[Skip to Content](https://www.sigmaaldrich.com#main-content) [![Merck](https://www.sigmaaldrich.com/static/logos/purple/merck.svg)](https://www.sigmaaldrich.com/CZ/en) Products Cart0 CZEN Products [Login / Register](https://www.sigmaaldrich.com/oidc-sign-in) [Order Lookup](https://www.sigmaaldrich.com/CZ/en/order-lookup) [Quick Order](https://www.sigmaaldrich.com/CZ/en/quick-order) Cart0 [Home](https://www.sigmaaldrich.com/CZ/en)[Solid Phase Extraction (SPE)](https://www.sigmaaldrich.com/CZ/en/applications/analytical-chemistry/sample-preparation/solid-phase-extraction)Online SPE/LC-MS Method for the Rapid Analysis of Thyroid Hormones in Plasma # Online SPE/LC-MS Method for the Rapid Analysis of Thyroid Hormones in Plasma __Xiaoning Lu, David Bell__ ### Using Ascentis® Express Fused-Core® Columns for Trapping and Separation The power of bonded phase selectivity in both sample prep and separation is demonstrated here. The Ascentis Express RP-Amide was shown to be a better trapping phase than conventional C8, and the Ascentis Express Phenyl-Hexyl phase provided baseline resolution of the closely-related compounds. The technique uses standard hardware and is a viable approach to solve other clinical and bioanalytical challenges. ## Introduction Thyroid hormones play critical roles in the regulation of biological processes such as growth, metabolism, protein synthesis, and brain development. Specifically, thyroid hormones 3,3´,5,5´-tetraiodo- L-thyronine (thyroxine or T4) and 3,3´,5-triiodo-L-thyronine (triiodothyronine or T3) are essential for the development and maintenance of normal physiological functions. For a clinical laboratory, measurements of total T4 and total T3, along with estimates of free T4 (FT4) and free T3 (FT3) are important for the diagnosis and monitoring of thyroid diseases. The physiological role and need for testing of 3,3´,5´-triiodo-L-thyronine (reverse triiodothyronine or rT3) is debated in clinical research circles. ## Analytical Challenges of Thyroid Hormone Measurement Thyroid hormones are typically measured by an immunoassay technique that is subject to assay interference and easily perturbed by changes in protein levels that alter the free hormone availability1. Liquid chromatography with mass spectrometric detection (LCMS) has been reported to offer superior specificity and speed over immunoassays for the determination of thyroid hormones in biological matrices, such as serum, plasma, and tissue. Nevertheless, the reported sample preparation procedures, typically by liquid-liquid extraction followed by solid phase extraction (SPE), involve multiple time-consuming steps, and are less compatible with automation2,3. ## Study Goal and Overview of Approach The present work exploited online SPE with LC-MS for the rapid determination of T4, T3, and rT3 from plasma. The structures of these compounds appear in __Figure 1__. A 100 µL aliquot of rabbit plasma was spiked with T4, T3, and rT3. Protein was precipitated by addition of 25 µL ZnCl2, and 200 µL methanol, vortex agitation for 1 minute, and centrifugation at 9,000 g for 3 minutes. The resulting supernatant was collected and injected into the online SPE/LC-MS system described below. The LC-MS conditions are described in __Table 1__. ![Chemical Structures of Thyroid Hormone Analytes](https://www.sigmaaldrich.com/content/dam/cms-commons/sigmaaldrich/marketing/global/images/technical-documents/protocols/analytical-chemistry/solid-phase-extraction/t214001-thyroid-hormones-fig-1.gif "Chemical Structures of Thyroid Hormone Analytes") __Figure 1.__Chemical Structures of Thyroid Hormone Analytes | | | |------------------|-------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------| | Instrument | Shimadzu LCMS-8030 | | Trapping Column | Ascentis Express RP-Amide (Product No. 53514-U) or Ascentis Express C8 guard cartridges (Product No. 53509-U), both 5 mm x 2.1 mm I.D., 2.7 µm particles; Ascentis Express guard cartridge holder (Product No. 53500-U) | | HPLC Column | Ascentis Express Phenyl, 5 cm x 2.1 mm I.D., 2.7 µm particles (Product No. 53334-U) | | Mobile Phase | (A) water; (B) methanol, both with 0.1% acetic acid | | Column Pressure | 1800 psi (88 bar) | | Detection | MS, ESI(+), MRM mode (777.7/731.8 for T4; 651.8/605.5 for T3 or rT3) | | Injection Volume | 5 µL | Table 1. Online SPE LC-MS Method ## Online SPE/LC-MS Program The online SPE/LC-MS system shown in __Figure 2__ comprises a trapping column, a switching valve and an LC-MS instrument. The program consists of three distinct steps outlined in __Table 2__. The valve switches between position 1 for loading/washing (__Step 1__) and equilibration (__Step 3__), and position 