carmine. The recoveries and precision of the method were determined using candy, yogurt, and juice samples fortified with carmine at three different concentration levels (10, 50 and 100 lg g 1). For each concentration level, triplicates were prepared and injected once. The recovery of carmine was obtained by calculating the peak area of the chromatogram. The precision expressed as percent relative standard deviation (RSD%) was also determined for carmine using spiked candy, yogurt, and juice samples. The intra-day precision was assessed by performing the five repetitions during a single day and inter-day precision by three repetitions per day over three different days. The LOD was evaluated as the concentration giving a signal equal to three times of noise (S/N = 3) and the LOQ was determined as the concentration giving a signal equal to ten times of noise (S/ N = 10). 3. Results and discussion 3.1. Method development 3.1.1. Liquid chromatographic conditions A good separation of carmine and carminic acid can be achieved with 15% methanol and phosphate buffer (pH 6.0) as the mobile phase system under the isocratic elution condition described above. Since carmine has the similar absorption spectrum of carminic acid at the range of 200–500 nm, several C18 columns were tested for the separation of carmine and carminic acid. Using the conventional analytical column (NovaPak C18, Waters, 150 3.9 mm, 5 lm) at flow rate of 0.8 ml min 1, the separation of carmine and carminic acid was accomplished (Fig. 1). The analysis was completed within
Fig. 1. Chromatogram of a standard solution (100 lg ml carminic acid; (2) carmine.
1
523
7 min. Carmine was monitored at 281 nm, nearest to the maximum absorption by a photodiode array detector. The absorbance spectra for carmine and carminic acid were similar. Typical separation of a mixed standard solution show retention time of 4.5 and 5.6 min for carminic acid and carmine, respectively. Plots of solvent combination rate vs. retention time were made for testing the most suitable mobile phase. 15% methanol was the most suitable phase for application to carmine as shown in Fig. 2. The high-performance liquid chromatography with diode array detector and C18 column has been successfully applied for determining aluminium lake dyes in food matrix (Alves, Brum, Branco de Andrade, & Pereira Netto, 2008; Ma, Luo, Chen, Su, & Yao, 2006; Yang & Shao, 2011; Yoshioka & Ichihashi 2008). However, no research has been reported in the literature for the determination of carmine by HPLC. 3.1.2. Solvents for the extraction of carmine Carmine is an aluminium lake or aluminium-calcium lake dye. Although carmine is insoluble in water and organic solvents, it can be dissolved by strong alkaline or acid solutions such as sodium hydroxide and hydrochloric acid. As hydrochloric acid is used, carminic acid is released from carmine (Marshall & Horobin 1974). Carmine also could be dissolved by dimethyl sulfoxide solution, an aprotic solvent which dissolves both polar and nonpolar compounds. However, the separation of carmine and carminic acid was not achieved by this solvent. Therefore, sodium hydroxide solution was selected for the extraction of carmine in food samples. It is also supported by the reports that aluminium lake dyes in foods and drugs were efficiently extracted with 0.02 M sodium hydroxide solution (Ishikawa et al., 2003; Yang & Shao, 2011). The effects of different sodium hydroxide concentrations of carmine on peak area were investigated using the standard solution. The concentration of NaOH solution was studied at 0.01, 0.02, 0.03, 0.04, 0.05, and 0.06 M (Fig. 3). The peak area of carmine increased when NaOH concentration increased to 0.05 M. On the other hand, when the concentration of NaOH reached 0.06 M, the area of carmine significantly decreased by 16%, which might have occurred as a result of the loss of carmine due to hydrolysis with the solution of high pH value. Optimum area value was obtained with concentration of 0.05 M. 3.2. Method performance 3.2.1. Linearity, limit of detection and limit of quantification The linearity of the method was determined by the injection of various standard concentrations. The method was linear in the
) separated by liquid chromatography with detection by a photodiode array set at 281 nm. Peaks identity: (1)
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Fig. 2. Plots of retention time and peak area of carmine on C18 column vs. methanol content (%) for optimisation of mobile phase.
