Kırmızı Pul Biber Örneklerinde HPLC İle Aflatoksin Türlerinin Analizi

Öz

Aspergillus flavus, Aspergillus parasiticus, Aspergillus nomius ve Penicillium ve Rhizopus küflerinin ürettiği aflatoksinler bir mikotoksin türevi olup insan sağlığına zararlıdır. Bu çalışmada, Kayseri semt pazarında satılan aflatoksin B1, B2, G1 ve G2 türleri kırmızı pul biber örneklerinde yüksek performanslı sıvı kromatografisi (HPLC) ile belirlenmiştir. Öncelikle aflatoksin türleri 30 oC sıcaklıkta metanol ve su (80:20 v/v) karışımında batch yöntemiyle ekstrakte edilmiştir. Kromatografik ayırma, Floresan Dedektörü ile ODS-2 üzerinde, ör: 360 nm, em: 440 nm, 30 oC'de 30 dakika boyunca gerçekleştirildi. Aflatoksin B1, B2, G1 ve G2'nin tayini için, bilinen konsantrasyondaki analitlerin standart çözeltileri, resmi yöntem AOAC 999.07'ye göre hazırlandı. Kalibrasyon doğrusu standartlara göre hazırlanmış ve kalibrasyon doğrusuna göre sonuçlar verilmiştir. Algılama limiti B1 ve G1 için 0,216 µg L-1 ve B2 ve G2 için 0,0648 µg L-1 olarak belirlendi. En yüksek konsantrasyon AFB1 için 7,32, AFB2 için 2,39 ve AFG1 için 1,23 μg L-1 olarak analiz edilirken, örneklerde AFG2 konsantrasyonunun miktar sınırının altında kaldığı gözlendi. Gözlenebilme sınırı, aflatoksin B1 ve G1 için 0.720 μg/L ve B2 ve G2 için 0.216 μg L-1 olarak hesaplandı. Geri kazanım çalışmaları %90-104 aralığında bulundu. %RSD ≤ %5 (n=11) olarak hesaplandı.

Anahtar Kelimeler

• Aflatoksin B1 (AFB1)
• Aflatoksin B2 (AFB2)
• Aflatoksin G1 (AFG1)
• Aflatoksin G2 (AFG2)
• Kırmızı Pul Biber
• HPLC

Analysis of Aflatoxin Types in Red Pepper Flakes Samples by HPLC

Abstract

Aflatoxins produced by Aspergillus flavus, Aspergillus parasiticus, Aspergillus nomius and Penicillium and Rhizopus molds are a mycotoxin derivative and are harmful to human health. In this study, the aflatoxin B1, B2, G1 and G2 types sold in Kayseri street market were determined in red flakes pepper samples by high performance liquid chromatography (HPLC). Firstly, the aflatoxin types were extracted by shaking technique. A mixture of methanol and water (80:20 v/v), at the 30 oC temperature. The chromatographic separation was conducted on ODS-2 with Fluorescence Detector, ex: 360 nm, em: 440 nm at 30 oC for 30 min. For the determination of aflatoxin B1, B2, G1 and G2, standard solutions of analytes at known concentration were prepared according to the official method AOAC 999.07. The calibration line was prepared according to the standards and the results were given according to the calibration line. The detection limit was determined as 0.216 µg L-1 for B1 and G1 and 0.0648 µg L-1 for B2 and G2. While the highest concentration was analyzed for AFB1 as 7.32, for AFB2 as 2.39 and for AFG1 as 1.23 μg L-1, it was observed that the concentration for AFG2 remained below the quantification limit in samples. The quantification limits were 0.720 μg L-1 for aflatoxins B1 and G1, and 0.216 μg L-1 for B2 and G2. Recovery studies are in the range of 90-104%. The %RSD was calculated as ≤ 5% (n=11).

Keywords

• Aflatoxin B1 (AFB1)
• Aflatoxin B2 (AFB2)
• Aflatoxin G1 (AFG1)
• Aflatoxin G2 (AFG2)
• Red Pepper Flakes
• HPLC

