Analysis of Digoxin and Metildigoxin in Whole Blood Using Solid-Phase Extraction and Liquid Chromatography Tandem Mass Spectrometry

A simple and rapid UPLC/MS/MS method has been developed and validated for the analysis of digoxin and metildigoxin in whole blood. Samples were prepared by SPE extraction with Oasis HLB columns. Separation was achieved with an ACQUITY UPLC HSS T3 column ( ; 1.8？μm), at 35°C. The mobile phase consisted of acetonitrile (70%) and ammonium formate 5？mM (30%). Total run time was 1.5？min operating at isocratic mode with a flow rate of 0.3？mL/min. Mass spectrometry detection was performed by positive mode electrospray, on the ammonium adducts with two transitions for each analyte and one for the IS (d3-digoxin). The method proved to be specific and linear over the range (0.3–10)？ng/mL. This technique also showed high sensitivity with a 0.09？ng/mL LOD and a 0.28？ng/mL LOQ, for both substances. Percentage recovery ranged from 83 to 100% for digoxin and from 62 to 94% for metildigoxin. The intra- and interday precision CV were ≤10%. 1. Introduction Digitalis glycosides are the drugs of choice for the treatment of congestive heart failure and certain disturbances in cardiac rhythm, producing a positive inotropic activity and increase, myocardial contractility [1]. Some authors state that digoxin is clinically the most commonly used cardiac glycoside [2]. Nevertheless, in Portugal, metildigoxin is also a common cardiac glycoside, being the second more frequently used [3]. Digoxin (Figure 1) is metabolised mainly in the liver [2], by stepwise removal of the sugar moieties to form digoxigenin, which is further metabolised to inactive metabolites which may be excreted in free or conjugated forms. Reduction to dihydrodigoxin, relatively inactive, also occurs [4]. However, digoxin is metabolised in man in a small percentage [5]. Effectively, digoxin is excreted mainly unaltered in the urine, with up to 80% of a dose excreted in urine in 7 days being 27% of the dose excreted in the first 24？h [4]. Digoxin can be found as the result of its administration or due to metabolism since it is a metabolite of deslanoside, digitoxin, lanatoside C and metildigoxin [4]. Metildigoxin (Figure 2) is metabolized by demethylation into digoxin and by hydrolysis into the bis- and monoglycosides. About 75% of a dose is excreted in urine over a several days period, with about 25–30% excreted in the first 24？h. About 30–50% of the material excreted in urine corresponds to unchanged drug, being 15% conjugated bis- and monoglycosides and the remains are digoxin [4]. Figure 1: Chemical structure of digoxin (C 41H 64O 14). Figure 2: Chemical structure of metildigoxin (C 42H 66O 14). These