2 for LC-MS analysis (__Step 2__). In Step 1, samples are loaded onto the trap and washed with mobile phase containing low percent organic and high flow rate to remove salts and other interferences which are directed to the waste. In Step 2, the analytes are eluted from the trap, separated and detected by the LC-MS at optimum flow for both chromatographic separation and MS signals. In Step 3, the system returns back to valve position 1 for re-equilibration under the sample loading/washing conditions. ![Configuration of the LC-MS System and Valve Positions for Online SPE/LC-MS](https://www.sigmaaldrich.com/content/dam/cms-commons/sigmaaldrich/marketing/global/images/technical-documents/protocols/analytical-chemistry/solid-phase-extraction/t214001-thyroid-hormones-fig-2.gif "Configuration of the LC-MS System and Valve Positions for Online SPE/LC-MS") __Figure 2.__Configuration of the LC-MS System and Valve Positions for Online SPE/LC-MS Final Online SPE/LC-MS ProgramMinFlow (mL/min)ValvePump B% 1\. Sample loading/washing0.00 2.79 2.800.4 0.4 0.251 1 15 5 5 2\. LC-MS/MS3.00 5.00 8.000.25 0.25 0.252 2 25 70 70 3\. Equilibration8.01 8.09 8.10 12.00.25 0.25 0.4 0.41 1 1 170 70 5 5 Table 2.Online SPE/LC-MS Program ## Choosing the Trapping Column The choice of the right trapping column is critical for successful online SPE/LC-MS method development as it affects both trapping efficiency (e.g., the recovery of the analytes) and the downstream LC separation. As a rule of thumb, the trapping column should be less retentive than the analytical column to avoid possible peak broadening. Two guard cartridges (traps), Ascentis Express Fused- Core RP-Amide and C8, of the same dimensions were evaluated. As can be seen in __Figure 3__, both trapping columns led to comparable sharp peak shape (high separation efficiency). However, the RP-Amide delivered higher peak responses than the C8 trap under the trapping conditions with 5% methanol (top panel) as the washing solvent. Further increase of the methanol content to 20% in the washing solvent (__Table 3__) resulted in a 50% signal decrease on the C8 traps, but almost no change in signals with the RP-Amide traps (__Figure 3, bottom panel__). These results indicate better washing can be achieved with minimal sample loss with RP-Amide traps. ![Comparison of the LC-MS Chromatograms with Online Trapping with C8 and RP-Amide Guard Cartridges](https://www.sigmaaldrich.com/content/dam/cms-commons/sigmaaldrich/marketing/global/images/technical-documents/protocols/analytical-chemistry/solid-phase-extraction/t214001-thyroid-hormones-fig-3.gif "Comparison of the LC-MS Chromatograms with Online Trapping with C8 and RP-Amide Guard Cartridges") __Figure 3.__ Comparison of the LC-MS Chromatograms with Online Trapping with C8 and RP-Amide Guard Cartridges LC Program:MinFlow (mL/min)ValvePump B% 1\. Sample loading/washing0 0.5 2.80.4 0.4 0.251 1 15 20 20 2\. LC-MS/MS3.0 5.0 8.00.25 0.25 0.252 2 220 70 70 3\. Equilibration8.0 8.1 12.00.25 0.4 0.41 1 170 5 5 Table 3. Online SPE/LC-MS Conditions (high organic sample wash) ## Summary An online SPE/LC-MS method was developed and described here for the determination of the thyroid hormones T4, T3, and rT3 from plasma. The method uses an Ascentis Express RP-Amide cartridge to trap the analytes, and an Ascentis Express Phenyl-Hexyl column to resolve them. Compared to the commonly used C8 trap, the RP-Amide trap gave higher analyte signals and was compatible with 20% methanol washing. Another advantage of the RP-Amide traps is their compatibility with 100% aqueous mobile phases and samples. This is due to the hydrophilic nature of the amide-embedded phase, which makes it very useful for polar analytes. __Legal Information__ Ascentis is a registered trademark of Sigma-Aldrich Co. LLC Fused-Core is a registered trademark of Advanced Materials Technology, Inc. ## Materials Sorry, an unexpected error has occurred Response not successful: Received status code 500 ### References 1\. Kahric-Janicic N, Soldin SJ, Soldin OP, West T, Gu J, Jonklaas J. 2007. Tandem Mass Spectrometry Improves the Accuracy of Free Thyroxine Measurements During Pregnancy. Thyroid. 17(4):303-311. [https://doi.org/10.1089/thy.2006.0303](https://doi.org/10.1089/thy.2006.0303) 2\. Tai SS, Sniegoski LT, Welch MJ. 2002. Candidate Reference Method for Total Thyroxine in Human Serum. 48(4):637-642. [https://doi.org/10.1093/clinchem/48.4.637](https://doi.org/10.1093/clinchem/48.4.637) 3\. Wang D, Stapleton HM. 2010. Analysis of thyroid hormones in serum by liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem. 397(5):1831-1839. 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