Kaynakça

1. Moss, M. O. (1992). Secondary Metabolism and Food Intoxication-Moulds. Journal of Applied Bacteriology Symposium Supplement, (73), 80–88.
2. Goldblatt, L. A. (1969). Aflatoxin Scientific Background, Control and Implications. New York: London, 472p.
3. Heperkan, D. (2014). Gıdalarda Mikotoksinler (1.Baskı). İzmir, Sidas Yayınları, 29–42.
4. Topdaş, E. F., Başlar, M., ve Ertugay, M.F. (2011). Elmaların ozmotik kurutulması üzerine ultrases işleminin etkisi. Akademik Gıda, 9(5), 6–10.
5. Roberts, B.A., Patterson, D.S.P. (1976). Mycotoxins: Detection of Twelve Mycotoxins in Mixed Animal Feedstufs. Using a Novel Membrane Procedure. Journal - Association of Official Analytical Chemists. (58), 1178–1181.
6. Romer, T.R. (1973). Determination of Aflatoxins in Mixed Feeds. Journal - Association of Official Analytical Chemists, (56), 1111–1115.
7. Demircioğlu, S., Filazi, A. (2010). Türkiye’de Üretilen Kırmızıbiberlerde Aflatoksin Kalıntılarının Araştırılması. Veteriner Hekimler Dergisi, 81(2), 63–66.
8. Yıldız, G., İzli, G. Gıdaların Kurutulmasında Mikrodalga Ve Sıcak Hava Yöntemleri. İksad Yayınevi, Ankara, Türkiye, 128s.

9. CAST, (2003). Mycotoxins: Risks in Plant, Animal and Human Systems, Task Force Report No. 139. Council for Agricultural Science and Technology, Ames, Iowa, USA.
10. Desjardins, A., Maragos, C., Norred, W., Pestka, J., Philips, T., Vardon, P., Whitaker, T., Wood, G., Egmond, H. (2003). Mycotoxins: Risks in Plant, Animal and Human Systems Council. USA: Iowa, 199p.
11. Magan, N., Olsen, M. (2004). Mycotoxins in Food: Detection and Control. New York: Woodhead Publishing Ltd, 471p.
12. European Commission RASFF Portal, Notification details-2018.3783. file:///C:/Users/tesli/Desktop/aflatoksin%20proje/RASFF%20Portal.html. Last Access Date: 04 February 2022.
13. FAO, (1987). Sayfa 15. Current Limits and regulations on Mycotoxins. Joint FAO/WHO/UNEP Second International Conference on Mycotoxins. Bangkok, Thailand, 28 September - 3 October.
14. FAO, (1987). Sayfa 27. Distribution of Mycotoxins-an Analysis of Worldwide Commodities Data, Including Data from FAO/WHO/UNEP Food Contamination Monitoring Programme. Joint FAO/WHO/UNEP Second International Conference on Mycotoxins Bangkok, Thailand 28 September - 3 October.
15. Turner, N.W., Subrahmanyam, S., Piletsky, S. A. (2009). Analytical methods for determination of mycotoxins. Analytica Chimica Acta, (632), 168–180.
16. Trucksess, M., Brumley, W., Nesheim, S. (1984). Rapid quantitation and confirmation of aflatoxins in corn and peanut butter, using a disposable silica gel column, thin layer chromatography, and gas chromatography/mass spectromety. Journal of the Association of Official Analytical Chemists, 67(5), 973–975.
17. Zhao, Y., Huang, J., Ma, L., Wang, F. (2017). Development and validation of a simple and fast method for simultaneous determination of aflatoxin B1 and sterigmatocystin in grains. Food Chemistry, (221), 11–17.
18. Hamed, M.A., Moreno-Gonzalez, D., García-Campana, A.M., Gamiz-Gracia, L. (2017). Determination of Aflatoxins in Yogurt by Dispersive Liquid–Liquid Microextraction and HPLC with Photo-Induced Fluorescence Detection. Food Analytical Methods, (10), 516–521.
19. Li, Q., Lv, S., Lu, M., Lin, Z. Dianping Tang, D. (2016). Potentiometric competitive immunoassay for determination of aflatoxin B1 in food by using antibody-labeled gold nanoparticles. Microchimica Acta, 183 (10), 1–8.
20. Pagkali, V., Petrou, P.S., Makarona, E., Peters, J., Haasnoot, W., Jobst, G., Moser, I., Gajos, K., Budkowski, A., Economou, A., Misiakos, K., Raptis, I., Kakabakos, S.E. (2018). Simultaneous determination of aflatoxin B1, fumonisin B1 and deoxynivalenol in beer samples with a label–free monolithically integrated optoelectronic biosensor. Journal of Hazardous Materials, (359), 445–453.
21. AOAC (2007) AOAC (999.07). Official methods of analysis, Association of official analytical chemists. 18th edition, Washington D C.
22. Kanbur, M., Liman B. C., Eraslan, G., Altınordulu, Ş. (2006). Kayseri’de Tüketime Sunulan Kırmızı Biberlerde Aflatoksin B1’in Enzim İmmunoassay (EIA) ile Kantitatif Analizi. Journal of Faculty of Veterinary Medicine, Erciyes University, (3), 21–24.