Biol. Pharm. Bull. 30(9) 1653—1656 (2007)

toxin, have been commonly used for the treatment of congestive heart failure and other cardiac diseases. In addition to the narrow therapeutic range of the serum digitalis glycosides, their bioavailability is easily affected by kidney or liver failure and the dosing of other medicines. Therefore, it is important to accurately measure the digitalis glycosides in sera from digitalized patients. Dg has been more commonly used than digitoxin and its therapeutic range in serum is 0.8—2.0 ng/ml. Immunoassay is routinely used for therapeutic drug monitoring (TDM) of Dg. However, the commercially available antibodies show a high cross-reactivity with Dg metabolites, such as digoxigenin bisdigitoxoside (Bis-Dggenin), digoxigenin monodigitoxoside (Mono-Dggenin) or digoxigenin (Dggenin) (Fig. 1), which were formed by the successive cleavage of the sugar moiety, as well as with endogenous digitalis-like immunoreactive substances (DLIS). These cause a false-elevation or -lowering of the serum Dg concentration due to the inappropriate prescriptions of Dg dosages by physician. To overcome this problem, Ikeda et al. designed a new hapten of Dg and developed a radioimmunoassay (RIA) for Dg in human serum, which can clearly discriminate Dg from the Dg metabolites (Bis-Dggenin, Mono-Dggenin and Dggenin), but not DLIS. Liquid chromatography/mass spectrometry (LC/MS) also seems to be suitable for this purpose because of its high sensitivity, specificity and without interfering from DLIS. Recently, the determination of digitoxin or Dg using LC/MS has been reported, but its application to real samples obtained from digitalized patients followed by comparison with the data obtained from immunoassay has only been done in our report for determining digitoxin. In this study, we developed a determination method for Dg in human serum using stable isotope dilution LC/electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) and applied this method to real samples. The data obtained in this study were compared to those obtained by using RIA with the anti-serum clearly discriminating Dg from its metabolites.