Thyreostats (TSs) are thioamide anti-thyroid drugs. They act by inhibiting production of hormones by the thyroid gland, which results in weight gain caused by increased filling of the gastro-intestinal tract and water retention in edible tissues.
European Union (EU) regulations prohibit the use of TSs as growth promoters in food-producing animals (Council Directive 81/602/EC) [1] because of the potential teratogenic and carcinoge-nic effects of TSs. Council Directive 96/23/EC [2] classifies TSs as compounds with anabolic properties (group A2), and their control is mandatory for slaughter animals. In Poland, the authorities monitor TSs as part of the official control of veterinary drug residues. TS residues have been found mainly in the urine and thyroid gland tissue of bovine animals. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods for determining TSs in these target matrices have been developed and validated according to the requirements of Commission Decision 2002/657/EC [3].
The isolation of TSs from urine is problematic because they are amphoteric, highly polar, low-molecular-weight molecules, which undergo rapid tautomerization and oxidation, which makes them difficult to isolate and analyse using LC-MS/MS. The majority of currently available analytical methods circumvent these difficulties by using derivatization before analysis, which increases the molecular weight, lowers the polarity, and stabilizes the molecule in a single tautomeric form. Several LC-MS/MS methods have been reported for determining TSs in various biological matrices (urine, thyroid gland and muscle tissues) [4-6]. Most of these methods involved a derivatization step with 3-iodobenzylbromide [6-8]. These methods differed in terms of sample clean-up (silica solid-phase extraction cartridges or gel permeation chromatography).
The aim of this study was to develop and validate a UHPLC-MS/MS method for the determination and confirmation of tapazole (TAP), thiouracil (TU), methylthiouracil (MTU), propylthiouracil (PTU) and phenylthiouracil (PhTU) in bovine urine without a derivatization step. In 2007, the Community Reference Laboratories recommended a mass concentration of 10 μg/L in urine for control purposes [2]. This means that in EU laboratories the decision limit (CCα) and detection capability (CCβ) of a method must be lower than 10 μg/L.
Few LC-MS/MS and UHPLC-MS/MS methods have been reported for determining TSs in urine without derivatization. The first LC-atmospheric pressure chemical ionization (APCI)-MS/MS method was introduced by Blanchflower et al. [9]. Samples were extracted with ethyl acetate and the extracts were cleaned-up using silica solid-phase cartridges. Mercaptoethanol was used to reduce protein binding and improve recoveries. Limits of detection (LOD) were in the region of 25 μg/L, which is above the recommended mass concentration (10 μg/L).
Chiesa et al. [10] reported an LC-ESI-MS/MS method for determining TSs. Salting-out-assisted liquid-liquid extraction (LLE) was used for urine samples. The method was validated in accordance with Commission Decision 2002/657/EC [3]. The CCα and CCβ values were 6.9-7.3 μg/L and 8.5-9.7 μg/L, respectively.
A method for TS conformation based on UPLC-ESI-MS/MS was developed by Vanden et al. [11]. This method involved a reduction step with dithiothreitol under denaturation conditions at 65 ℃, followed by LLE with ethyl acetate. This analytical procedure was validated according to the EU criteria (Commission Decision 2002/657/EC [3]). The CCα and CCβ values ranged from 1.1 to 5.5 μg/L and from 1.7 to 7.5 μg/L, respectively.
In this study, the matrix proteins were denatured to disrupt protein-TS interactions, as reported by Blanchflower et al. [9]. The samples were extracted/cleaned-up with ChemElut extraction cartridges. Validation was performed in agreement with the criteria set in Commission Decision 2002/657/EC [3].
