Convergence of Three Methods to Resolve Discrepant Immunoassay Digitoxin Results

Abstract

We have observed significant analytical discrepancies (20% to 220%) in digitoxin (Crystodigin; DTN) immunoassay results for 11 serum samples from patients taking digitoxin. We used three methods—HPLC, immunoassay, and liquid chromatography electrospray mass spectrometry (LC/MS)—to investigate the possible sources of these discrepancies. DTN is indicated for treatment of heart failure, atrial flutter, and supraventricular tachycardia (1). This drug is the most slowly excreted cardiac glycoside, with a half-life of 4 to 10 days (2). Administration and therapeutic monitoring of DTN are more popular in European countries than in the US. Plasma concentrations of 15–25 μg/L are considered therapeutic (3), and values >35 μg/L have been associated with toxicity in 80% of patients (4). Biotransformation of DTN is complex. It is oxidatively cleaved to bisdigitoxoside (bis-DTN) in the liver, catalyzed by a specific cytochrome P-450 enzyme (5). Further deglycosylation to monodigitoxoside (mono-DTN) and digitoxigenin has been documented in mice, rabbits, and guinea pigs (5). DTN can be hydroxylated at position 12 to form digoxin. Glucuronidation of mono-DTN and, to a lesser extent, of digitoxigenin to form polar conjugates has also been documented (6). In isolated guinea pig atria, the positive inotropic actions of DTN and its metabolites have been ranked in order of decreasing activity as follows: mono-DTN, bis-DTN, digoxin, DTN, digitoxigenin (7). Discrepant patients’ samples had been analyzed by the following immunoassays: ACS:180® (Chiron Diagnostics), which utilizes a monoclonal antibody with chemiluminescence detection; TDx® (Abbott Labs.), which utilizes a polyclonal antibody and fluorescence polarization detection; and DPC (Diagnostic Products Corp.), which uses a polyclonal antibody with RIA technology. Patients’ samples had been collected in Germany in accordance with the institutional human studies regulations of that country. All immunoassays had been performed according to their manufacturers’ instructions. Initially, we expected the deglycosylated congeners of …