Indo-Am. J. Agric. & Vet. Sci., 2014 Ashok Kumar ISSN D and 2321 9602 E Sunil anand www.iajavs.com Kumar, 2014 Vol. 2, No. 1, March 2014 2014 Meghana Publications. All Rights Reserved Research Paper DETERMINATION OF MYCOTOXIN FROM FEED SAMPLE BY THIN LAYER CHROMATOGRAPHY Ashok Kumar D 1 * and E Sunil anand Kumar 1 *Corresponding Author: Ashok Kumar D ashok9901@gmail.com Thin Layer Chromatographic (TLC) method is described for the determination of Citrinin in feeds. Citrinin is extracted from feed with acetonitrile and hexane. The solution is filtered and extracted with chloroform to remove most of the interfering materials. The extracted chloroform residue is diluted and spotted on the thin layer chromatographic plate, which is developed in Toluene-Ethyl acetate-formic acid with a ratio of 5:4:1 in equilibrated chamber. The Citrinin is viewed under ultra violet transilluminator after running the TLC plate. The proposed method can eliminates the streaks and poor sensitivity of citrinin on TLC plate and permits an accurate method for determination of citrinin by using Thin Layer Chromatographic plate from feed samples. Keywords: Thin Layer Chromatography, Quantification, Determination, Mycotoxin, Citrinin, Feed sample INTRODUCTION Citrinin is a toxic secondary fungal metabolite and isolated as a pure compound from a culture of Pencillium citrinum by Hetherinton and Raistrick (1931) later, yellowish coloured rice, imported from Thailand to Japan in 1951 was found to contaminated with Pencillium citrinum. Citrinin was reported to be a food contaminant in tribal areas of Medak District in Andhra Pradesh (Reddy et al., 1986). Citrinin was identified in compounded feed and various feed ingredients used in poultry rations in Namakkal area Tamilnadu (Ahamad and Vairamuthu., 2000). Dietary ingestion of grains contaminated by citrinin caused kidney and liver damage and alterations in morphology of the intestinal villi in broiler chicken (Ames et al., 1976 and Witlock et al., 1977) and also seen in rats, dogs and rabbits (Ramadoss et al., 1973). Citrinin was detected colorimetrically by using a ferric chloride complex of citrinin during the yellowish coloured rice problem in post war japan, later a fluorometric method for detecting citrinin in rice was developed by (Kawashiro et al., ). The first Thin Layer Chromatography (TLC) method for screening the citrinin was developed by (Scott 1 Department of Veterinary Biochemistry, College of Veterinary Science, Rajendranagar, Hyderabad, Andhra Pradesh 500 030, India. 2 Department of Veterinary Gynaecology & Obstetrics, College of Veterinary Science, Rajendranagar, Hyderabad, Andhra Pradesh 500 030, India. 23
Indo-Am. J. Agric. & Vet. Sci., 2014 Ashok Kumar D and E Sunil anand Kumar, 2014 et al., 1970) but the citrinin appeared as streaks on the TLC plate and could not be quantified. Although several methods have been described with poor sensitivities were reported for citrinin. This paper presents a technique that eliminates the streaks and poor sensitivity of citrinin on TLC plate and permits an accurate method for determination of citrinin by using Thin Layer Chromatography. MATERIALS AND METHODS The structure of citrinin was proposed by Brown et al (1989), C 13 H 14 O 5. Molecular weight 250, forms lemon yellow needles (melting point 175 0 C) when crystallized from absolute enthanol or benzene-chlorohexane. It is soluble in hot enthanol, ethyl acetate, benzene, acetone and chloroform and insoluble in water (spector et al., 1957) About 10 g of ground maize sample was weighed and transferred to blender jars and blended at high speed for 3 minutes with 36 ml of acetonitrile, 4 ml of KCl and 0.8 ml of 5 N HCl solutions. Filtered the blended solution through Whatmann filter paper No:1. Take 20 ml of filterate and then transferred into 250 ml separation funnel. 