High levels of mycotoxin contamination have been reported in various food commodities in Pakistan, however, there has been no exposure assessment study using multiple mycotoxins' biomarkers. This study aimed to simultaneously assess the exposure to the five major mycotoxins: aflatoxin B 1 (AFB 1 ), deoxynivalenol (DON), fumonisin B 1 (FB 1 ), ochratoxin A (OTA) and zearalenone (ZEN) in a Pakistani population using an integrated approach of human biomonitoring. Human urine samples ( n = 292) were analyzed by a super-sensitive liquid-chromatography tandem mass spectrometry (LC-MS/MS) method. Rice and wheat were also collected and analyzed for mycotoxins by the LC-MS/MS method. Food consumption data were collected using a 24 h recall method. A high prevalence of urinary AFM 1 (66%, mean ± SD 20.8 ± 41.3 pg/mL) and OTA (99%, 134.7 ± 312.0 pg/mL) were found, whilst urinary DON, FB 1 and ZEN levels were low. The probable daily intake (PDI) derived from the urinary biomarkers revealed that 89% of the participants had exposure to OTA exceeding the established tolerable daily intake (TDI = 17 ng/kg bw/day). The average PDI of AFB 1 for the studied population was 43 ng/kg bw/day, with rice as the main source of AFB 1 exposure. In summary, exposure to AFB 1 and OTA are of health concern and require further management.
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