Identification and quantification of metabolites of the fungicide tebuconazole in human urine

Tebuconazole (TEB) is a fungicide used in agriculture; the objective of this work was to identify and quantify TEB metabolites in human urine. Samples from seven vineyard workers exposed to TEB were submitted to liquid chromatography interfaced with a triple quadrupole mass spectrometer, equipped with an electron spray source, and a linear ion trap to gain a profile of candidate metabolites. Based on the presence of the ion m/z 70 in the MS/MS spectra, which corresponds to protonated triazole (a specific moiety of TEB), and the isotopic pattern of the molecular ions, typical of molecules with one chlorine atom, hydroxyl and carboxyl derivatives of TEB, that is, TEB-OH and TEB-COOH, were identified as major metabolites, both as free molecules and as glucuronide (Glc) conjugates. The mean molar fractions were 0.67, 0.13, 0.13, and 0.07 for TEB-O-Glc, TEB-OH, TEB-COO-Glc, and TEB-COOH. Urine samples were submitted to hydrolysis with β-glucuronidase, and the free compounds were quantified in the presence of deuterated TEB (TEB-d₆) as the internal standard (IS), by multiple reaction monitoring (MRM) mode. The assay was linear in the ranges of 0.2-600 μg/L and 0.1-240 μg/L for TEB-OH and TEB-COOH, respectively; precision, accuracy, and the limit of quantification (LOQ) were 6 controlled these sources of bias. The urinary levels of TEB-OH and TEB-COOH in specimens collected from farmers exposed to TEB ranged from 10 to 473 and from 3 to 159 μg/L, respectively.