Fig. 3. Plots of peak area of carmine vs. NaOH concentration for optimisation of extraction solvents.
range of concentrations from 1.0 to 100 lg ml 1 (r = 0.9999). This result indicates that the standard curve could be effectively applied to quantify carmine in food samples. LOD was evaluated as the mass giving a signal equal to three times the noise (S/N = 3); LOQ was determined as the mass giving a signal equal to ten times that of the blank (S/N = 10). The calculated LOD and LOQ using standard solutions were 0.4 and 1.0 lg ml 1, respectively. LOD and LOQ conform with AOAC guideline (AOAC, 2013). The analysis of carmine via high-performance liquid chromatography can be useful in quality control during and after production of food products containing carmine. 3.2.2. Accuracy and precision Recovery was calculated as the percentage of foods recovered from three replicate determinations of three different solutions
containing 10, 50, and 100 lg ml 1. Fig. 4 shows a chromatogram of carmine extracted from candy, yogurt, and juice filtered and then chromatographed using the methanol-phosphate buffer (pH 6.0) mobile phase. Recoveries ranged from 90.8 ± 2.7% to 96.2 ± 4.5% in candy, yogurt, and juice as shown in Table 1, demonstrating that the method is accurate within the desired range. The precision, evaluated as the repeatability of the method, was studied by calculating the relative standard deviation (RSD) for three determinations of the concentrations 10, 50, and 100 lg ml 1 performed on the same day and under the same experimental conditions. The RSD values obtained ranged from 2.8% to 6.7% (Table 1). The inter-day precision was assessed by calculating the RSD for determining three levels of concentration on five different days. The RSD values obtained were below 7% for three samples. The accuracy and precision of the method conform with AOAC guideline.
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Fig. 4. Chromatograms of carmine recovered from candy, yogurt and juice spiked at 100 lg ml
3.3. Analytical applications To assess the reliability of the proposed method described above, it was applied to determine carmine in a variety of commercially available food products retailed in Korea. One hundred-twenty-four samples of food products retailed in Korea were analysed using this method. Quantification of carmine was carried out using standard calibration curves and confirmed by comparing absorption spectra and retention time of the samples with carmine standard. The concentrations of carmine in food samples are shown in Table 2. It was detected from total of thirty food products and its contents ranged from N.D. to 973.9 ± 2.8 lg g 1.
1
, respectively: (A) candy; (B) yogurt; (C) juice.
The average content of carmine was the highest (973.9 ± 2.8 lg g 1) in the imported beverage. The carmine contents in imported confectionery and sugaring product were 865.0 ± 4.7 and 754.7 ± 2.8 lg g 1, respectively. In EU, carmine usage in some foods, such as red marbled cheese, jam, and chorizo sausage are specified from 100 to 200 mg kg 1 (European Commission, 1994). Usage in a wide variety of foods in CODEX is set from 50 to 500 mg kg 1 except for fresh eggs which are set up as good manufacture practise (CODEX Alimentarius, 2013). However, the carmine usage is not specified in Korea. For this reason, carmine levels in foodstuffs retailed in Korea may be higher than the allowed levels established by EU and CODEX.
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Table 1 Recoveries of carmine from candy, yogurt and juice sample spiked at three different concentrations (n = 3). Sample
Spiked level (lg ml 1)
Recovery (%)
Candy
10 50 100
yogurt
Beverage
Intra-day
Inter-day
90.8 ± 2.7 94.6 ± 1.9 95.2 ± 2.5
3.1 2.8 4.6
5.2 3.6 4.7
10 50 100
90.4 ± 3.4 95.9 ± 2.4 95.2 ± 2.1
5.1 6.7 5.3
4.3 5.6 4.2
This research was supported by a Grant (12161-MFDS-113) from Ministry of Food and Drug Safety in 2012.
10 50 100
93.8 ± 2.7 95.1 ± 3.8 96.2 ± 4.5
4.3 4.2 3.4
6.8 5.5 4.4
References
Table 2 Concentration (lg g
a
Precision (RSD%)
method that can be applied to various food products such as beverages, confectionery, and candies. This study is the first report on HPLC method to differentiate carmine and carminic acid in foods. Thus, this method is applicable for the effective surveillance of carmine in food products.
1
) obtained for the samples analysed (n = 3).
