Validation of a UHPLC–MS/MS method for quantification of zearalenone, α-zearalenol, β-zearalenol, α-zearalanol, β-zearalanol and zearalanone in human urine

• A method to determine zearalenone and its five metabolites in human urine is proposed.
• The method involves LLE with ethyl acetate/formic acid and UHPLC–MS/MS detection.
• The method was completely validated and LODs ranged from 0.03 to 0.3 ng mL−1.
• The method was applied to urine samples from healthy women who live in Tunisia.

Humans can be exposed to mycotoxins through the diet. Evaluation of exposure levels to mycotoxins can be performed by direct determination in urine. The present work proposes a sensitive ultrahigh-performance liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method for the determination of zearalenone (ZON) and its five metabolites (α-zearalenol [α-ZOL], β-zearalenol [β-ZOL], α-zearalanol [zeranol, α-ZAL], β-zearalanol [teranol, β-ZAL] and zearalanone [ZAN]) in human urine samples. The method involves the enzymatic hydrolysis of the samples, extraction of the analytes using liquid–liquid extraction (LLE) with ethyl acetate/formic acid (99:1 v/v) and a cleanup step using hexane, prior to their quantification by UHPLC–MS/MS, using an electrospray ionization (ESI) interface in the negative mode. Zearalenone-d6 (ZON-d6) was used as surrogate. The limits of detection and the limits of quantification ranged from 0.03 to 0.3 ng mL−1 and from 0.1 to 1.0 ng mL−1, respectively. The method was validated using matrix-matched calibration and a spike recovery assay. Recovery rates for spiked samples ranged from 96% to 104%, with relative standard deviations lower than 8.5%. This method was satisfactorily applied to 42 urine samples from Tunisian women for the determination of zearalenone and its five metabolites.