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36. DETERMINATION OF AFLATOXINS BL AND ML IN CHICKEN LIVER BY HPLC Kalantari H., Zandmoqadam A. and Abdolahi Lorestani S.

School of Pharmacy, Ahwaz University of Medical Sciences Ahwaz Iran ABSTRACT Mycotoxins are toxic or carcinogenic secondary metabolites produced by fungi on agricultural commodities. The presence of mycotoxins in the food and feed stuffs is a result of a complex series of interaction among the causative fungi, the contaminated products, the various environmental factors and the intoxicated host. aflatoxinis produced generally by Aspergillus species that contaminates food and feed. Epidemics due to these toxins have occasionally killed many domestic animals.In this work 100 samples of chicken liver collected from poultry producers and liver were analyzed to determine aflatoxin Bl and Ml both qualitatively by TLC and quantitatively by HPLC. The samples were extracted with dichloromethane and a column of silica gel was used to remove fats and other impurities. Then it was eluted by dichloromethane and acetone in the ratio (4:1) solvent was evaporated and the residue was dissolved in known amount of dichloromethane for further experiments. Preliminary results by TLC showed that about 43 % of the samples were contaminated with aflatoxins and then were separated and quantified by HPLC. The results on HPLC showed that the highest levels of aflatoxin Bl and Ml were 0.71% and 1.1 g/100 grams of samples respectively. On the other hand the minimum amount of aflatoxin B 1 and Ml which were detected by TLC were 0.11 and 0,14 g/100 grams of samples as judged by HPLC. According to the previous reports these levels of aflatoxins are considered as safe but should be noted that the production of aflatoxins may be accelerated by improper production and handling of feeds. INTRODUCTION The impact of mycotoxins on human and animal health is well recognized. Mycotoxin entry to the human and animal dietary systems is mainly by ingestion takes place and exhibit a wide range of adverse biological effects and individual mycotoxin can be mutagenic, carcinogenic, embryo toxic, teratogenic or estrogenic. Establishing a causal relationship between mycotoxins exposure and human disease is complicated(l). Mycotoxins are chemical compounds produced by variety of fungi that can cause illness in human and animals. The adverse effects of fungal products have caused mass poisoning in both man and animals in many countries. As far as public health problems are concerned, aflatoxin is well known as one of the most important environmental toxicants, since its potent hepatotoxicity has been demonstrated in various experimental animals and its natural occurrence in cereals and grains has been shown by chemical analysis (2, 3). The presence of mycotoxin producing fungi have been well demonstrated as a natural pollutant in several plant products including cereals, grains and foodstuffs in many countries of Europe, Russia, USA, Canada, and several Asian countries (4). The aim of this investigation was to determine qualitatively and quantitatively the aflatoxin Bl and aflatoxin Ml in chicken liver by high performance liquid chromatography (HPLC). MATERIALS AND METHODS Liver tissues were collected from local poultry producers. aflatoxins Bl and Ml were obtained from Sigma CO. The HPLC eluent solvent and other chemicals were purchased from Merck. The liver samples were homogenized and then to the 100 grams of homogenized liver 10 mL of 20 % citric acid were added and mixed well then 200mL of dichloromethane were added and kept in automatic shaker for about 30 minutes .The mixture was filtered and the filtrated materials were evaporated under vacuum ( 5). Then column was packed with silica gel and the extracted materials were poured to the top of the gel in column and eluted with hexane to remove fats. Other impurities were removed by a mixture of hexane , ether and ,acetonitrile in the ratio of 1 : 3 : 6 respectively. Then column was eluted with a mixture of dichloromethane and acetone. Aflatoxins were obtained after evaporation of the organic solvent and again the sample was dissolved in a known volume of dichloromethane and it was prepared for thin layer chromatographic and high performance liquid chromatographic determination. Thin layer chromatography has been a convenient and simple method for analysis. The solvent system employed was isopropanol, acetone and chloroform ( 8 : 10: 82 ) and aflatoxins Bl and Ml were detected qualitatively under UV after sprayed with 50 % sulfuric acid . For quantitatively determination we employed HPLC which was equipped by UV-detector at 244nm and the mobile phase was actonitrile, methanol and water in the ratio of 45:25:30 respectively RESULTS AND DISCUSSION

With regards to the method validity those samples which were positive in TLC were analyzed by HPLC and the results are shown in Table 1. The HPLC chromatograms of standard aflatoxin Bl and aflatoxin Ml are shown in fig. 1.In fig 2. the HPLC chromatogram of a contaminated sample is shown. In this study 100 liver samples were collected from different local poultry and were analyzed for aflatoxins Bl and Ml contamination by TLC and HPLC and emphasis was given to HPLC method. The impact of mycotoxins on human and animal health is well understood and to obtain a clear view on food products it is worthy to carry out the best sensitive and accurate method of analysis because aflatoxin B 1 is recognized as a potent toxic carcinogenic substance ( 6, 7 ) and its metabolite aflatoxin M! in the biological fluids. The results from this investigation showed some contamination but it is not in the range of high toxic level as shown in Table 1. Results on HPLC showed that the highest levels of aflatoxins Bl and Ml were 0.71 and 1.1 g/100 grams of samples respectively. On the other hand the minimum concentration of aflatoxin Bl and Ml which were detected by TLC were 0.11and0.14ug/100gramsofsamplesasjudged by HPLC. According to the previous reports these levels of aflatoxin Bl and Ml are considered as safe but it should be noted that the production of aflatoxins may be accelerated by improper production and handling of feeds. REFERENCES 1 ) Smith JE, Solomons G, Lewis C and Anderson S. The role of mycotoxins in human and animal nutrition and health. Nat. Toxins 1995 ,3(4); 187- 192 2 ) Betina , V . Mycotoxin production, isolation , separation and purification .Elsevier Scientific publishers , Amsterdam 1984 . 3 ) Ciegler A , Vesonder R, F . CRC handbook of foodborn diseases of biologic origin Ed. M, Fechcigl ,CRC press Inc. , Boca Raton , Florida 1983 , 57- 166 4 ) Smith ,J, E. and Moss M , 0 . Mycotoxins formation ,analysis and significance, John Wiley and sons Ltd. 1985 . 5 ) Williams S . 1984 Official Methods of Analysis ( AOAC ) 14th ed. Virginia USA 1141 p. 6 ) Coker , R ,D . and Jones ,B , D . Determination of mycotoxins 1988 pp .335 375 Academic press New York . 7 ) Ramos A, J. and Hernandez ,E . Prevention of aflatoxicosis in farm animals by means of hydrated sodium alumininosilicate addition to feed stuffs ; a review. Animal Feed Science Technology 1997, 65 , 197-206 .

FIGURES AND TABLES Figure 1. HPLC Chromatogram of standard aflatoxin Bl and Ml

Figure 2. HPLC Chromatogram of contaminated sample.

Figure 3. Column chromatogram.

Table 1: Liver samples were analysed for Aflatoxin Bl and Ml by HPLC Aflatoxin Total Positive Maximum Minimum samples samples Conc. g/100r Conc. g/100g Bl Ml 100 100 43 43 0.71% 101% 0.11 0.14

Mean conc. of positive sample g/100g 0.46 0.6

SD

0.17 0.28