Food type
Number of sample
Detected sample
Carmine level Range ± SD
Processed milk Confectionery Functional food Processed fruit vegetable product Tea Sugaring product Meat product Fish meat product Processed cheese Beverage Fermented soybean paste Salted seafood Seasoned food Kimchi Chocolate Coffee
4 50 20 4
1 22 – 2
NDa–46.7 ± 0.7 ND–865.0 ± 4.7 ND ND–149.9 ± 4.0
1 5 4 7 4 10 2 5 4 2 1 1
– 1 – 1 1 2 – – – – – –
ND ND–754.7 ± 2.8 ND ND–28.9 ± 0.3 ND–19.0 ± 0.1 ND–973.9 ± 2.8 ND ND ND ND ND ND
ND: not detected means below limits of quantification (1.0 lg g
1
).
Twenty-eight samples among thirty detected samples were imported food products. All detected samples were also under the regulation of Korea Food Additives Code. Carmine was not detected in functional food, tea, meat product, fermented soybean paste, salted seafood, seasoned food, Kimchi, chocolate, and coffee. The analytical methods of carmine as carminic acid by hydrochloric acid hydrolysis have a disadvantage that carmine or carminic acid in foods could not be distinguished (Carvalho & Collins 1997; Merino et al., 1997). However, as the proposed method in this study was applied to samples, retention time and spectrum of carmine were different from carminic acid, which was detected in a sample. Carmine and carminic acid didn’t occur together in all examined foods. Carmine and carminic acid are not used at the same time due to the similar colour (from red to dark red colours). 4. Conclusions A rapid and simple analytical method suitable for carmine has been successfully developed and validated in our study. The accuracy and precision obtained were satisfactory for carmine after optimisation of experimental conditions. This method is useful for distinguishing carmine from carminic acid, which is a major component of cochineal extract. In addition, it is a quantitative
Acknowledgements
Alghamdi, A. H., Alshammery, H. M., & Ahdalla, M. A. (2009). Determination of carmine food dye (E120) in foodstuffs by stripping voltammetry. Journal of the Association of Analytical Chemists International, 92, 1454–1459. Alves, S. P., Brum, D. M., Branco de Andrade, E. C., & Pereira Netto, A. D. (2008). Determination of synthetic dyes in selected foodstuffs by high performance liquid chromatography with UV-DAD detection. Food Chemistry, 107, 489–496. AOAC (Association of Official Analytical Chemistry) (2013). Guidelines for single laboratory validation of chemical methods for dietary supplements and botanicals. Accessed 23.10.13. Carvalho, P. R. N., & Collins, C. H. (1997). HPLC determination of carminic acid in foodstuffs and beverages using diode array and fluorescence detection. Chromatographia, 45, 63–66. CODEX Alimentarius. Codex general standard for food additives. CODEX STAN 1921995. (2013). Accessed 24.10.13. European Commission. (1994). Commission Directive 94/36/EC on colours for use in foodstuffs. Official Journal of the European Community, L237, 13–29. FDA (U.S. Food and Drug Administration) (2012). Color Additives Listed for Use in Food (From the Code of Federal Regulations). Accessed 20.10.13. Feller, L. (1986). Artists’ pigments, a handbook of their history and characteristics (Vol. 1). London, UK: Cambridge University Press. Ishikawa, F., Shigeoka, S., Nagashima, M., Takahashi, M., & Kamimura, H. (2003). Simultaneous determination of eleven dyes and their aluminum lakes in drugs. Journal of the Association of Analytical Chemists International, 86, 215–221. JECFA (Joint FAO/WHO Expert Committee on Food Additives) (2001). Evaluation of certain food additives and contaminants, WHO Technical Report Series, No. 901. Geneva: World Health, Organization [pp. 10–12]. Kalvoda, R., & Kopanica, M. (1989). Adsorptive stripping voltammetry trace analysis. Pure and Applied Chemistry, 61(1), 97–112. Korea Ministry of Food and Drug Safety (MFDS). (2013a). Korea food additives code. Seoul: Korea Food Industry Association. Korea Ministry of Food and Drug Safety (MFDS). (2013b). Korea food sanitation act. Seoul: Korea Food Industry Association. Lancaster, F. E., & Lawrence, J. F. (1996). High-performance liquid chromatographic separation of carminic acid, alpha- and beta-bixin, and alpha- and betanorbixin, and the determination of carminic acid in foods. Journal of Chromatography A, 732, 394–398. Liu, H., Zhu, T., Zhang, Y., Qi, S., Huang, A., & Sun, Y. (1995). Determination of synthetic