Determination of thyreostats in urine and thyroid gland by ultra high performance liquid chromatography tandem mass spectrometry
Introduction
In recent years highly sensitive method for the determination of thyreostats in urine and other matrices has been reported [1], [2], [3], [4]. The method is based on the derivatisation of compounds with 3-iodobenzylbromide (3-IBBr) prior to purification. This strategy presents obvious advantages: (1) after derivatisation the chemical structure of thyreostats is stabilized under a single tautomeric form which makes the subsequent extractions more amenable, (2) the more apolar nature of the resulting compounds leads to improved retention and separation characteristics on the reversed-phase support, (3) the molecular mass of these analytes is significantly increased making the low molecular mass noise less significant and (4) halogenated derivatives ionize very intensively, enabling high measurement sensitivity and low detection limits to be achieved.
The aim of this study was to develop and validate in accordance with Commission Decision 2002/657/EC [5] a routine and robust method for the determination and confirmation of thiouracil (TU), methylthiouracil (MTU), propylthiouracil (PTU), phenylthiouracil (PhTU), tapazole (TAP) and mercaptobenzimidazole (MBI) in urine and thyroid gland samples. The method is based on the above approach. The recommended concentrations of TU, MTU, PTU and TAP in urine and thyroid gland samples have been lowered now to 10 μg kg−1 [6]. This means that in EU laboratories decision limit (CCα) and detection capability (CCβ) values of the method should be lower than 10 μg kg−1.
Lately new studies, employing different analytical strategies to determine thyreostats in some tissues have been published. Asea et al. [7] have developed a method which is able to detect many thyreostatic drug residues in thyroid gland and muscle at a concentration of 5 μg kg−1. However, this method is applicable only to screening purposes. The methods developed by Abuin et al. [8], [9] are based on the detection of underivatised thyreostats and therefore suffer for unacceptable values of CCα and CCβ for many compounds, especially for TAP. Moreover, a very recent review by van den Bussche et al. [10] gives an insight into all the important methods developed during the last 35 years which are based on the TLC, GC and LC separation to determine thyreostats in various matrices.
Section snippets
Standards and reagents
The reagent grade diethyl ether was from Riedel-de Haen (Seelze, Germany). All other organic solvents were of HPLC grade and purchased from Rathburn Chemicals (Walkerburn, Scotland). 3-IBBr was obtained from Aldrich (St. Louis, MO, USA). Reagent grade hydrochloric acid (37%) was obtained from Riedel-de Haen. Potassium dihydrogen phosphate, sodium hydroxide and di-sodium hydrogen phosphate were of analytical grade and were purchased from Merck (Darmstadt, Germany). Anhydrous acetic acid
UPLC and MS parameters
During the instrument tuning step, in addition to acetic acid, formic acid and ammonium acetate based solutions were also evaluated as candidate mobile phase additives but were discarded because of the considerably lower ion current signals obtained. The less viscous acetonitrile was introduced as an organic modifier of the mobile phase instead of methanol used in the original study [1]. This change was done mainly because of high pressure concerns under UPLC conditions. Also different
Conclusions
The aim of the study was to optimise and validate UPLC–MS/MS methods for the quantification and confirmation of six thyreostats in urine and thyroid gland samples. Sensitive methods were also made reproducible by the use of 2H-labelled internal standards. The fit-for-purpose CCα and CCβ (as well as in-house reproducibility) values were obtained for routine surveillance purposes. The UPLC instrumentation used enabled shorter analysis times and, consequently, a higher throughput to be achieved.
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