Abstract
A rapid hybrid solid phase extraction (HybridSPE®) protocol tailored to liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI–MS/MS) analysis, was developed for the determination of four thyroid hormones, L-Thyroxine (T4), 3,3′,5-triiodo-L-thyronine (T3), 3,3′,5′-triiodo-L-thyronine (rT3) and 3,3′-diiodo-L-thyronine (T2) in blood plasma from Glaucous gulls (Larus hyperboreus) and Baikal seals (Phoca sibirica). The use of target analyte specific 13C internal standards allowed quantification to be performed through the standard solvent calibration curves and alleviated the need to perform quantification with matrix match curves. The relative recoveries were 100.0–110.1 % for T4, 99.1–102.2 % for T3, 100.5–108.0 % for rT3, and 100.5–104.6 % for T2. The matrix effects ranged from −1.52 to −6.10 %, demonstrating minor signal suppression during analysis. The method intra-day precision (method repeatability, RSD %, N = 5, k = 1 day) and inter-day precision (method reproducibility, RSD %, N = 10, k = 2 days) at the 1 ng/mL concentration of fortification were 8.54–15.4 % and 15.4–24.8 %, respectively, indicating acceptable chromatographic peak stabilities for all target THs even at trace level concentrations. The method limit of detection (LOD) for T4, T3, rT3 and T2 was 0.17, 0.16, 0.30 and 0.17 ng/mL, respectively. The HybridSPE® protocol was simple and rapid (<1 min) upon application, while the HybridSPE® cartridge did not require (as in classical SPE cartridges) any additional equilibration nor conditioning step prior sample loading. A total of 46 blood plasma samples, 30 samples collected from Glaucous gulls and 16 samples collected from Baikal seals, were analyzed for thyroid hormones to demonstrate the applicability of the developed method in these wildlife species. The concentrations of T4 and T3 in blood plasma from the Glaucous gulls were 5.95–44.2 and 0.37–5.61 ng/mL, respectively, whereas those from Baikal seals were 3.57–46.5 and 0.45–2.07 ng/mL, respectively. In both species, rT3 demonstrated low detection rate, while T2 was not detected. Furthermore, cross-array comparison between the HybridSPE®-LC-MS/MS protocol and an established routine radioimmunoassay (RIA) kit-based method was performed for T4 and T3 concentrations from selected Baikal seal plasma samples.
Highlights
Thyroid hormones (THs) are endocrine molecules that regulate a wide range of biological processes in vertebrates, including growth, energy metabolism, neurodevelopment, protein synthesis and mito chondrial action [1,2]
The T4 and T3 concentrations that were determined in the blood plasma from Baikal seals by the HybridSPE® method, were cross-array compared with those con centrations determined by an established routine RIA kit-based method to further demonstrate measurement validity
The low uncertainty of the TH absolute recoveries with the HybridSPE® protocol was attributed to the rapid sample preparation that does not impact the stability of THs, and to the lack of an evaporation and reconstitution step that introduces by-default higher uncertainties [29]
Summary
Thyroid hormones (THs) are endocrine molecules that regulate a wide range of biological processes in vertebrates, including growth, energy metabolism, neurodevelopment, protein synthesis and mito chondrial action [1,2]. The transformation of T4 to T3 has a strong impact on T3 concentrations, which are significantly (even 2 orders of magnitude) lower than those of T4 in the blood of vertebrates [19] Another major challenge in LC-MS/MS bioanalysis of THs is the uncertainty of quantification due to matrix effects that derive mainly from the high endogenous protein and phospholipid content of serum and plasma in contrast to the natural occurring trace concentrations of THs [13,16]. The advantage of the HybridSPE® technique is that the extracted biological matrix is rapidly processed, and the gross amounts of proteins and phospholipids are removed, significantly decreasing the impact of the matrix during analysis [20] In this context, three different sample preparation techniques, including liquid–liquid (LLE), solid phase (SPE) and HybridSPE® extraction were assessed in ultrapure water and bovine blood serum. The T4 and T3 concentrations that were determined in the blood plasma from Baikal seals by the HybridSPE® method, were cross-array compared with those con centrations determined by an established routine RIA kit-based method to further demonstrate measurement validity
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