colourant food additives by capillary zone electrophoresis. Journal of Chromatography A, 718, 448–453. Ma, M., Luo, X., Chen, B., Su, S., & Yao, S. (2006). Simultaneous determination of water-soluble and fat-soluble synthetic colorants in foodstuff by highperformance liquid chromatography-diode array detection-electrospray mass spectrometry. Journal of Chromatography A, 1103, 170–176. Marshall, P. N., & Horobin, R. W. (1974). A simple assay procedure for carmine and carminic acid samples. Stain Technology, 49, 19–28. Merino, L., Edberg, U., & Tidriks, H. (1997). Development and validation of a quantitative method for determination of carmine (E120) in foodstuffs by liquid chromatography: NMKL collaborative study. Journal of the Association of Analytical Chemists International, 80, 1044–1051. Tripathi, M., Khanna, S. K., & Das, M. (2004). A novel method for the determination of synthetic colors in ice cream samples. Journal of the Association of Analytical Chemists International, 87, 657–663. Yang, Y., & Shao, B. (2011). Simultaneous determination of five aluminum lake dyes in chewing gum by HPLC with photodiode array detection. Food Additives and Contaminants, 28, 1159–1167. Yoshioka, N., & Ichihashi, K. (2008). Determination of 40 synthetic food colors in drinks and candies by high-performance liquid chromatography using a short column with photodiode array detection. Talanta, 74, 1408–1413.
Contents lists available at ScienceDirect
Food Chemistry journal homepage: www.elsevier.com/locate/foodchem
Review
Quantitative determination of carmine in foods by high-performance liquid chromatography Ho-Soo Lim, Jae-Chon Choi, Sung-Bong Song, Meehye Kim ⇑ Food Additives and Packages Division, Ministry of Food and Drug Safety, Osong Health Technology Administration Complex, 187 Osong, Chungcheongbuk-do 363-951, Republic of Korea
a r t i c l e
i n f o
Article history: Received 11 November 2013 Received in revised form 29 January 2014 Accepted 19 February 2014 Available online 3 March 2014 Keywords: Carmine Carminic acid Cochineal extract HPLC
a b s t r a c t A simple and rapid method has been developed and validated for the determination of carmine in foods. Samples were homogenised and extracted with 0.05 M NaOH, followed by centrifugation. The resulting solution was filtered and injected to HPLC. Carmine was separated by HPLC using an NovaPak C18 column coupled to a photodiode array detector. The contents of carmine were finally quantified using corresponding calibration curves over ranges of 1.0–100 lg ml 1, with good correlation coefficients (r2 = 0.9999). The recoveries of carmine from foods spiked at levels of 10, 50, and 100 lg g 1 which ranged from 90.4% to 96.2% with relative standard deviations between 2.8% and 6.8%. Limit of detection and limit of quantification of carmine were 0.4 and 1.0 lg ml 1, respectively. This method was found to be useful to distinguish carmine from carminic acid, a major component of cochineal extract. The method has been successfully applied to various foods. Ó 2014 Elsevier Ltd. All rights reserved.
Contents 1. 2.
3.
4.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Chemicals and reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Preparation of standard solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Sample collection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.4. Preparation of samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. High performance liquid chromatography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Method validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Method development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.1. Liquid chromatographic conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2. Solvents for the extraction of carmine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Method performance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1. Linearity, limit of detection and limit of quantification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2. Accuracy and precision . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Analytical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
⇑ Corresponding author. Tel.: +82 043 719 4351; fax: +82 043 719 4350. E-mail address: [email protected] (M. Kim). http://dx.doi.org/10.1016/j.foodchem.2014.02.122 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.
