Kırmızı toz biberlerde aflatoksin miktar tayininde kullanılabilecek üç farklı analiz metodunun karşılaştırılması

Abstract

Bu çalışmada aflatoksin miktar tayininde, mevcut ekstraksiyon ve temizleme metotları ve kromatografik metotlar içinde, gerekli modifikasyonlar da yapılarak kırmızı toz biberler için en uygun olanının saptanması amaçlanmıştır. Materyal olarak seçilen 8 değişik kırmızı toz biber örneğine belirli konsantrasyonlarda aflatoksin standartları ilave edilerek, standart ilavesinden önce ve sonra, CB, BF ve ST ekstraksiyon ve temizleme metotları kullanılarak aflatoksin ekstraktları elde edilmiştir. Bunlardan yalnız Stoloff-Trucksess (ST) metodu ile tek boyutlu TLC'ye izin veren çok temiz, renksiz, berrak ekstraktlar elde edilmiş ve kantitatif tayin yapılabilmiştir. BF ve CB metoduna ait ekstraktlar ise bulanık ve kırmızımsı-sarı renkli olup, ancak Steiner metodu ile kantitatif tayine olanak sağlamıştır. HPLC analizi için kalibrasyon eğrisi hazırlanarak çalışılmıştır. Sonuçta ST ve CB metoduna ait ekstraktlarda aflatoksin Bt tayinleri yapılmış, BF metoduna ait ekstraktlarda ise kirliliğin yoğun olması ve aflatoksin B! ile yakın alıkonma süresine sahip başka girişim yapıcı maddelerin bulunması nedeniyle sağlıklı sonuçlar elde edilememiştir. Ayrıca bu girişim yapıcı maddeler TLC analizinde yüksek okumalara neden olabildiğinden, HPLC sonuçları TLC sonuçlarından daha düşük bulunmuştur. Varyasyon analizi (ANOVA) sonunda çalışılan örneklerde metotlar arası farklılık (a=0,05 düzeyinde) önemli bulunmamakla birlikte, en yüksek "geri kazanım"ın TLC ve HPLC için sırasıyla %98 ve %73 ile ST metoduna ait olduğu, metotların "tekrar edilebilirliği"nin analizinde ise en düşük varyasyon katsayısının (TLC için:0,02 ve HPLC için:0,19) yine ST metoduna ait olduğu saptanmıştır. Kromatografik metotların birbiriyle karşılaştırılmasında HPLC'nin tekrar edüebükliğinin TLC'den daha düşük bulunmuş olmasının, bu çalışmada kullanılan HPLC sistemi ile metodunun limitasyonları ve yapılmış zorunlu modifikasyonlardan kaynaklandığı düşünülmüştür. Bu sonuçlara dayanarak, kırmızı toz biberlerde aflatoksin Bj tayini için en uygun prosedürün "Stoloff-Trucksess" ekstraksiyon ve temizleme metodunun "tek boyutlu TLC" ile birlikte kullanılması olduğu sonucuna varılmıştır.

