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Abstract: The widespread contamination of foodstuffs and beverages by mycotoxins, such as ochratoxin A (OTA), has made the monitoring of human contamination levels essential. By using a sensitive, accurate and speedy method that combines extraction with 5% NaHCO3, immunoaffinity column clean-up and HPLC with fluorescence detection, the human exposure to OTA through urine analysis can be monitored. This method is less invasive than blood monitoring and has the potential to be a good marker of human exposure. The limit of quantification of the method was 0.007ng/mL of urine, with recoveries of OTA, from urine samples spiked at levels between 0.02 and 0.1ng/mL, higher than 91% with RSD lower than 15.5%. This study evaluated OTA contamination levels in human urine sample fractions, collected in the morning and afternoon, in two populations, one from Coimbra city, in Portugal, and another from the Valencian community, in Spain. In the Coimbra population, 60 samples from 30 healthy individuals were analyzed, levels of OTA in 13 morning samples and 14 afternoon samples having been detected, with concentrations ranging from 0.011 to 0.208 and 0.008 to 0.11ng/mL respectively. In the Valencia population, 62 samples from 31 healthy individuals were analyzed, with OTA being detected in 25 morning samples and 26 afternoon samples. The concentrations varied between 0.007 and 0.124ng/mL in the morning samples, and 0.008 and 0.089ng/mL in the afternoon samples. Significant differences were found between the morning levels of OTA from both populations (P=0.033). For afternoon samples, significant differences were not found, P value=0.163. [Copyright &y& Elsevier]