Abstract
BACKGROUND: Milk can sometimes carry harmful mycotoxins, which pose health risks, especially in hot and humid areas. We collected samples from both farms and households in Khyber Pakhtunkhwa (KP), Pakistan, to see how common these toxins are, whether the genes that make them are present, and which environmental factors might make contamination worse. METHODS: We tested raw milk for aflatoxins (AFM1, AFM2), ochratoxin A (OTA), and zearalenone (ZEN) using TLC, HPLC, and UHPLC-MS/MS. All testing procedures were validated following ICH Q2(R2), ISO 17025, and FDA guidance. PCR was applied to check for the genes aflC, otaA, and zen1. We then combined chemical, molecular, microbial, and environmental data and used multivariate statistics and PLS-DA modeling to find the main factors driving contamination. RESULTS: Milk from farms had higher mycotoxin levels than household milk. Average AFM1 concentrations were [Formula: see text]g/kg in farm milk and [Formula: see text]g/kg in domestic milk (p < 0.001). AFM2 and OTA showed similar patterns, while ZEN was below detection in all samples. UHPLC-MS/MS confirmed the HPLC findings and offered greater sensitivity. The genes aflC and otaA were found in 68% of farm samples and were strongly linked to AFM1 and OTA levels, while zen1 was absent. High temperature (over 28°C) and humidity (over 75%) were associated with increased contamination. PLS-DA modeling effectively distinguished high- and low-risk samples (AUC = 0.92), highlighting AFM1 concentration, aflC presence, and humidity as key predictors. CONCLUSIONS: Combining chemical testing, gene screening, and environmental monitoring provides a practical way to detect and evaluate mycotoxin risk in milk. Farm milk showed higher contamination than household milk, emphasizing the need for targeted monitoring and preventive measures. Identifying environmental thresholds and risk factors can support early interventions to improve food safety and protect public health.