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1. Introduction Synthetic food dyes are food additives that can impart colour when added to foods. These substances must be preapproved by the Korea Ministry of Food and Drug Safety and listed in Korea Food Additives Code (MFDS, 2013a) in order to be legally used in food products retailed in Korea. The MFDS lists 18 permitted synthetic colour additives in synthetic substances part of the Code. The use of food colours is strictly controlled by laws and regulations (MFDS, 2013b). Carmine, one of the synthetic food dyes, is authorised for use in Korea, USA (FDA, 2012), and European Union (European Commission, 1994) except for Japan. The acceptable daily intake (ADI) value of carmine, which is formulated by Joint FAO/WHO Expert Committee on Food Additives, is 5 mg kg 1 based on weight (JECFA, 2001). As the Committee’s conclusion, carmine in foods may initiate or provoke allergic reactions in some individuals. Carmine is a lake pigment used in many different products such as juice, candies, and confectioneries. It is usually produced by precipitating carminic acid in aluminium hydroxide in the form of aluminium or aluminium-calcium salt. Carminic acid is obtained from dried bodies of the female insect Cocus cacti L., which lives on various cactus plants (Feller, 1986). Carminic acid also is a major component of cochineal extract, which is used as a natural food colour in Korea. Carmine and cochineal extract permitted in Korea should not be used for the food items as followings: natural food (meat, seafood (whale meat included), fruits, vegetables, marine algae, bean and their simply processed food (peeled or cut)); tea and coffee; red pepper powder or shredded red pepper; Kimchi; Gochujang (fermented red pepper pastes) or seasoned Gochujang (seasoned soybean paste with red peppers); vinegars; spice products (limited to products containing red pepper or red pepper powder). In addition, the permissible level is not established in Korea Food Additive Code. Carmine might be present as a non-permitted colour additive in food products imported to Korea and illegally added to food products by manufacturers to improve appearance and colour. Cochineal extracts also may be marked on food products where carmine was actually used. Accordingly, monitoring of carmine in foods is necessary to ensure food safety and consumer confidence. Most research has been related to the analysis of carminic acid, a major component of cochineal extract, using high-performance liquid chromatography (Ishikawa, Shigeoka, Nagashima, Takahashi, & Kamimura, 2003; Lancaster & Lawrence, 1996; Merino, Edberg, & Tidriks, 1997), capillary electrophoresis (Liu et al. 1995), and spectrophotometric methods (Tripathi, Khanna, & Das, 2004). Recently, a method used to determine carmine itself in foodstuffs by stripping voltammetry was reported (Alghamdi, Alshammery, & Ahdalla, 2009). This method had an advantage that is suitable for the analysis of very diluted samples and decrease the real time of determination. However, the proposed method was applied to determine carmine only in spiked commercial ice cream and soft drinks. This electrochemical procedure is known to be influenced by factors such as temperature, pH, and organic substances (Kalvoda & Kopanica, 1989). The stripping voltammetry is also not widely used in the analysis of food additives. To the best of our knowledge, no chromatographic method which can separate carmine from carminic acid in foods, has been reported in the literature for the determination of this dye by highperformance liquid chromatography. This is due to insolubility in water and organic solvents. For this reason, carmine cannot be effectively extracted from food samples with usual liquid extraction. Thus, the development of a specific method for the quantification of carmine in foods is required. In the study described in this paper, we have developed a simple and quantitative method for the analysis of carmine in food products by high-performance liquid chromatography coupled to
