Rapid solid-phase-extraction for the quantitative analysis of serum iodothyronine profiles
Carolin Hoefig, Franziska Wohlgemuth, Jeannette Klein, Oliver Blankenstein, Ulrich Schweizer & Josef Köhrle
A novel solid-phase-extraction (SPE) method followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) was established for the simultaneous detection and quantification of thyroid hormone profiles in one sample of human or mouse serum.
Recovery during preanalytical steps was monitored by addition of an internal deuterated standard (d5-T4) to 200 μl serum, which was deproteinized by adding a precipitating agent consisting of 1% formic acid in acetonitrile (1:3, v/v). After vortexing precipitated protein was removed by centrifugation and the resulting supernatant was transferred to a HybridSPE cartridge. After applying vacuum, the resulting eluent was concentrated in a speedvac prior to LC-MS/MS analysis. This new SPE cartridge offers the advantage of removing proteins and phospholipids thus reducing ion-suppression in LC-MS/MS measurements.
LC-MS/MS analyses were performed using a Shimadzu UFLC system and a 4000QTRAP triple-quadrupole tandem mass spectrometer equipped with TurboIonSpray interface. The detection was performed using positive electrospray ionization (ESI+) in the multiple reaction monitoring (MRM) mode. Chromatographic separation of all iodothyronines was achieved using a Synergi Polar-RP 80-Æ column (150×2 mm) with Guard Cartridge using a gradient elution program at a flow rate of 300 μl/min. The optimized elution parameters were generated by a gradient of mobile phase A (wateracetonitrileacetic acid) and mobile phase B (acetonitrilewateracetic acid).
Using this rapid SPE method it is possible to quantitatively and simultaneously detect T4, T3, rT3 and 3,5-T2 in biological concentrations within a single sample of human as well as in mouse serum in good correlation to antibody based routine procedures. T4, the most abundant iodothyronine with the highest avidity to serum distributor proteins showed the lowest recovery (66%). Further standardization and validation experiments are required for measurement of clinical cohorts and samples of animal models.
Supported by Graduate College 1208, Deutsche Forschungsgemeinschaft (DFG).