LC/MS with Online Solid Phase Extraction (SPE-LC/MS) of Thyroid Hormones in Plasma on Ascentis® Express Phenyl-Hexyl using RP-Amide Traps

LC/MS with Online Solid Phase Extraction (SPE-LC/MS) of Thyroid Hormones in Plasma on Ascentis® Express Phenyl-Hexyl using RP-Amide Traps

Conditions

sample preparation SPE (Solid Phase Extraction)
column Ascentis Express Phenyl-Hexyl, 5 cm x 2.1 mm I.D., 2.7 µm particles (53334-U)
column temp. ambient
mobile phase [A] water; [B] methanol, both with 0.1% acetic acid
gradient 5% B for 3 min; to 70% B in 2 min, hold 3 min
pressure 1800 psi
sample protein precipitated rabbit plasma spiked with desired amount of T4, T3 and rT3
injection 5 µl
detector MS, ESI(+), MRM mode (777.7/731.8 for T4; 651.8/605.5 for T3 or rT3)

Description

Analysis Note An online SPE-LC/MS method is shown here for the determination of thyroid hormones in blood plasma. The method exploits RP-Amide or C8 as the trapping column and a phenyl-hexyl phase as the analytical column, respectively. Under the optimized conditions, both RP-Amide and C8 effectively trapped the thyroid hormones extracted from spiked rabbit plasma sample which had been protein precipitated. However, the RP-Amide traps were shown to be superior to the commonly used C8 traps because they provided higher analyte signals and recoveries. Additionally, RP-Amide traps are more flexible with washing solvent as they are compatible with 100% aqueous mobile phases and can be used with up to 20% methanol with minimal sample loss. Separation was carried out using an Ascentis Express 2.7 μm Phenyl-Hexyl column. The highest grade solvents provided clean, robust operation. Cerilliant CRMs provided reliable quantification.
Categories Analytical Chromatography, SPE Applications, Hormones, Immunosuppressants
Featured Industry Clinical
Life Science and Biopharma
Legal Information Ascentis is a registered trademark of Sigma-Aldrich Co. LLC
suitability application for LC-MS, application for SPE

Materials

     
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