a diode array detector. The new method separates carmine from carminic acid in food samples. The procedure is quantitatively characterised and linearity, accuracy, limits of detection and quantification are satisfactory. 2. Materials and methods 2.1. Chemicals and reagents Carmine and carminic acid used as standards were purchased from Sigma–Aldrich (St. Louis, MO, USA). All other chemicals used in extraction and preparation of mobile phase, such as sodium hydroxide, sodium phosphate dibasic, and sodium phosphate monobasic were of analytical reagent grade and also supplied by Sigma–Aldrich. Water for all applications in our study was obtained from an Milli-Q ultra-pure water system (Millipore, Bedford, MA, USA) with resistivity equal to or higher than 18.2 MO cm. 2.2. Preparation of standard solutions The stock solution (1000 lg ml 1) of carmine was prepared by transferring 100 mg of carmine into a 100 ml beaker and adding 0.05 M NaOH solution to give a final volume of 100 ml. Calibration standard solutions at serial concentrations of carmine were obtained by mixing subsequent dilution (1–100 lg ml 1) with water. 2.3. Sample collection A total of 124 samples were purchased from retail stores in big cities including Seoul, Incheon, Daejeon, Gwangju, Daegu and Busan in South Korea. The samples were categorised into 16 food types. Processed milk (4), confectionery (50), functional food (20), processed fruit vegetable product (4), tea (1), sugaring product (5), meat product (4), fish meat product (7), processed cheese (4), beverage (10), fermented soybean paste (2), salted seafood (5), seasoned food (4), Kimchi (2), chocolate (1) and coffee (1) in commercial products were purchased after checking the labels and colours written on the products. All samples were stored at 4 °C. 2.4. Preparation of samples A 5.0 g of ground food samples was transferred into a 100 ml beaker and 0.05 M NaOH solution was added to give a final volume of 50 ml. After it was mixed by a homogenizer for 2 min, the sample was shaken mechanically for 10 min with a shaking rate of 300 min 1. The extract was centrifuged for 5 min at 5000 rpm. The 10 ml supernatant was then filtered with 0.45 lm membrane filter for injection into HPLC. 2.5. High performance liquid chromatography HPLC analysis was performed on an Agilent HPLC 1200 series (Santa Clara, CA, USA) coupled to a photodiode array detector. The LC system consisted of degasser, binary pump, autosampler, and column oven. An NovaPak C18 column (150 3.9 mm, 5 lm) purchased from Waters Corporation (Milford, MA, USA) was used for chromatographic separation. All separations were carried out isocratically at room temperature with a mobile phase consisting of methanol-phosphate buffer (pH 6.0) at ratios of 15:85 (v/v). The flow-rate was maintained at 0.8 ml min 1 and a 20 ll sample volume was injected into HPLC. Carmine, eluted from the column, was monitored by photodiode array detector set at 281 nm. The absorption spectra of carmine
H.-S. Lim et al. / Food Chemistry 158 (2014) 521–526
were recorded between 200 and 500 nm. Peak identification was performed by comparing the retention time and absorption spectra of samples with the standard solution. Separation of carmine from carminic acid, a major component of cochineal extracts, was carried out in the same conditions as above. 2.6. Method validation The method was validated for linearity, accuracy (recovery), precision, limit of detection (LOD) and limit of quantification (LOQ) using carmine standard solutions and control samples. The linearity was determined with working standard solutions of carmine over ranges of 1.0–100 lg ml 1 (1, 10, 20, 40, 60, 80, 100 lg ml 1). Integrated peak areas of the absorbing wavelength of carmine at 281 nm were used to construct seven-point calibration curves, which were applied for quantification of carmine. The recoveries and precision of the method were determined using candy, yogurt, and juice samples fortified with carmine at three different concentration levels (10, 50 and 100 lg g 1). For each concentration level, triplicates were prepared and injected once. The recovery of carmine was obtained by calculating the peak area of the chromatogram. The precision expressed as percent relative standard deviation (RSD%) was also determined for carmine using spiked candy, yogurt, and juice samples. The intra-day precision was assessed by performing the five repetitions during a single day and inter-day precision by three repetitions per day over three different days. The LOD was evaluated as the concentration giving a signal equal to three times of noise (S/N = 3) and the LOQ was determined as the concentration giving a signal equal to ten times of noise (S/ N = 10). 3. Results and discussion 3.1. Method development 3.1.1. Liquid chromatographic conditions A good separation of carmine and carminic acid can be achieved with 15% methanol and phosphate buffer (pH 6.0) as the mobile phase system under the isocratic elution condition described above. Since carmine has the similar absorption spectrum of carminic acid at the range of 200–500 nm, several C18 columns were tested for the separation of carmine and carminic acid. Using the conventional analytical column (NovaPak C18, Waters, 150 3.9 mm, 5 lm) at flow rate of 0.8 ml min 1, the separation of carmine and carminic acid was accomplished (Fig. 1). The analysis was completed within
Fig. 1. Chromatogram of a standard solution (100 lg ml carminic acid; (2) carmine.
