Isotope dilution-liquid chromatography/electrospray ionization-tandem mass spectrometry for the determination of serum thyroxine as a potential reference method

A new candidate reference method is presented for the determination of thyroxine in serum. The method is based on isotope dilution‐liquid chromatography/tandem mass spectrometry using electrospray for ionization. The internal standard used was 13C6‐thyroxine, sample pretreatment consisted of protein precipitation and a two‐step liquid/liquid extraction procedure, HPLC was performed on a C‐18 column with an eluent containing methanol/water/formic acid (60:40:0.1, by volume), and finally thyroxine and its isotopically labeled analogue were measured in the selected reaction monitoring mode for the transitions m/z 777.7 → 731.7 and m/z 783.7 → 737.7, respectively. The detection limit for thyroxine was 6 pg, the within‐run coefficient of variation was 1.1%. The samples were measured in six‐fold: in duplicate on three independent days. The mean overall coefficient of variation of the method was 1.6%. The new method was evaluated by measuring nine control sera previously determined by an existing ID‐GC/MS method. The differences between the results of the two methods ranged from −1.6% to +3.3%, with a mean of +0.2%. © 1998 John Wiley & Sons, Ltd.