Ingestion of commodities contaminated with mycotoxins may have carcinogenic, mutagenic and teratogenic effects. The most potent mycotoxins are aflatoxins, which are the toxic metabolites of Aspergillus flavus and Aspergillus parasiticus occurring frequently in nature. Aflatoxins are characterized with the fleuorescent colors they exhibit under ultraviolet light. Their nomenclature as well as their analyses with chromatographic techniques are based on this property: Aflatoxins B: and B, giving blue fluorescence as opposed to the green fluorescence of aflatoxins G, and G,. Their Rf values on thin layer chromatographic plates depend on their relative polarities and gradually increase is the order of B^ B2, G1} and G2. Due to their proven toxic effects, aflatoxins have been of major world concern because of both public health and economical loss implications and there fore, many countries have already established legal action levels for aflatoxins in foods and feeds. Turkey first met with the aflatoxin problem in 1967, when 10 tons of Turkish hazelnuts exported to Canada were rejected with the claim of aflatoxin contamination. The same situation arouse repeatedly in 1971, with Turkish pistachionuts being returned from USA, in 1972 with Turkish figs being rejected from Denmark, and in 1976 with hazelnuts being returned from West Germany with similar claims. The last incidence has been with the Turkish red peppers exported to Germany in 1995. The action level for aflatoxin contamination in human foods is 20 jug/kg (ppb). The very low levels and heterogenicity of contamination require very precise and highly sensitive analytical methods for quantifications. The key to success and reliability of any survey or research conducted for studying aflatoxins in foods is the analytical methodology being used. In the available-literature, there exist many methods developed for the extraction and clean up of aflatoxins from specific types of food commodities, since each may offer advantages or disadvantages as to their applicabilities to different matrix systems. However, no official method has yet been accredited for spices including red pepper. This present study aimed at selecting the most recommendable method for extraction and clean-up of aflatoxins from red peppers as well as the chromatographic method for quantification. For this purpose, red pepper samples were collected randomly from Istanbul "Mısır Çarşısı" and "Rami" markets. 3 kg lots from each of the 8 samples taken were placed in cloth bags and kept refrigerated till analyzed. On these samples, the following physical and chemical analyses were performed: 1. Moisture analyses (TS 2134) 2. Ash content (TS 2131) 3. Non volatile extractables (TS 2137) 4. Sieve analyses (TS 3479 and 3495) Later, known concentration of each of the four aflatoxins (16 |ag/lt (ppb) B^ 21,6 ug/lt B,, 31,2 ug/lt Gt and 12.8 ug/lt G,) were added as internal standards to a sub-set of all 8 of the samples. Both the 8 unspiked and 8 spiked sample-sets were subjected to the following three different extraction and clean-up procedures: 1. BF procedure (AOAC, 1990, 970.45.D) 2. CB procedure (AOAC, 1990, 968.22.D) 3. Stoloff and Trucksess (ST) procedure (Awe et al., 1981) The initial qualitative screening analysis for aflatoxins showed that all of the unspiked samples naturally contained aflatoxin Bt but none of the other three aflatoxins (B,, Gt, and G2). These chromatographic plates were later subjected to the following confirmation tests: 1. Observation under short wavelength UV-light (TS 4672) 2. Sulfuric acid test (TS 4672) After confirmation of the spots for the presence of aflatoxin B, the samples were analyzed by three different quantitative thin layer chromatographic methods for aflatoxin Bv These quantitative assays were performed on both spiked and unspiked sample extracts by the following methods: xi 1. One-dimensional TLC methods (AOAC, 1990, 968.22.F.c) 2. Two-dimensional TLC methods (AOAC, 190, 978.15.E) 3. Steiner Method (Steiner et al., 1988) 4. HPLC method (Stubblefield and Shotwell, 1977) The one-dimensional thin layer chromatographic quantitative method was applied to all samples but gave no dedectable results, except for the sample extracts obtained with Stoloff and Trucksess extraction and clean-up methods, due to high level of interfering substances present in samples obtained with BF and CB methods. These sample extracts containing relatively higher amounts of interfering substances were subjected to a more sensitive quantitative TLC method developed by Steiner and friends (1988) for the determination of aflatoxin Bv In the view of available literature, certain substances having similar Rf value and fluorescence properties to aflatoxins may interfere the analysis. Therefore all three extracts of sample 1 were subjected to a two-dimensional TLC technique to investigate the presence of such interfering substances. As a result, the extracts from CB and BF methods when analyzed with two-dimensional TLC yielded lower aflatoxin Bj values than those obtained with Steiner method. This was interpreted as impurities in the aflatoxin B, spots obtained with Steiner method, and, when two- dimensional TLC was applied, these impurities were significantly removed in the second direction of development. But this method could not be applied to all three replicates of the other spiked and unspiked samples due to the economical reasons. Furthermore, the extracts of sample No.l obtained with Stoloff-Trucksess method when analyzed with one-dimensional TLC, yielded very similar results with those obtained from two-dimensional TLC. This also confirmed that Stoloff- Trucksess method was successful in removing significant levels of interfering impurities. The HPLC method was applied to all extracts for further quantification of aflatoxin Bt contents. For this purpose, the HPLC system (Waters Model 501) present in the ITU Food Engineering Department was used. However, due to limitations of the existing system and column (u Bondapak Clg), it was necessary to use only the reverse phase procedures. Following literature search for reverse phase quantitative HPLC methods for aflatoxins, the method developed by Stubblefield and Shotwell (1977) was adapted and modified to meet the existing systems requirements. A calibration graph which is shown in Figure 1, using a set of 8 different aflatoxin Bt standards with different concentrations (10, 20, 40, 80, 100, 200, 300 and 400 ug/lt (ppb)) was prepared, by taking peak areas as the basis for integration. The HPLC chromatogram obtained with this aflatoxin resolution standard solution is given in Figure 2. The HPLC studies yielded satisfactory results with sample extracts obtained by both CB and Stoloff-Trucksess methods, but not with BF method, due to presence xii * io1 peak response Figure 1. Calibration Curve for aflatoxin B, standars 2.5? -I 2.54. 2.3a > 2.SCH 3 2.494 2. 46 2.44 a. 4» x/vsv^t^^^ 0.00 0.50 x i0x Minute Figure 2. The HPLC chromatogram of aflatoxin resolution standard (aflatoxin Bj, B;,, Gt and G,) solution xui of higher amounts of interfering substances in the latter, resulting in non-integratable peaks. Therefore, the results of HPLC quantification with BF method were not included in the statistical evaluation. The three methods used in this study for the quantitative analysis of aflatoxin Bt in red peppers were subjected to a final evaluation based on their accuracy, repeatibility, ease, speed and cost to determine the most suitable and effective method. The analysis of variance (ANOVA) with Microsoft Excel 5.0 package program on the recovery values of the three extraction and clean-up procedures yielded no significant differences among the methods at a=0,05 level. Furthermore, the recovery values of the Stoloff-Trucksess and CB methods, when subjected to t- test yielded no significant difference at oc=0,05 level. On the other hand, when accuracy is evaluated being based solely on recovery rates, the Stoloff-Trucksess method ranked the first, with 98% recovery in TLC and 73% recovery in HPLC. The highest repeatibility, when based on the smallest of variation coefficient percent, was also observed in the Stoloff-Trucksess method (V.C.% for Stoloff- Trucksess TLC=0,02; V.C.% for Stoloff-Trucksess HPLC=0,19). However, the lower repeatibility of the HPLC quantification may have been due to the limitations and obligatory modifications of the individual HPLC method used in this study. Based on the results, the most recommendable method for aflatoxin Bl analysis in red peppers is the Stoloff-Trucksess extraction and clean-up procedure coupled with one-dimensional TLC quantification.