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Aflatoxin M1 (AFM1) and ochratoxin A (OTA) are highly toxic mycotoxin metabolites that are found as food pollutants, posing health risks to humans and animals. The objective of the current study is to establish a sensitive, reliable method for determining AFM1 and OTA using high-performance liquid chromatography (HPLC) and attempting to assess the efficacy of bentonite, date pit, and chitosan nanoparticles for AFM1 and OTA detoxification from contaminated milk. As revealed, AFM1 was found in 65.7% of analyzed samples ranging from 4.5 to 502 ng/L, while 25.7% of examined samples contained OTA ranging from 1.45 to 301 ng/L. Furthermore, for AFM1 and OTA. The advanced procedure was thoroughly validated by evaluating linearity (R2?>?0.999), LOD (0.9615 and 0.654 ng/L), and LOQ (2.8846 and 1.963 ng/L), recovery (93–95% and 87–91%), as well as precision (≤?1%RSD). The experimental data revealed a higher removal efficiency of bentonite and date pit than chitosan nanoparticles in the case of AFM1 (68%, 56%, and 12%) and OTA (64%, 52%, and 10%), respectively with slight change in nutritional milk components like fat, protein, and lactose. Eventually, it is concluded that bentonite and date pit can be considered efficient adsorbing agents to extract AFM1 and OTA from contaminated milk.

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