Quantification of aflatoxins in animal feed in Punjab, Pakistan, using HPLC-FLD and confirmation by LC-MS/MS

Animal feedstuffs are prone to fungal contamination due to their protein content and high moisture levels at harvest and storage, often leading to mycotoxin production. This study investigated the occurrence of aflatoxins in animal feed and the feed ingredients cottonseed cake and maize oil cake, collected from livestock farms in Okara, Punjab, Pakistan (2021-2022). Aflatoxins were extracted with an acetonitrile - water mixture, purified with Mycosep columns, quantified by HPLC with fluorescence detection and confirmed by mass spectrometry. Aflatoxins were detected in 74% of the animal feed samples, in 87% of the cottonseed cake samples and in 63% of the maize oil cake samples. Notably, 33% of animal feed and 70% of oilseed cake samples exceeded the maximum limit of 20 µg/kg. These findings revealed a high level of aflatoxin contamination, particularly in oilseed cakes, with statistically significant results (<i>p</i> < 0.05), highlighting potential animal health risks.