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

The use of thyreostats in livestock is strictly forbidden by European legislation since 1981. The investigation of thyreostats is commonly performed by their detection as derivatives with 3-iodobenzylbromide. Although it has advantages, the derivatisation procedure can generally cause a decrease in analyte concentrations. With the aim of simplifying the analysis of five thyreostats in both bovine urine and in thyroid glands, two methods were developed without the derivatisation step. Salting-out assisted liquid–liquid extraction was carried out for both matrices, followed by high-performance liquid chromatography coupled with triple-quadrupole mass spectrometry analysis. The methods were validated in agreement with the guidelines of Commission Decision 2002/657/EC. For all the thyreostats evaluated, satisfactory results were achieved; the recovery was within 96–104 % for both the matrices, while precision (coefficient of variation) was less than 20 % for urine and 21 % for thyroid glands. The limits of decision and capacities of detection for all the compounds were lower than the recommended values of 10 μg L−1 and 10 μg kg−1, respectively. In urine, the limits of decision ranged from 6.9 to 7.3 μg L−1, and the capacities of detection ranged from 8.5 to 9.7 μg L−1, while in thyroid glands these values varied from 6.6 μg kg−1 to 7.4 μg kg−1 and from 8.0 μg g−1 to 9.7 μg kg−1, respectively. The results obtained show that the methods described are suitable for the direct detection of thyreostats in bovine urine and thyroid glands.


9
Thyreostats are drugs that interfere with the mechanism involved in the synthesis of thyroid hormones and cause a condition of deficiency of circulating thyroxine (T4) and triiodothyronine (T3) [1,2], whose production and release are 11 controlled by the hypothalamus-anterior pituitary axis. The hypothalamus secretes thyrotropin-releasing hormone (TRH), 2-mercapto-imidazole (tapazole, TAP) [4-6]. The chemical structures of these substances are shown in Figure 1.

23
The fraudulent use of thyreostats produces low quality meat. Moreover, the edible tissues derived from treated 24 animals might represent a potential risk to the consumer's health due to the presence of residues and their teratogenic and  Thyreostats analyses typically consist of separation methods based on gas or liquid chromatography associated 38 with a mass spectrometry system of detection. Normally, the extraction of the substances is carried out by using polar 39 solvents more suitable to the chemical characteristics of the thyreostats, such as methanol, acetonitrile or ethyl acetate.

40
Further steps of purification or clean-up with different kinds of solid-phase extraction (SPE) have been reported. Due to 41 the low molecular mass and high polarity of the thyreostats, several authors have proposed a derivatisation step before or 42 after the clean-up, mainly by using 3-iodobenzylbromide (3-IBBr) in the case of HPLC-MS/MS analysis [6]. In the case 43 of GC methods, derivatisation is an unavoidable step in order to convert the analytes into volatile compounds. When

44
HPLC is applied as the separation technique, analytes may be derivatised and, in the analysis of thyreostats, this procedure 45 induces the stabilisation of the chemical structure of the molecule in a specific and single tautomeric form, the reduction 46 of the molecular polarity in order to increase the separation characteristics on the reversed-phase column in the case of 47 HPLC-MS detection, and an increase in the molecular mass [20]. The low molecular mass, particularly, could be disturbed 48 by the chemical noise. In term of sensitivity, the derivatisation leads to an improvement of the signal to noise ratio, and 49 subsequently of the detection capabilities [21]. Despite these advantages, the derivatisation procedure can generally cause 50 a loss in analyte concentrations. Furthermore, removing derivatisation step simplifies, shortens and makes cheaper the 51 whole analysis procedure [22,23]. Based on these observations, we developed the extraction without derivatisation of the 52 five above-mentioned thyreostats in bovine urine and thyroid glands followed by a sensitive, specific and reproducible

79
NaCl (2 g) was added to the solution to mixture as a salting-out reagent.

80
The extraction was performed by twice repeating these steps: addition of 5 mL tert-butyl methyl ether,

102
The mobile phase consisted of 0.1% aqueous formic acid (solvent A) and methanol (solvent B), and the flow rate was set 103 at 200 L/min. The gradient program is shown in Table 1

120
The method validation parameters were determined with fortified blank urine and thyroid gland samples at three 121 concentration levels (5.0, 10, 15 μg L -1 and μg kg -1 ) in six replicates on three different days (6 samples × 3 concentration 122 levels × 3 series = 54 analyses). Method recovery and precision were evaluated using the matrix curves; recovery is 123 calculated as ratio between the measured concentration to fortified concentration, corrected by internal standard and 124 expressed in percentage; precision is calculated in terms of intra-and inter-day repeatability expressed as the coefficient 125 of variability (CV). The same data from the matrix calibration curves were used to calculate the decision limit (CCα) and

Results and Discussion
Despite the diversity of the matrices analysed, we carried out two similar methods to prepare urine and thyroid glands in 132 order to have the same steps for each matrix.
a reducing agent, such as DTT. Differently from the above mentioned study, which considered only urine, we adopted 136 this step for both urine and thyroid glands, with a ten-time-lower concentration of DTT.

137
The polarity of the thyreostats requires the use of an organic polar solvent to extract them from the matrices: we             PhTU 6.9 6.6 8.5 8.0