Abstract

With increasing knowledge of the persistence of OTA in the food chain, exposure to this mycotoxin is a potential human health hazard to humans, and evaluating its presence in populations has become highly important.

A sensitive and accurate analytical method for the determination of ochratoxin A in urine was validated, since is less invasive than blood monitoring. It involves extraction with 5% NaHCO₃, immunoaffinity column (IAC) for clean-up and high performance liquid chromatography with fluorescence detection (HPLC-FD). The limit of quantification was 0.02 ng/mL of urine (1.3 ng/mL of the extract injected) and recovery of ochratoxin A from urine samples spiked at the three fortification levels, were higher than 90% with RSD lower than 9%. The identification of OTA was confirmed by methyl ester derivatization and then HPLC analysis.

Based in ours first results we can assume that OTA conjugation with glucuronic acid in human urine occurs. In the present study, we follow up OTA levels in 60 urine samples of inhabitants from Coimbra city, Portugal, in order to evaluate population contamination, and the presence of OTA was found in 42 samples, at concentrations above the LOQ, ranged between 0.021 and 0.105 ng/mL.

Keywords: Food safety; Mycotoxins; Ochratoxin A; Urine; HPLC; Fluorescence detection

Abbreviations: AOAC; BEN, Balkan endemic nephropathy; FD, fluorescence detection; HPLC, high performance liquid chromatography; IAC, immunoaffinity column; IARC, International Agency for Research on Cancer; MS, mass spectrometry; LOQ, limit of quantification; OTA, ochratoxin A; RSD, relative standard deviation; UTT, urinary tract tumors

Article Outline

1. Introduction

2. Experimental

2.1. Solvents and materials

2.2. HPLC system and chromatographic conditions

2.3. Extraction and clean-up

2.4. Sampling

2.5. Chemical confirmation of OTA by methyl ester formation

2.6. Assay with β-glucuronidase

2.7. Statistical analysis

3. Results and discussion

3.1. HPLC conditions optimization

3.2. Extraction and clean-up optimization

3.3. Analytical method validation

3.4. Confirmation by methylation of OTA

3.5. Stability study

3.6. Study with β-glucuronidase

3.7. Evaluation of OTA in urine samples

4. Conclusions

Acknowledgements

References