BackgroundAflatoxin is a toxic cancerous fungal secondary metabolite produced by Aspergillus species. Consumption of aflatoxin-contaminated cereals and their products can cause several health-related effects including acute and chronic aflatoxicosis. This study aims to evaluate the total aflatoxin content in “Cheka” and its major ingredients using the Immuno-Affinity Column Clean-up technique by sensitive HPLC (High-Performance Liquid Chromatography) with fluorescence detection.MethodsThis study followed a completely randomized experimental design to evaluate the level of aflatoxin in traditionally brewed “Cheka” and its major ingredients (maize) by HPLC from November 2019 to June 2020. Traditionally brewed 6 “Cheka” beverages and 6 maize seed samples were collected randomly from Konso and transported to Bless Agri Food Laboratory in Addis Ababa for laboratory evaluation of aflatoxin in samples. The data were analyzed by SPSS version 25.ResultsIn the present study aflatoxin contamination was detected in all maize and “Cheka” samples. A mean total aflatoxin level of 6.78 ± 8.28 μg/kg and 10.76 ± 4.50 μg/kg were observed in maize and “Cheka” samples respectively. The maize taken from above-ground storage (“Gotera”) and underground storage (pitfall) showed a mean total aflatoxin level of 10.23 ± 10.12 μg/kg and 3.35 ± 5.79 μg/kg respectively. The samples taken from “Cheka” prepared from maize of above-ground storage (“Gotera”) and underground storage (pitfall) showed a mean total aflatoxin level of 11.34 ± 3.06 μg/kg and 10.19 ± 6.35 μg/kg respectively. Aflatoxin G family was not detected in both maize and “Cheka” samples while aflatoxin B1 and B2 contributed to the total aflatoxin level. AFB1 predominantly contributed to the contamination of all samples. Although there was no significant difference while comparing between mean total aflatoxin of any other sample types, a significant difference was observed for the AFB1 mean level of pitfall maize and their respective “Cheka” samples (P = 0.033).ConclusionThe mean level of AFB1 in pitfall maize differs from a respective “Cheka” sample. As most of the samples evaluated for aflatoxin contamination showed levels above healthy cut-off points of aflatoxin set by different authorities like the European Union, CODEX, and Ethiopian Standards Agency, there must be a protracted interventional approach to reduce aflatoxin contamination in maize and its products in the region.