UV-Vis spectrometry, including elaboration of the 1 and 2 derivative, was employed for the detection of red colorants in saffron extracts prepared according to the ISO 3632 –1993 standard. The sensitivity of the method was improved when saffron naturally encountered colorants, the crocins, were removed from the analytical sample. For this reason, thermal treatment of the extract at low pH was necessary. In this way even low levels of artificial red colorants (30g /kg saffron powder) were detectable. Moreover, tristimulus colorimetry was used as a screening method to control the authenticity of saffron samples prior to sample pretreatment. It seems that the chromatic parameters a* and h offer some preliminary evidence as to whereas red colorants are present in a saffron aqueous extract. INTRODUCTION Saffron commands a rather high value in the international spice trade that results in its frequent adulteration. One of the most common means of deception is the addition of artificial colorants. Such a practice is expected to improve the appearance of the dried stigmas or even to give rise to the coloring strength of the aqueous extract, expressed as E 440nm. Misleading of the consumer, or misclassification of a sample at a higher commercial quality are not the only consequences of such an illegal practice as the latter may also confront to food safety principles. The aim of the present study was to examine the potential of UV-Vis derivative spectroscopy along with color measurement in aqueous extracts of saffron for the detection of a series of red colorants. Though various spectrometric (Basker and Negbi, 1985; Sujata et al., 1992; Orfanou and Tsimidou, 1996) and chromatographic methods (Pfander and Rychener, 1982; Himeno and Sano, 1987; Sujata et al., 1992; Tarantilis et al., 1994, 1995; Alonso et al., 2001) have been developed to estimate the coloring strength or determine the natural colorants of saffron, the literature concerning detection of adulteration with synthetic ones is rather limited (Alonso et. al., 1999). In the current version of the ISO standards 3632 (1993) such a methodology is absent whereas in the revision under discussion since 2000, the application of a laborious protocol for the detection of the adulterants is proposed. The latter takes advantage of the solid phase extraction to preconcentrate the adulterant (e.g. red colorant) prior to ion pair HPLC. In an effort to enhance the quality control system of the medium size enterprises, which are responsible for the trade of saffron within the European Union, less sophisticated procedures were employed in the present study to detect the lowest attainable amounts of red colorants. The colorants examined were allura red AC (E 129), amaranth (E 123), azorubine (E 122), carminic acid (E 120), erythrosine (E 127), ponceau 4R (E 124) and red 2G (E 128). MATERIALS AND METHODS Twenty-two pure saffron samples were obtained from the Cooperative of Saffron Growers (Kozani, Greece). Equal amounts of each sample were used to prepare a representative saffron sample that was used in further analysis. Allura red AC, amaranth, azorubine, carminic acid, erythrosine, ponceau 4R and red 2G were purchased from Sigma-Aldrich Chemie GmbH (Steinhem, Germany). The water used was HPLC grade. The solutions were acidified with HCl 37 % (Riedel de Haën, Seelze, Germany) (for analysis). The spectrophotometer used for the analysis was Shimadzu UV 1601 (Kyoto, Proc. I IS on Saffron Eds: J.-A. Fernández & F. Abdullaev Acta Hort 650, ISHS 2004 332 Japan) equipped with the software UVPC 1601 (Personal Spectroscopy Software, v.3.9, Shimadzu). Color measurements were obtained using a MiniScan TM XE Plus (Hunter Assoc. Inc., Reston, Virginia, USA) colorimeter with reference to D65 illuminant and 10 angle of vision. UV-Vis Spectrometric Procedure for the Detection of Red Colorants 1. Preparation of Saffron Extracts. Aqueous extracts of saffron were prepared according to the ISO 3632-2 (1993) method. Saffron powder (0.25 g) was transferred with water in a 500 mL volumetric flask. The crocins were extracted by rigorous agitation for 1 hour at ambient temperature away from direct sunlight. The working solutions were prepared by dilution of the extracts with distilled water (1:10 v/v). 2. Preparation of the Solutions of Red Colorants. A series of seven solutions for each colorant in the range of 1-10 μg/mL was obtained from stock standard solutions (200 μg/mL). 3. Preparation of Aqueous Extracts of Adulterated Saffron Samples. The admixtures were prepared by adding specific volume of each colorant solution to the saffron powder. Then extraction was as described in 1. The working solutions were prepared by dilution of the extracts with water (2:3, v/v). 4. Elimination of Crocins. The solutions of saffron mixtures with (or without) colorants were acidified to pH 0.1 and heated at 90C for 30 min. The solutions were filtered through a Gooch filter no 4 and then the spectrum of the filtrate was obtained in the UV-Vis region. 5. UV-Vis Spectra Recording. The UV-Vis spectra of all solutions were recorded in the region 200-600 nm. Measurements in triplicate were taken for each solution. The spectra were stored and the derivatives were calculated using the software facilities. Evaluation of the Chromatic Parameters a*, b*, C* and h Two ceramic plates, black and white, were used for the calibration of the colorimeter. The working solutions were the same as for the spectrometric analysis. A black plastic ring was placed into a glass container (d=6.5 cm), which was then filled with 20 ml of each solution and sealed with a white ceramic plate. A mean value of nine readings was taken as the overall value for each case. The CIEL*a*b* coordinates a* (red-green component) and b*(yellow-blue component) along with the parameters C*(chroma) and h (hue angle) were recorded and used to evaluate the apparent color of the solutions. RESULTS AND DISCUSSION One of the expected difficulties in ensuring the authenticity of natural products is the variability in the chemical composition of the authentic products. In the case of saffron natural variability could be attributed to the genetic material, the origin of the sample as well as to the processing conditions. Therefore, in order to exploit the potential of spectrometry to detect adulteration with red colorants, it was considered necessary to analyze first a considerable number of authentic samples from the same region (Kozani area), processed by different producers to establish the characteristics of the UV-Vis spectrum of aqueous saffron extracts. The spectrum of the extracts for the 22 samples and the 1 and 2 spectrum derivative revealed several characteristics (Figure 1, Tables 1 & 2) that were repeatable in all cases, indicating, thus, a typical UV-Vis profile for Greek authentic saffron samples. Taking into account preliminary results presented by Alonso et al. (1999) and the levels of adulteration reported therein, we decided to work at a much lower level of adulteration. For the selected adulteration levels of 30g colorant/kg saffron powder no significant difference in the values of % 1 440nm E between an authentic saffron sample and an adulterated one was found (Table 3). Moreover, UV-Vis zero-order spectra of the aqueous extracts of adulterated samples did not differ from that of an authentic one
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