Validation of a quantitative method using liquid chromatography coupled to multiple mass spectrometry for thiouracil in feedstuffs used in animal husbandry

Abstract

The use of thyreostatic drugs, like thiouracil (TU), in animal production has been banned for over three decades by the European Union, due to potential teratogenic and carcinogenic effects of its residues upon human consumption. Besides, thyreostats induce water retention in livestock, causing fallacious weight gain and deterioration of meat quality. Development of more competent analytical methods gave rise to sporadic TU detection in urine of untreated animals, questioning the actual synthetic origin TU. Research showed that TU can be formed upon digestion of Brassicaceae feeds in vivo and in vitro, which called for a means of differentiation between endogenous formation of TU and illicit administration. Therefore, in the present study, a routinely applicable liquid chromatography (LC) ion trap multiple mass spectrometry (MS(2)) method for TU analysis in animal feeds was optimised and validated, according to CD 2002/657/EC. A fractional factorial Plackett-Burman design was used to optimise the extraction procedure for TU from Brassicaceae and non-Brassicaceae feeds. This resulted in the discrimination of five influential factors (amount of feed, myrosinase, pH 7 buffer, 3-iodobenzyl bromide and elution solvent), for which the most optimal conditions were perfected. The limit of quantification for TU amounted 0.5 ng g(-1). The individual recoveries for TU ranged between 90.9 and 99.7%. Good results for repeatability and intra-laboratory reproducibility (RSD%) were observed, i.e. ≤6.0 and ≤5.2%, respectively, for TU. Excellent linearity was proven based on determination coefficient (R(2) ≥ 0.99) and lack-of-fit test (F test, α = 0.05). Subsequently, a selection of feeds sampled during European national monitoring campaigns were evaluated with the present method showing concentrations ranging from 0.32 to 20.60 ng g(-1), demonstrating the relevance of the method in the analysis of TU from animal feeds.

Determination of Thyreostats in Bovine Urine and Thyroid Glands by HPLC–MS/MS

“…However, a relationship between the presence of Brassicaceae in feed and thiouracil in urine has been demonstrated by Pinel et al [9], Vanden Bussche et al [14] and Kiebooms et al [15,16]. The Community Reference Laboratories (CRLs) in 2007 proposed a recommended concentration of 10 μg L −1 in urine and 10 μg kg −1 in thyroid tissue for the purpose of control, as "low concentrations of thiouracil have been detected in bovine animals fed with cruciferous plants, however, there is scientific evidence showing that levels above 10 ppb in urine cannot be linked to natural origin due to this contamination" [17].…”

Determination of Thyreostats in Urine Using Supported Liquid Extraction and Mixed-Mode Cation-Exchange Solid-Phase Extraction: Screening and Confirmatory Methods

“…The liquid chromatography-tandem mass spectrometry (LC-MS/ MS) is the most suitable technique to analyze thyreostats in complex matrices at ≤RC (10)(11)(12)(13)(14). The four thyreostats mentioned above are polar and weak basic compounds (logP: −0.62-1.39, pK a : 4.41-8.…”