1
523
7 min. Carmine was monitored at 281 nm, nearest to the maximum absorption by a photodiode array detector. The absorbance spectra for carmine and carminic acid were similar. Typical separation of a mixed standard solution show retention time of 4.5 and 5.6 min for carminic acid and carmine, respectively. Plots of solvent combination rate vs. retention time were made for testing the most suitable mobile phase. 15% methanol was the most suitable phase for application to carmine as shown in Fig. 2. The high-performance liquid chromatography with diode array detector and C18 column has been successfully applied for determining aluminium lake dyes in food matrix (Alves, Brum, Branco de Andrade, & Pereira Netto, 2008; Ma, Luo, Chen, Su, & Yao, 2006; Yang & Shao, 2011; Yoshioka & Ichihashi 2008). However, no research has been reported in the literature for the determination of carmine by HPLC. 3.1.2. Solvents for the extraction of carmine Carmine is an aluminium lake or aluminium-calcium lake dye. Although carmine is insoluble in water and organic solvents, it can be dissolved by strong alkaline or acid solutions such as sodium hydroxide and hydrochloric acid. As hydrochloric acid is used, carminic acid is released from carmine (Marshall & Horobin 1974). Carmine also could be dissolved by dimethyl sulfoxide solution, an aprotic solvent which dissolves both polar and nonpolar compounds. However, the separation of carmine and carminic acid was not achieved by this solvent. Therefore, sodium hydroxide solution was selected for the extraction of carmine in food samples. It is also supported by the reports that aluminium lake dyes in foods and drugs were efficiently extracted with 0.02 M sodium hydroxide solution (Ishikawa et al., 2003; Yang & Shao, 2011). The effects of different sodium hydroxide concentrations of carmine on peak area were investigated using the standard solution. The concentration of NaOH solution was studied at 0.01, 0.02, 0.03, 0.04, 0.05, and 0.06 M (Fig. 3). The peak area of carmine increased when NaOH concentration increased to 0.05 M. On the other hand, when the concentration of NaOH reached 0.06 M, the area of carmine significantly decreased by 16%, which might have occurred as a result of the loss of carmine due to hydrolysis with the solution of high pH value. Optimum area value was obtained with concentration of 0.05 M. 3.2. Method performance 3.2.1. Linearity, limit of detection and limit of quantification The linearity of the method was determined by the injection of various standard concentrations. The method was linear in the
) separated by liquid chromatography with detection by a photodiode array set at 281 nm. Peaks identity: (1)
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Fig. 2. Plots of retention time and peak area of carmine on C18 column vs. methanol content (%) for optimisation of mobile phase.
Fig. 3. Plots of peak area of carmine vs. NaOH concentration for optimisation of extraction solvents.
range of concentrations from 1.0 to 100 lg ml 1 (r = 0.9999). This result indicates that the standard curve could be effectively applied to quantify carmine in food samples. LOD was evaluated as the mass giving a signal equal to three times the noise (S/N = 3); LOQ was determined as the mass giving a signal equal to ten times that of the blank (S/N = 10). The calculated LOD and LOQ using standard solutions were 0.4 and 1.0 lg ml 1, respectively. LOD and LOQ conform with AOAC guideline (AOAC, 2013). The analysis of carmine via high-performance liquid chromatography can be useful in quality control during and after production of food products containing carmine. 3.2.2. Accuracy and precision Recovery was calculated as the percentage of foods recovered from three replicate determinations of three different solutions
containing 10, 50, and 100 lg ml 1. Fig. 4 shows a chromatogram of carmine extracted from candy, yogurt, and juice filtered and then chromatographed using the methanol-phosphate buffer (pH 6.0) mobile phase. Recoveries ranged from 90.8 ± 2.7% to 96.2 ± 4.5% in candy, yogurt, and juice as shown in Table 1, demonstrating that the method is accurate within the desired range. The precision, evaluated as the repeatability of the method, was studied by calculating the relative standard deviation (RSD) for three determinations of the concentrations 10, 50, and 100 lg ml 1 performed on the same day and under the same experimental conditions. The RSD values obtained ranged from 2.8% to 6.7% (Table 1). The inter-day precision was assessed by calculating the RSD for determining three levels of concentration on five different days. The RSD values obtained were below 7% for three samples. The accuracy and precision of the method conform with AOAC guideline.