Standard TU, TAP, MTU, PTU, PhTU samples, and dimethylthiouracil (DMTU) for use as an internal standard (IS) were obtained from Sigma Aldrich (St. Louis, MO, USA). The purity of PhTU was higher than 95%, the purity of other standards were higher than 99%. IS was higher than 99%. Acetonitrile (LC-MS grade) and ethyl acetate (LC grade) were provided by Baker (Deventer, Netherlands). 2-Mercaptoethanol was obtained from Sigma Aldrich (St. Louis, MO, USA). Sample extraction and clean-up were performed using Chem Elut-3 mL unbuffered cartridges from Agilent Technologies (USA). Ultrapure water was obtained using a Milli-Q system (Millipore, Bedford, MA, USA). The filters for extract filtration were obtained from Millipore (Millex GV, 0.45 μm).
Phosphate-buffered saline (PBS, pH 7) was prepared by 1.2 g dissolving disodium hydrogen phosphate, 0.2 g potassium dihydrogen phosphate, and 0.2 g potassium chloride in 900 mL Milli-Q water. The pH was adjusted to 7 with 2 mol/L hydrochloric acid. The solution was made up to 1 L.
Individual stock solutions of reference compounds of mass concentration 1 g/L were prepared in methanol (stable for at least 3 months). Stock solutions of TU, TAP, MTU, PTU, and PhTU were combined and diluted in methanol to prepare working standard solutions of mass concentration 0.5 g/L (stable for at least 1 month). The working standard solutions were added to urine samples in appropriate microliter aliquots corresponding to 5, 10, and 15 μg/L.
Stock solutions of DMTU were combined and diluted in methanol to prepare a working IS solution of mass concentration 0.5 mg/L (stable for at least 1 month). All standard solutions were prepared in amber volumetric flasks and stored at 4 ℃.
Urine samples were obtained from the Polish residue control plan farm. The urine samples were centrifuged at 3 000 r/min for 10 min, filtered through a 0.45 μm poly(vinylidene fluoride) (PVDF) membrane, and stored at -20 ℃.
Bovine urine (1 mL) was placed in a 10 mL glass tube, and 50 μL working IS solution (5 mg/L DMTU) and 10 μL 2-mercaptoethanol were added. The sample was vortexed and left for 5 min to equilibrate. A volume of 2 mL pH 7 PBS buffer was added, and the mixture was vortexed. The sample was loaded on the ChemElut cartridge and equilibrated for 10 min. Elution was performed with 15 mL tert-butyl methyl ether. The sample solution was then evaporated to dryness at 50 ℃ under a nitrogen stream. The residue was dissolved in 200 μL water containing 0.2% (v/v) formic acid and filtered through a 0.45 μm PVDF membrane into an amber vial.
UHPLC analysis was performed using an LC-20ADXR liquid chromatography system (Shimadzu Corporation, Japan). Chromatographic separation was performed on an Acquity UPLC SS T3 column (100 mm×2.1 mm, 1.3 μm). The column oven temperature was set at 40 ℃. The flow rate was set at 300 μL/min and the injection volume was 10 μL. The mobile phase consisted of solvent A (0.2% (v/v) formic acid in water) and solvent B (acetonitrile). The linear gradient was 0-0.1 min, 5% A; 0.1-8.0 min, from 5%A to 50%A; 8.0-9.0 min, from 50%A to 5% A; and 9.0-15.0 min, 5%A. The total run time was 15 min.
MS/MS was performed using an AB Sciex Triple Quad 5500 mass spectrometer (Concord, Ontario, Canada) in positive ESI mode. MS was performed in multiple reaction monitoring (MRM) mode. Two MRM transitions were monitored for each compound. The individual precursor and product ions for each analyte, with their respective collision energies, are listed in Table 1.
The other ESI parameters were as follows: ion spray voltage was 3 500 V; source temperature was 400 ℃; curtain gas was 2×105 Pa (30 psi); collisionally activated dissociation gas was medium, ion source gas 1 was 0.28 MPa and gas 2 was 0.41 MPa. Nitrogen served as both the turbo and collision gases. The dwell time was set at 100 ms. The Q1 and Q2 quadrupoles were both maintained at unit resolution. The Analyst 1.6.3 software controlled the UHPLC-MS/MS system and processed the date.
