The measurement of free thyroxine by isotope dilution tandem mass spectrometry.
BACKGROUND
Most clinical chemistry laboratories measure free thyroxine (FT4) by an analogue (direct) method. Nevertheless, the validity of analogue FT4 immunoassays has been questioned and patient's results using this approach frequently do not fit in with the clinical presentation. Because of these problems we routinely send out all direct free T4's that are < 2.5th percentile and many that are > 97.5th percentile for measurement by equilibrium dialysis, the gold standard method. In approximately half of these cases, the FT4 by equilibrium dialysis was normal. We developed a rapid, reliable free T4 method employing isotope dilution tandem mass spectrometry and compared our results with both the analogue (direct) free T4 and equilibrium dialysis procedures.


METHODS
An API-4000 tandem mass spectrometer (Sciex, Toronto, Canada) equipped with TurboIonSpray and Agilent HPLC system was used employing isotope dilution with deuterium labeled internal standard (IS=l-thyroxine-d2). Serum was filtered through the Centrifree YM-30 ultrafiltration device by centrifugation, IS added to the ultrafiltrate, and 400 microL injected onto a C-18 column. After washing, the switch valve is activated and a methanol gradient allows for elution of both T4 and the IS into the LC/MS/MS system. Quantitation was by MRM analysis in the negative mode. Equilibrium dialysis was performed by the Nichols method and analogue free T4 results were obtained on the Dade Dimension RxL.


RESULTS
The within-day and between-day CV's were < 7.1% at all concentrations tested. The results correlated well with equilibrium dialysis (Eq Dial=0.971 MS+0.041, n=68, Syx=1.381, r=0.954). A poor correlation was found with the analogue (direct) free T4 method (IA=0.326 MS+6.27, n=154, Syx=1.96, r=0.459).


CONCLUSIONS
Samples can be processed in batches of 30. The free T4 tandem MS method has a rapid turn-around-time vs the equilibrium dialysis procedure, with a chromatographic run time of 8 min per sample.
Soldin
Steven J
SJ
Bioanalytical Core Laboratory, Georgetown Clinical Research Center, Georgetown University, Washington, DC 20007-2197, USA. sjs44@georgetown.edu
Soukhova
Nadia
N
Janicic
Natasa
N
Jonklaas
Jacqueline
J
Soldin
Offie P
OP
D002853:Chromatography, Liquid / Q000379:methods / Q000592:standards; D003903:Deuterium; D005260:Female; D006801:Humans; D007201:Indicator Dilution Techniques; D013058:Mass Spectrometry / Q000379:methods / Q000592:standards; D011247:Pregnancy; D012016:Reference Values; D012680:Sensitivity and Specificity; D013696:Temperature; D013974:Thyroxine / Q000032:analysis* / Q000097:blood / Q000592:standards
D003903:Deuterium; D013974:Thyroxine