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Fig. 4. Chromatograms of carmine recovered from candy, yogurt and juice spiked at 100 lg ml
3.3. Analytical applications To assess the reliability of the proposed method described above, it was applied to determine carmine in a variety of commercially available food products retailed in Korea. One hundred-twenty-four samples of food products retailed in Korea were analysed using this method. Quantification of carmine was carried out using standard calibration curves and confirmed by comparing absorption spectra and retention time of the samples with carmine standard. The concentrations of carmine in food samples are shown in Table 2. It was detected from total of thirty food products and its contents ranged from N.D. to 973.9 ± 2.8 lg g 1.
1
, respectively: (A) candy; (B) yogurt; (C) juice.
The average content of carmine was the highest (973.9 ± 2.8 lg g 1) in the imported beverage. The carmine contents in imported confectionery and sugaring product were 865.0 ± 4.7 and 754.7 ± 2.8 lg g 1, respectively. In EU, carmine usage in some foods, such as red marbled cheese, jam, and chorizo sausage are specified from 100 to 200 mg kg 1 (European Commission, 1994). Usage in a wide variety of foods in CODEX is set from 50 to 500 mg kg 1 except for fresh eggs which are set up as good manufacture practise (CODEX Alimentarius, 2013). However, the carmine usage is not specified in Korea. For this reason, carmine levels in foodstuffs retailed in Korea may be higher than the allowed levels established by EU and CODEX.
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Table 1 Recoveries of carmine from candy, yogurt and juice sample spiked at three different concentrations (n = 3). Sample
Spiked level (lg ml 1)
Recovery (%)
Candy
10 50 100
yogurt
Beverage
Intra-day
Inter-day
90.8 ± 2.7 94.6 ± 1.9 95.2 ± 2.5
3.1 2.8 4.6
5.2 3.6 4.7
10 50 100
90.4 ± 3.4 95.9 ± 2.4 95.2 ± 2.1
5.1 6.7 5.3
4.3 5.6 4.2
This research was supported by a Grant (12161-MFDS-113) from Ministry of Food and Drug Safety in 2012.
10 50 100
93.8 ± 2.7 95.1 ± 3.8 96.2 ± 4.5
4.3 4.2 3.4
6.8 5.5 4.4
References
Table 2 Concentration (lg g
a
Precision (RSD%)
method that can be applied to various food products such as beverages, confectionery, and candies. This study is the first report on HPLC method to differentiate carmine and carminic acid in foods. Thus, this method is applicable for the effective surveillance of carmine in food products.
1
) obtained for the samples analysed (n = 3).
Food type
Number of sample
Detected sample
Carmine level Range ± SD
Processed milk Confectionery Functional food Processed fruit vegetable product Tea Sugaring product Meat product Fish meat product Processed cheese Beverage Fermented soybean paste Salted seafood Seasoned food Kimchi Chocolate Coffee
4 50 20 4
1 22 – 2
NDa–46.7 ± 0.7 ND–865.0 ± 4.7 ND ND–149.9 ± 4.0
1 5 4 7 4 10 2 5 4 2 1 1
– 1 – 1 1 2 – – – – – –
ND ND–754.7 ± 2.8 ND ND–28.9 ± 0.3 ND–19.0 ± 0.1 ND–973.9 ± 2.8 ND ND ND ND ND ND
ND: not detected means below limits of quantification (1.0 lg g
1
).
Twenty-eight samples among thirty detected samples were imported food products. All detected samples were also under the regulation of Korea Food Additives Code. Carmine was not detected in functional food, tea, meat product, fermented soybean paste, salted seafood, seasoned food, Kimchi, chocolate, and coffee. The analytical methods of carmine as carminic acid by hydrochloric acid hydrolysis have a disadvantage that carmine or carminic acid in foods could not be distinguished (Carvalho & Collins 1997; Merino et al., 1997). However, as the proposed method in this study was applied to samples, retention time and spectrum of carmine were different from carminic acid, which was detected in a sample. Carmine and carminic acid didn’t occur together in all examined foods. Carmine and carminic acid are not used at the same time due to the similar colour (from red to dark red colours). 4. Conclusions A rapid and simple analytical method suitable for carmine has been successfully developed and validated in our study. The accuracy and precision obtained were satisfactory for carmine after optimisation of experimental conditions. This method is useful for distinguishing carmine from carminic acid, which is a major component of cochineal extract. In addition, it is a quantitative
Acknowledgements
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