The method was validated according to the criteria in Commission Decision 2002/657/EC [3]. The LC-MS/MS identification criteria were verified throughout the validation study by monitoring the relative retention times, signal-to-noise ratios, and relative ion intensities. The LC-MS/MS identification criteria set out in the legislation were verified throughout validation of the method. The following performance characteristics were checked: specificity, linearity, accuracy/trueness (recovery), precision (repeatability and within-laboratory reproducibility), CCα, and CCβ.
To evaluate possible interferences in the method, the method specificity was verified by analysing 20 blank samples of different bovine urine.
For matrix calibration curve construction, blank urine samples were fortified with working standard solutions at five levels, corresponding to 0, 5, 10, 15, and 20 μg/L. The samples were analysed on three different days and the calibration curve linearity was expressed by the correlation coefficient.
The accuracy/trueness (IS corrected recovery) and precision/repeatability were assessed by fortifying six blank urine samples at each of four mass concentration levels, i. e. 5, 10, 15, and 20 μg/L. The samples were analysed on the same days using the same instrument and different operators. The accuracy was assessed based on the relative recovery. The relative standard deviation (RSD) was calculated to determine the repeatability.
Only the within-laboratory reproducibility was determined. Two series of six replicates of fortified samples at the same concentration levels of analysed compounds used for the repeatability analysis were analysed by different operators on different days. The overall RSD was calculated as the within-laboratory reproducibility.
To comply with Commission Decision 2002/657/EC [3], the CCα and CCβ were determined. CCα means the limit at and above which it can be concluded with an error probability of α=0.1% that the sample is non-compliant in case there is a non-zero test limit. CCβ means the smallest content of the substance that may be detected, identified or quantified in a sample with the error probability of β=95%.
The CCα and CCβ values were determined using a matrix calibration curve procedure according to case 1 in ISO 11843-2-constant standard deviation [12]. The CCα and CCβ values were calculated using two calibration curves (at five mass concentration levels 0, 5, 10, 15, and 20 μg/L) from six different experiments on different bovine urine matrices and different days. The curves were constructed using analyte/IS peak area ratios versus analyte concentrations.
Direct analysis of urine samples using LC-MS/MS often results in ion suppression caused by interference from endogenous compounds. Sample treatment is therefore often performed to avoid these adverse effects. In the determination of TSs (without derivatization) in urine, LLE is generally the technique of choice for producing clean extracts that can be directly injected into the LC-MS/MS system [9, 11, 13]. Because of the well-known drawbacks of LLE (i. e. emulsion formation and poor phase separation), in the present study, the TSs from urine were extracted/cleaned-up using supported LLE cartridges (ChemElut). Generally, extraction involves adsorption of aqueous samples on diatomaceous earth, followed by passing a non-miscible solvent through the cartridge, with further extraction and elution of analytes. The extraction efficiencies of TSs from spiked blank samples were determined. In our preliminary studies, ethyl acetate and tert-butyl methyl ether were compared. The bovine urine samples were spiked with TSs at 10 μg/L. The sample was passed through the cartridge. The TSs were eluted using ethyl acetate and tert-butyl methyl ether. The extraction was assessed from the recovery. The recoveries of the TSs were obtained by the ratio of the relative peak area of the analyte to IS in the spiking matrix with that of the standard sample in corresponding concentration.
Better recoveries for all compounds were obtained when tert-butyl methyl ether was used. The overall recoveries from urine were 47%, 47%, 63%, 102%, and 105% for TU, TAP, MTU, PTU, and PhTU, respectively. The lower recoveries for TAP, TU, and MTU reflect the difficulties in extracting these polar compounds from aqueous solutions. Tert-butyl methyl ether was used in this work.