20 ml of water and 20 ml of hexane were added and shake well. Collected the bottom acetonitrile phase into another 20 ml of hexane was added and shake well, then collect the bottom acetonitrile phase. The resulting acetonitrile phase is extracted with two 10 ml portions of chloroform into beaker the extracted portion of chloroform beaker was placed at 50-60 0 C in hot air oven for 30 min. So that the chloroform was evaporated completely and the extract is attached to the walls of beaker. Then the residue is dissolved in 0.2 ml of chloroform by proper mixing with glass rod. The commercially available (20 x 20 cm) TLC plate was collected and mark for spottings then place the TLC plate in hot air oven at 100 0 C for 5 min for activation then dip the activated TLC plate on 10% oxalic acid solution and allowed to dry. The 2,5,10,15 and 20 µl of chloroform residues were spotted on 10% oxalic acid dipped activated TLC plate with the citrinin standard spots (2,5,10,15 and 20 µl). The spotted plate was developed using Toluene-Ethyl acetate-formic acid with a ratio of 5:4:1 in equilibrated chamber. After running the mobile phase over the stationary phase, the TLC plate was dried and viewed under long wave length ultra violet transilluminator and the spots were identified. The intensity of the fluorescent spots of the samples were compared with that of standard spots (citrinin appeared as lemon yellow colour). The citrinin content was calculated by the formula S C D Citrinin (ppb) = 1000 T E where S = standard volume which matches with test volume in fluorescence intensity C = concentration of standard (5µg/ml) T = test volume which matches with standard volume in fluorescence intensity E = effective weight of sample (4 g for 10 g of sample). CONCLUSION Thin Layer Chromatography is widely used technique for determination of mycotoxins. The Citrinin is viewed under ultra violet transilluminator after running the TLC plate.the mobile phase used in this study was Toluene-Ethyl acetate-formic acid with a ratio of 5:4:1 in equilibrated chamber. This particular mobile phase can eliminates the streaks and more sensitivity and permits an accurate method for determination of citrinin by using Thin Layer Chromatographic plate from feed samples. 24
Indo-Am. J. Agric. & Vet. Sci., 2014 Ashok Kumar D and E Sunil anand Kumar, 2014 ACKNOWLEDGMENT The authors are thankful to Sri Venkateswara Veterinary Science, Tirupati; and Dr. G V Narasa Reddy, Retd. Professor and Head, Department of Veterinary Biochemistry, Hyderabad, for providing necessary facilities to carry out this work. REFERENCES 1. Ahmad D B and Vairamuthu S (2000), Occurrence of citrinin mycotoxin in compounded feeds and various feed ingredients of poultry ration in Namakkal area (Tamilnadu), Indian Journal of Environment and Toxicology, Vol. 10, No. 2, pp. 84-86. 2. Ames D D et al. (1976), Effect of Citrinin, on laying hens and young broiler chicks, Poultry Science, Vol. 55, pp. 1294-1301. 3. Brown T P, Manning R O, Fletcher O J and Wyatt R D (1989), The individual and combined effects of citrinin and ochratoxin A on renal ultra structure in layer chicks, Avian Diseases, Vol. 30, No. 4, p. 1634. 4. Hetherington A C and Raistrick H (1931), Studies in the biochemistry of microorganisms, Part XIV on the production and chemical constitution of a new yellow colouring matter, citrinin produced from glucose by pencillium citrinin, Phil. Trans. Roy. Socio economic, London, Vol. 20, pp. 267-269. 5. Kawashiro I, Tanabe H, Takeuchi H and Nishimura C (1955), Chem. Abstr, Vol. 50, p. 7229. 6. Ramadoss C S and shanmugasundaram E R B (1973). Effect of citrinin on liver metabolism in rabbits. Indian Jounal of Biochemistry and Biophysics 10: 296-297 7. Reddy A S and Reddy S M (1983), Current science, Vol. 52, p. 613. 8. Scott P M, Walbeck W, Harwig J and Fennel D I (1970), Canada Journal of Plant Science, Vol. 50, pp. 583-585. 9. Spector W S (1957), Toxicology II Antibiotics Hand book, Saunders company, Philadelphia, London, p. 56. 10. Witlock D R, Wyatt R D and Ruff M D (1977), Morphological changes in the avian intestine induced by citrinin and lack of effect of aflatoxin and T-2 toxin as seen with scanning electron microscopy, Toxicon, Vol. 15, pp. 41-44. 25