VALIDATED HPTLC METHOD FOR AFLATOXIN B1 DETECTION IN FEED INGREDIENT AND FEED SAMPLES

Abstract

High performance thin layer chromatographic method was developed and validated according to the protocol on “Validation of Analytical Procedures: Methodology, Veterinary International Cooperation on Harmonization (VICH)” with respect to linearity, sensitivity, precision and accuracy for determination of aflatoxin B1 in feed ingredients and feed. Chromatography was performed on thin layer chromatography (TLC) silica gel 60F254, aluminum sheets by Camag Linomat-5 applicator, with mobile phase condition acetone : chloroform (1 : 9). Analysis of samples viz. feed ingredients and feed, for aflatoxin B1was carried by HPTLC method and compared with TLC method. Extraction of aflatoxin was done as per AOAC method with screening and quantification by TLC and further quantification by HPTLC using reference standards. Out of 38 samples of nine types of feed ingredients analysed, samples of Bengal gram and rice bran & wheat bran mixture were negative by both methods. The other ingredients like cumbu/bajra, de-oiled rice bran, groundnut oil cake, maize, soyabean meal and sunflower oil cake, by HPTLC method wherein the Aflatoxin B1was found to be ranging from 1.61 ppb to 630.73 ppb of 77.42% positive samples, whereas by TLC method it was from 05 ppb to 140 ppb in 70.97% positive samples. While 4 samples of wheat bran analysed were all negative for Aflatoxin B1 by TLC method, whereas 50% (2 samples) found to be positive with HPTLC method with concentration ranging from 2.73 to 17.88.Similarly out of 59 feed samples analysed, 47 and 46 samples were positive for Aflatoxin B1 representing 79.66% and 77.97% of the samples, with concentration ranging from 0.54 ppb to 204.72 ppb and from 05 ppb to 710 ppb by HPTLC and TLC respectively. In the present study, the Limit of detection by HPTLC was 0.5 ppb whereas it was 5 ppb with TLC method.