The specificity of the method was tested by analysing 20 different blank urine samples and urine samples spiked at 10 μg/L to verify the absence of potential interfering compounds at the analyte retention times. All the chromatograms obtained in the validation study show good stability of the retention times for the spiked samples, with a relative deviation always better than ±2.5%. For each transition, the chromatograms show a significant increase in peak area and intensity at its specific retention time compared with those of the blanks, assuming a signal/noise ratio of at least 3. Figs. 1 and 2 show the UPLC-MS/MS chromatograms of blank urine samples and urine samples spiked at 10 μg/L.
For the blank samples, interfering peaks were observed at the TU retention times for the 129/112 and 129/70 ion transitions. However, a comparison of the chromatograms of the blank bovine urine samples with those of the bovine urine samples spiked at 10 μg/L clearly shows that the TU interference is low compared to that for a positive sample at low levels. TU is suspected to occur naturally. Pinel et al. [8] showed a correlation between cruciferous-based animal feed and the identification of TU in urine. The mass concentrations of TU eliminated in urine during that diet were low and never exceeded 10 μg/L [8]. An European Union Reference Laboratory guidance paper [14] acknowledged this by stating that TU mass concentrations below 10 μg/L could have a natural origin, arising from Brassicaceae consumption. In our work, the TU mass concentration was between 1.1 and 5.2 μg/L. Therefore blank urine samples with TU mass concentrations lower than 2 μg/L were selected for method validation.
The concentrations of the analytes in the samples were calculated by matrix calibration using an IS (DMTU). Calibration curves with 1/x weighting were plotted for each individual analyte. The matrix calibration curves were linear over the range 0-20 μg/L. The correlation coefficients were between 0.989 and 0.998.
The accuracy/trueness calculated from the spiked recovery, the repeatability and within-laboratory reproducibility, which indicated the precision, at four concentration levels were summarized in Table 2. The average recoveries of all the compound ranged from 92% to 108% and fulfil the criteria put forward in EC/2002/657 [3], which stated that a mass fraction ranging from 1 to 10 μg/L should give a mean recovery of 70% to 110%, and a mass concentration of 10 μg/L or more should give a mean recovery of 80% to 110%. The recoveries obtained using this analytical method were highly satisfactory.
Good repeatability and within-laboratory reproducibility were obtained for all TSs at all levels, with RSDs less than 15% and 26%, respectively. The precision (RSD) was not evaluated using the Horwitz equation; too high values would be obtained because of the low concentration range used. However, in accordance with Commission Decision 2002/657/EC [3], the RSD obtained for a mass fraction lower than 100 μg/L was as low as possible.
The calculated CCα and CCβ values for the TSs are listed in Table 3. The mean CCα values were 3.1-6.1 μg/L and the mean CCβ values were 4.0-7.4 μg/L. From the laboratory point of view, this means that the CCα values for conformation methods and CCβ values for screening methods should be lower than the recommended value (10 μg/L). The calculated CCα values were verified by spiking a minimum of six blank matrices at a concentration corresponding to the calculated decision limit. For all such spiked samples, the analytes were detected and correctly identified.
The present procedure was also used to analyse TSs in porcine urine. The UHPLC-MS/MS method was used without any modification. Porcine urine samples were spiked with TSs at the same concentration levels as the bovine urine samples. In all cases, the results obtained for parameters such as linearity, accuracy, repeatability, and reproducibility were virtually the same as those for bovine urine. Moreover, the CCα and CCβ values were similar to those obtained for bovine urine. The mean CCα and CCβ values were 2.9-5.4 μg/L and 4.0-6.9 μg/L, respectively. The accuracy, repeatability, and reproducibility results for porcine urine samples are presented in Table 4.
This UHPLC-MS/MS method enabled simultaneous determination of five TS residues in bovine and porcine urine. The method involves easy sample preparation using a ChemElut extraction cartridge, without a derivatization step. The method gives good accuracy and can be used to quantify these residues at levels below the recommended value. This method was validated in accordance with Commission Decision 2002/657/EC [3] and is used in routine analysis in our laboratory.
2017,
Vol. 35 Issue (10): 1048-1054
PDF
2017,
Vol. 35 Issue (10): 1048-1054
PDF